S1. Drug Transporters: Roles in New Drug Discovery and Development

Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Drug transporters are expressed in many tissues, such as the intestine, liver, kidney, and the brain, and play key roles in drug absorption, distribution and excretion. In this presentation, I will summarize the significant role played by drug transporters in drug disposition, focusing particularly on their potential use during the drug discovery and develop-ment process. The use of transporter function offers the possibility of delivering a drug to the target organ, avoiding distribution to other organs (thereby reducing the chance of toxic side-effects), controlling the elimination process, and/or improving oral bioavailability. It is useful to select a lead compound that may or may not interact with transporters, depending on whether such an interaction is desirable. The expression system of transporters is an efficient tool for screening the activity of individual transport processes. The changes in pharmacokinetics due to genetic polymorphisms and drug-drug interactions involving transporters can often have a direct and adverse effect on the therapeutic safety and efficacy of many important drugs. Vectorial transport across epithelial cells is involved in the absorption/uptake and elimination of drugs in the small intestine, liver and kidney. The vectorial transport of a large number of organic anions is achieved by uptake and efflux systems. We have established double-transfected MDCK II cells where hepatic uptake transporters and efflux transporters are expressed on the basal and apical membrane, respectively, as an in vitro model for hepatobiliary transport. This system is useful for drug discovery and development studies and for investigat-ing drug-drug interactions involving hepatobiliary transport. In this presentation, I will show you how to establish a physiologically based pharmacokinetic (PBPK) model that includes the transporter-mediated membrane transport processes and to investigate the effect of changes in transporter function on the pharmacokinetics and, ultimately, the pharmacological and/or toxicological effects. We constructed a PBPK model for statins and simulated the plasma and tissue concentrations in rats at various doses. The parameters of membrane transport were taken from our previous studies. The simulated data were comparable to the observed data that exhibited non-linear pharmacokinetics. We also tried to extrapolate from in vitro to in vivo in human taking thus obtained analyses in rats into consideration. This PBPK model can then be applied to predict changes of drug concentration in plasma and target organs caused by changes in transporter function and expression level.

References

(1) Giacomini K. M., Sugiyama Y., 2005. Membrane transporters and drug response. In: Brunton L. 11th Edition, McGraw-Hill Professional, New York, pp. 41-70.

(2) Maeda K. and Sugiyama Y. 2007 In vitro-In vivo Scale-up of Drug Transport Activities. In: “Drug Transporters” pp. 557-588, ed. by You G. and Morris M.E.,

(3) Kusuhara H and Sugiyama Y In vitro-in vivo extrapolation of transporter-mediated clearance in the liver and kidney. Drug Metab Pharmacokinet 24:37-52 (2009)

(4) Watanabe T, Kusuhara H, Maeda K, Shitara Y and Sugiyama Y Physiologically based pharmacokinetic modeling to predict transporter-mediated clearance and distribution of pravastatin in humans. J Pharmacol Exp Ther 328:652-662 (2009).

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S2. Use of Animal Models for the Study of ADRs

Jack P. Uetrecht

Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada

ADRs, in particular idiosyncratic ADRs (IDRs), represent a major challenge because of their unpredictable nature. A major impediment to predicting and preventing IDRs is the fact that a basic understanding of their mechanisms is lacking. Although reactive metabolites appear to be responsible for most IDRs and most appear to be immune-mediated, the evidence for these hypotheses is mostly circumstantial and it is likely that there are exceptions. IDR mechanistic studies are intrinsically difficult because prospective studies in humans are virtually impossible, in vitro studies cannot possibly duplicate their complexity, and IDRs are also idiosyncratic in animals. However, a better mechanistic understanding might lead to strategies to increase the incidence in animals, which would, in turn, confirm the hypotheses that led to these strategies. However, to be useful animal models, they must involve the same basic mechanism as the human IDR. Even a good model will not be a model for the IDR that occurs in everyone because different patients have different IDRs to the same drug. We have developed a rat model of nevirapine-induced skin rash that has very similar characteristics as the relatively mild rash that frequently occurs in humans. With this model we have been able to demonstrate that it is immune-mediated, the major cell type involved, and what metabolic pathway is responsible. It is likely that the less common and more severe reactions that nevirapine can induce involve the same initiating events but different effector cells. Another important animal model is penicillamine-induced autoimmunity in Brown Norway rats, which appears to involve direct binding of the drug to carbonyl groups on antigen presenting cells leading to their activation. Using current mechanistic hypotheses we have tried to develop animal models of idiosyncratic liver injury and agranulocytosis without much success. We have been able to reproduce a mild, delayed type liver injury with amodiaquine, but most strategies to increase an immune response do not lead to liver failure and are often protective, possibly because they lead to decreased bioactivation of the drug. Even negative studies provide important mechanistic clues and are essential to progress in solving this problem. This research was supported by CIHR and a Canada Research Chair.

S3. Understanding Idiosyncratic Hepatoxicity: From Man to Mouse to Computer

Paul B. Watkins

Hamner, University of North Carolina Institute for Drug Safety Sciences, Research Triangle Park, NC, USA

Drug Induced Liver Injury (DILI) remains the major adverse drug event that leads to termination of clinical development programs, or regulatory actions including failure to approve for marketing, restricted indications, and withdrawal from the marketplace. The type of DILI that is most problematic is “idiosyncratic” meaning that only a very small fraction of treated patients are susceptible to the DILI. Current preclilnical models, even “humanized” ones, do not reliably identify molecules that are safe for the liver and, conversely predict liabilities in molecule that are in fact safe for the liver. For example, cur-rent preclinical testing does not detect the DILI potential of xymelagatran but would predict that the liver risk is high for acetaminophen even when taken as directed. Reliable preclinical testing will probably not be developed until there is greater understanding of the mechanisms underlying idiosyncratic DILI. The Hamner-UNC Institute for Drug Safety Sciences has been building novel research programs that are designed to bridge the gap in knowledge between preclinical models and patients. Research programs to be discussed include the Collaborative Cross strains of inbred mice to identify specific genes and pathways that underlie DILI susceptibility and that can be used to generate hypotheses that are testable in the human DILI gene banks assembled by the Severe Adverse Events Consortium (SAEC) and the Drug Induced Liver Injury Network (DILIN). A second research project involves clustering drugs by structural descriptors and cell based biological responses as a surrogate for mechanisms to generate samples sizes sufficienct to perform genome-wide association studies in the human DILI gene banks. Finally, the center is collaborating with Entelos and the FDA to develop DILI-sim, a computer-based platform consists of simulations of interacting pathways that underlie susceptibility to DILI. When completed, it will improve understanding of many aspects of DILI, including species differences in DILI susceptibility.

S4. Genetic Risk Factors for ADRs

M. Pirmohamed

Pharmacology and Therapeutics, University of Liverpool, Liverpool, AK, United Kingdom

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Adverse drug reactions (ADRs) are common, with many predisposing factors, both environmental and genetic. Genetic factors predispose to both on-target (type A) and off-target (type B) ADRs. Over the years, many genetic predispos-ing factors have been reported for a variety of ADRs, but very few have made it into clinical practice. There are many reasons for that including inconsistency in phenotyping, small sample sizes, lack of replication, and lack of demon-stration of clinical utility. With the completion of the human genome, the availability of comprehensive genotyping technologies, and the development of international consortia, these issues are slowly being resolved. This has had the benefit of identifying genetic predisposing factors for both type A and type B reactions which may be amenable for clinical implementation (or already have been). For example, with warfarin, a drug that causes type A reactions, many studies have now shown that variation in CYP2C9 and VKORC1, together with age and BMI, can account for at least 50% of the variation in dose requirement. Several randomised controlled trials are on-going globally which will provide evidence of clinical utility. For type B reactions, striking associations have been demonstrated with HLA alleles for a number of drugs that cause hypersensitivity reactions. For example, HLA-B*5701 is strong genetic predis-posing factor for abacavir hypersensitivity, which has been implemented into clinical practice, and has been shown to reduce the incidence of hypersensitivity. Recent data on liver injury caused by a number of drugs, for example, flucloxacillin, co-amoxiclav, lumiracoxib and lapatinib, has also resulted in the demonstration of HLA associations using genome wide scanning techniques. Over the next few years, many more genetic predisposing factors are likely to be identified which may be utilisable in clinical practice (depending on their predictive accuracy) or provide new insights into mechanisms.

S5. ADR Prediction: An Evolving Industry Perspective

Bruce D. Car1 and W. Griff Humphreys2

1Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Inc., Princeton, NJ, USA,2Department of Biotransformation-PCO, Bristol-Myers Squibb, Princeton, NJ, USA

The correct prediction of adverse drug reactions (ADRs) is a continually evolving pharmaceutical science spanning approximately 50 years. An array of preclinical biochemical, cellular, and animal-based technologies is now employed to identify the potential for ADRs. The predictiveness of the spectrum of AEs ranges from robust for certain cardiovascular events, to moderate for organ-based toxicities, poor for many idiosyncratic events, to near random for behavioral AEs, this spectrum likely reflecting an increasing complexity of the underpinning biochemical events. Of greatest predic-tive robustness is the battery of cellular electrophysiologic and telemetered conscious animal studies of parent and metabolites used to predict channelopathies in man. As the most common ADRs seen in up to 50% of programs during preclinical SAR, non-clinically predicted channelopathies may be observed harmlessly in their nascent, non-adverse forms in man. A considerable body of research underpins the approaches aimed at minimizing the potential for hepa-totoxicity. These approaches are moderately successful with non-idiosyncratic hepatotoxicity and largely unsuccessful with idiosyncratic events, though GWAS approaches when employed retrospectively, suggest a subset of these events may have specific MHC Class II associations. Frequently not considered in an ADME-centric universe are the poorly predicted, consistently rare behavioral AEs of seizure and suicidal ideation, the molecular pathologies of which are among the least well understood, complex drug-associated events. The uncertain potential for such rare behavioral or idiosyncratic events renders the determination of a benefit-risk equation for compounds or targets possibly carrying such serious liabilities highly problematic.

S6. Strategic Application of Toxicogenomics in Early Drug Discovery

James Stevens

Toxicology, Lilly Research Laboratory, Greenfield, IN, USA

Genomic technologies (e.g. toxicogenomics) and other systems approaches have been in use for drug safety assessment for over a decade, but the impact on the process of safety assessment and the integration of systems approaches into safety decisions has been less than initially expected. Although many companies have applied toxicogenomic technolo-gies strategically to address specific problems, no single application has emerged as an integrated component of the standard preclinical safety assessment paradigm. This presentation will focus on current applications of toxicogenomic

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technologies during the drug discovery process. Experience with predicting in vivo outcomes from in vitro transcript profiles using a toxicogenomic database will be discussed. The presentation will also highlight the need to integrate toxicogenomics, and other systems approaches, into an over arching strategy for incorporating early safety predictions into early safety decisions.

S7. Toxicogenomics: A Predictive Tool in Toxicology and Drug Development

Jean-Christophe Hoflack, Adrian B. Roth and Laura Suter.

Early and Investigative Toxicology, F Hoffmann La Roche AG, Basel, Switzerland

Robotized platforms and mature microarray platforms offer quick transcriptome-wide screens over large sample sets, whereas analytical workflows are in place to deliver shortly sound statistical and biological results.

Therefore, transcriptome profiling is today a standard way to perform fast and reliable molecular screens in early Toxicology studies, where timelines are tight and candidate molecules still numerous. Currently, these transcriptome screens remain mainly hypothesis-building methods and represent a starting point for specific mechanistic interpretations. The findings often suggest tailored follow-up experiments such as immunohisto-chemistry or functional assays. In addition there is interest in early Toxicology to achieve predictive results in a timely manner. A means toward this end is the use of databases of transcriptome profiles with pathological anchoring. Derived algorithms and gene lists help to predict liver, kidney and heart toxic liabilities. We will describe a few case examples highlighting the advantages as well as the limitations of transcriptomic screening in early Toxicology.

In later-phase Toxicology studies along the drug development, the transcriptome profiling may be considered optional, used when unexpected adverse events occur. The transcriptomic analysis address the mode of action with emphasis on mechanistic aspects that might provide additional information characterizing a type of toxicity. In these cases, transcriptomics is also a powerful tool for investigative toxicology.

S8. Epigenomics - Impact for Drug Safety Sciences

Jonathan Moggs

Investigative Toxicology, Preclinical Safety, Translational Sciences, Novartis, Basel, Switzerland

Recent advances in the mapping and functional characterisation of mammalian epigenomes, generate a wealth of new opportunities for exploring the relationship between epigenetic modifications, human disease and the therapeutic potential of pharmaceutical drugs. Epigenetics describes changes in genome function that occur in the absence of a change in DNA sequence. The principle ways in which epigenetic information is stored and propagated is via DNA methylation and chromatin modifications. Specific patterns of epigenetic marks form the molecular basis for developmental stage- and cell type-specific patterns of gene expression that are hallmarks of distinct cellular phenotypes. Importantly, epigenetic marks can be stably transmitted through mitosis and cell division. Thus, a unique opportunity arising from the application of epigenomic profiling technologies in drug safety sciences is the potential to gain novel insight into the molecular basis of long-lasting drug-induced cellular perturbations. We have evaluated the utility of integrated genome-wide epigenomic & transcriptomic profiling in tissues from preclinical animal models with particular emphasis on the identification of early mechanism-based markers for non-genotoxic carcinogenesis, a key issue for the safety profiling and assessment of new drugs. A well characterized mouse model for phenobarbital-induced non-genotoxic liver carcinogenesis, in which extensive perturbations of the epigenome have been previously described, has been used to evaluate the utility of combining genome-wide and locus-specific DNA methylation, chromatin modification, mRNA and microRNA profiling assays in target (liver) and non-target (kidney) tissues. The application of this integrated molecular profiling approach for identifying early mechanism-based markers of nongenotoxic carcinogenesis may ultimately increase the quality of cancer risk assessments for candidate drugs and ensure a lower attrition rate during late-phase development. Epigenomic profiling has great potential for enhancing toxicogenomics-based mechanistic investigations within drug safety sciences.

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S9. Biomarkers for Adverse Drug Reactions: Proteomic and Metabolomic Strategies

B. Kevin Park

MRC Centre for Drug Safety Science, University Of Liverpool, Liverpool, United Kingdom

Adverse drug reactions (ADRs) are a major complication of drug therapy and an impediment to drug development. Recent developments in Molecular Toxicology have enabled a more fundamental approach to understanding of the role of drug metabolism in adverse drug reactions.

Excessive drug accumulation and/or the formation of chemically reactive metabolites have been implicated in a number of off-target (including idiosyncratic) ADRs. Studies with model compounds and drugs such as paracetamol (acetaminophen) have helped to define the response of target organs to chemical stress and drug bioactivation with respect to various biological outcomes. These include adaptation (cell defence), apoptosis, necrosis, inflammation and activation of the innate and adaptive immune systems. In order to assess the impact of such events in drug safety there is a need to develop mechanism-based and organ/cell selective biomarkers. There has been significant progress in the establishment of protein biomarkers for drug-induced kidney damage such as kidney-injury molecule-1, trefoil factor 3 and albumin. With respect to drug-induced liver injury (DILI), High Mobility Group Box 1 (HMGB1) and keratin 18 (K18) have been proposed as biomarkers of apoptosis and necrosis for use with conjunction in more organ-selective established biomarkers such as serum transaminases. Metabolomic platforms such as NMR and mass spectrometry have revealed the potential of sensitivity to low molecular weight endogenous compounds, such as ophthalmic acid and taurine to be used for the prediction and the assessment of DILI. Similarly, pyruvate levels prior to the administration of Ximelagatran were a predictor of liver damage. However, metabolomic data may be heavily influenced by the gut microflora, therefore further work is required to qualify this approach and these particular biomarkers.

Important considerations for the interpretation of the results obtained with any biomarker are cell-selectivity, mecha-nism of formation, mechanism of release, half-life, disposition and secretion. Correct interpretation of biomarker data is crucial in the translation across in vitro, in vivo animal and the clinic to prevent unnecessary attrition.

S10. Analysis of P450 Structure-Function Relations in Drug Metabolism Using X-ray Crystallography

Eric F. Johnson, An Wang, Uzen Savas and C. David Stout

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA

P450 2D6 plays a prominent role in human drug metabolism, and in many cases P450 2D6 substrates are likely to be positively charged at neutral pH. Substrate binding often requires that P450s are open to allow substrate access, and the resulting complex can exhibit adaptations to accommodate the bound substrate. The structure complex of P450 2D6 with a potent inhibitor, prinomastat, was determined by x-ray crystallography. The structure exhibits a closed cavity with significant changes in shape from that observed for an open structure determined, previously for P450 2D6 in the absence of a bound ligand (PDB: 2f9q). These differences reflect significant reorganization of the quadrant of the protein comprising helix A, beta sheet 1, the helix B-C region, the helix F-G region and C-terminal beta sheet region. In the case of P450 2D6, the reverse amphipathicity of helix F′ directs strongly polar and potentially charged amino acid side-chains into the substrate binding cavity. This creates an environment conducive for binding positively charged substrates and is likely to contribute to large differences between the water- and substrate-occupied active site cavities. The results suggest that adaptive changes to the P450 2D6 binding site will be important consideration in understanding substrate binding. In contrast, a newly determined x-ray crystal structure for human P450 1B1 complexed with a-naphthoflavone (ANF) reveals a remarkably conserved active site architecture within the CYP1 family. Human CYP1B1 exhibits less than 40% amino acid sequence identity when compared to CYP1A1 or CYP1A2 but exhibits a substrate profile that is similar to those of the P450 1A enzymes. Although the sequences of CYP1B1 and CYP1A2 have diverged significantly and result in differences for 10 of 24 amino acid side-chains in the substrate binding site, the two enzymes exhibit narrow, largely hydrophobic active site cavities, which is likely to contribute to similarities in the substrate profiles. The maintenance of this active site architecture reflects evolutionary conservation of key residues that shape the cavity. Nevertheless, ANF binds in a distinctly different orientation in CYP1B1 from that observed previously for CYP1A2 (PDB:2hi4) due to differences in the shapes of the respective active site cavities and amino acid differences for substrate contact residues. (Supported by NIH Grant GM031001 and Pfizer Global Research & Development)

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S11. Dynamics of Multi-step Cytochrome P450 Reactions

F. Peter Guengerich, Christal D. Sohl and Goutam Chowdhury

Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA

Numerous cytochrome P450 (P450) enzymes catalyze multi-step reactions. At least five occur in physiological steroid oxidations, including androgen oxidation to estrogens by P450 19A1, the steroid aromatase. Such reactions are also known with xenobiotic substrates. Oxidation of N,N-dimethyl- or diethylnitrosamine by P450 2A6 yields formaldehyde or acetaldehyde, which is further oxidized to formic or acetic acid, respectively. The conversion of the nitrosamine to acid in each case is more efficient than predicted by combining the catalytic efficiencies of the two individual oxidation steps, and pulse chase experiments indicate that little exchange of the intermediate aldehydes occurs; i.e. the overall reactions are processive. Human P450 19A1, the aromatase, has been expressed in Escherichia coli and purified. P450 19A1 converts androstenedione (A), 19-hydroxyA, and 19-aldehyde (19=O) to estrone. A and the two intermediates (oxidation products) bind to P450 19A1 in 2-step reactions. Pulse chase experiments indicate free exchange of inter-mediates with the medium. Kinetic models have been developed to obtain individual rate constants for major steps of both the P450 2A6 and 19A1 reactions. The behavior of P450 2A6 is reminiscent of previous studies on the oxidation of ethanol to acetic acid by P450 2E1, and the basis of proclivity for handling intermediate aldehydes is still unclear. (Supported in part by USPHS grant R37 CA0090426 and P30 ES000267.)

S12. Structural Studies of Cholesterol-Metabolizing P450s

Irina A. Pikuleva1, Natalia Mast1, Andrew J. Annalora2, Casey Charvet1 and C. David Stout2

1Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, USA,2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA

Cytochrome P450 enzymes (CYP or P450) play key roles in cholesterol elimination from different organs. CYP46A1 catalyzes cholesterol 24S-hydroxylation, the first step in the major pathway of cholesterol removal from the brain, whereas CYP11A1 converts cholesterol to pregnenolone, the first step in the overall biosynthesis of steroid hormones. Previously we determined crystal structures of the ligand-free and substrate-bound CYP46A1, and in the present work we will describe crystal structures of CYP46A1 in complex with four different therapeutic agents (tranylcypromine, thiop-eramide, voriconazole, and clotrimazole). The crystal structure of CYP11A1 in complex with the physiological substrate 22R-hydroxycholesterol will also be presented. Structures of CYP46A1 with drugs reveal ligand-specific conformational changes and distinct features of each co-complex. These structures also demonstrate how the enzyme adapts to bind structurally distinct compounds and provide an explanation for the observed tight binding and differences in the P450 spectral response. CYP11A1 shares less than 25% amino acid sequence identity with CYP46A1, yet metabolizes the same substrate cholesterol. The CYP11A1 structure reveals specific interactions between the enzyme and high affinity substrate 22R-hydroxycholesterol and demonstrates a unique mode of interaction of the sterol 3?-hydroxyl group with the protein. This structure also provides mechanistic insight into the 3-step reaction catalyzed by this mitochondrial P450. A structural basis for the similarities and differences between the two cholesterol-metabolizing P450s CYP46A1 and CYP11A1 will also be discussed. Supported in part by NIH grant GM62882.

S13. Structure of P-glycoprotein

Geoffrey Chang1, Andrew Ward2, Alexandra Caya1, Stephen G. Aller1, Jodie Yu1, Yue Weng1, 3, Srini-vas Chittaboina1, Patina M. Harrell4, Rupeng Zhuo4, Yenphuong T. Trinh4, Qinghai Zhang1 and Ina L. Urbatsch4

1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA,2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA,3College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, China,4Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA

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P-glycoprotein (Pgp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance in the treatment of cancers. Substrate promiscuity is a hallmark of Pgp activity, thus a structural description of polyspecific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo-Pgp at 3.8 Å reveals an internal cavity of ∼6,000 Å3 with a 30 Å separation of the two nucle-otide binding domains (NBD). Two additional Pgp structures with cyclic peptide inhibitors demonstrate distinct drug binding sites in the internal cavity capable of stereo-selectivity that is based on hydrophobic and aromatic interactions. Apo- and drug-bound Pgp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding. We will present our latest findings on P-glycoprotein and present strategy on obtaining other conformations, extending the diffraction resolution, and new co-crystal structures with inhibitors/drugs.

S14. Therapeutic Implications of OATP1B1 Polymorphisms

Mikko Niemi

Department of Clinical Pharmacology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland

Organic anion transporting polypeptide 1B1 (OATP1B1) is an influx transporter expressed on the basolateral membrane of human hepatocytes. It facilitates the uptake of its substrates from portal blood into the liver. Several clinically used drugs (e.g., many statins and angiotensin receptor antagonists) and endogenous compounds (e.g., certain bile acids) are substrates of OATP1B1. Genetic variability in the SLCO1B1 gene encoding OATP1B1 can result in marked interindividual differences in drug pharmacokinetics. A common single nucleotide polymorphism of SLCO1B1, c.521T>C (p.Val174Ala), decreases the transport function of OATP1B1. As a result, the plasma concentrations of many OATP1B1 substrates are increased in carriers of this variant, most notably in those with the homozygous c.521CC genotype. For example, the plasma AUC of active simvastatin is increased more than 3-fold in individuals with the homozygous c.521CC genotype (2). This translates into a markedly increased risk of simvastatin-induced myopathy during continuous treatment with a high dose of simvastatin (3). The SLCO1B1 c.521C allele also raises the plasma concentrations of many other, but not all (fluvastatin), statins and, e.g., those of the antidiabetic drug repaglinide (4,5). Interestingly, genetically impaired OATP1B1 activity also reduces the hepatic uptake of methotrexate, decreasing the risk of gastrointestinal toxicity during high-dose intravenous methotrexate therapy (6). On the other hand, the SLCO1B1*1B allele (c.388G-c.521T) is associated with increased hepatic uptake and reduced plasma concentrations of certain OATP1B1 substrates, such as pravastatin and repaglinide (7,8). The frequencies of functionally important SLCO1B1 variants differ markedly between ethnic groups (9).The impaired activity c.521C allele has a frequency of about 15-20% in Caucasians, 10-15% in Asians and about 2% in Sub-Saharan Africans or African Americans (9). The frequency of the *1B haplotype is about 25% in Caucasians, 40% in South/Central Asians, 60% in East Asians, and as high as 80% in Sub-Saharan Africans or African Americans (9). Thus, the effects of SLCO1B1 polymorphisms on drug pharmacokinetics are broad and of established clinical significance.

References

1. Kalliokoski A & Niemi M. Br J Pharmacol 2009;158:693-705.2. Pasanen MK, et al. Pharmacogenet Genomics 2006;16:873-9.3. SEARCH Collaborative Group. N Engl J Med 2008;359:789-99.4. Niemi M. Clin Pharmacol Ther 2010;87:130-3.5. Niemi M, et al. Clin Pharmacol Ther 2005;77:468-78.6. Treviño LR, et al. J Clin Oncol 2009;27:5972-8.7. Maeda K, et al. Clin Pharmacol Ther 2006;79:427-39.8. Kalliokoski A, et al. Pharmacogenet Genomics 2008;18:937-42.9. Pasanen MK, et al. Pharmacogenomics 2008;9:19-33.

S15. Cardiovascular Drug Transport

Heyo K. Kroemer

Department of Pharmacology, Research Center of Pharmacology and Therapeutics, Ernst Moritz Arndt University Greifswald, Greifswald, Germany

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Cardiovascular diseases are the leading cause of death worldwide. The hallmark of treatment is chronic drug therapy. In order to reach their therapeutic targets (eg, heart, blood vessels or peripheral cells) cardiovascular drugs have to pass multiple biomembranes. Successful therapy by the oral route, for example, requires uptake into the enterocyte, transfer to the vascular system and subsequent uptake into target tissue. Within the last decade, a significant number of in vitro experiments, animal studies and clinical trials pointed out that drug-transporting proteins are important modifiers along the entire process of a cardiovascular drug reaching its site of action.

Following oral administration drugs reach the intestinal wall as its first absorption barrier. The process of drug absorp-tion is rather complex and modulated by active and passive uptake processes, mucosal metabolism and efflux transport. One of the first studies addressing the influence of the ABC-transporter P-gp on pharmacokinetics of digoxin was carried out by Greiner et al (1) and indicated that intestinal expression of P-gp predicts oral bioavailability of digoxin.

Other cardiovascular compartments have not been thoroughly investigated with respect to the role of intracellular pharmacokinetics and its modification by transport proteins. In human heart, P-gp and bcrp have been identified in the endothelial wall of both cardiac arterioles and capillaries (2). Unlike P-gp and bcrp, MRP5 is expressed not only in the endothelium, but also in smooth muscle cells and cardiomyocytes. Moreover, it has been demonstrated that its expression is enhanced by ischemic cardiomyopathy. (3). Several uptake transporters have been identified in cardiac tissue. For example, the cation transporter OCTN2 has been localized in the endothelial wall of cardiac blood vessels (4). Another SLC transporter expressed in human heart is OATP2B1 and its cardiac expression is again mainly restricted to the endothelium (5). Regulation of OATP2B1 is in part driven by PKC-mediated internalization (6).

Peripheral cells of the cardiovascular system express transport proteins. Jedlitschky et al identified MRP4 in human platelets by immuno blotting and immuno fluorescence (7). These data indicate a function of MRP4 in platelet mediator storage giving rise to various forms of MRP4 associated platelet dysfunctions (8).In summary, drug transporters are expressed throughout the entire cardiovascular system thereby modulating intracellular concentrations and hence actions of drugs.

References

1) Greiner, B.; Eichelbaum, M.; Fritz, P.; Kreichgauer, H. P.; von Richter, O.; Zundler, J.; Kroemer, H.K. The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J. Clin. Invest 1999, 104, 147-153.

2) Fromm, M. F.; Kauffmann, H. M.; Fritz, P.; Burk, O.; Kroemer, H. K.; Warzok, R. W.; Eichelbaum, M.; Siegmund, W.; Schrenk, D. The effect of rifampin treatment on intestinal expression of human MRP transporters. Am. J. Pathol. 2000, 157, 1575-1580.

3) Dazert, P.; Meissner, K.; Vogelgesang, S.; Heydrich, B.; Eckel, L.; Bohm, M.; Warzok, R.; Kerb, R.; Brinkmann, U.; Schaeffeler, E.; Schwab, M.; Cascorbi, I.; Jedlitschky, G.; Kroemer, H. K. Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart. Am. J. Pathol. 2003, 163, 1567-1577.

4) Grube, M.; Meyer zu Schwabedissen, H. E.; Prager, D.; Haney, J.; Moritz, K. U.; Meissner, K.; Rosskopf, D.; Eckel, L.; Bohm, M.; Jedlitschky, G.; Kroemer, H. K. Uptake of cardiovascular drugs into the human heart: expression, regulation, and function of the carnitine transporter OCTN2 (SLC22A5). Circulation 2006, 113, 1114-1122.

5) Grube, M.; Kock, K.; Oswald, S.; Draber, K.; Meissner, K.; Eckel, L.; Bohm, M.; Felix, S. B.; Vogelgesang, S.; Jedlitschky, G.; Siegmund, W.; Warzok, R.; Kroemer, H. K. Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart. Clin. Pharmacol. Ther. 2006, 80, 607-620.

6) Köck, K.; Koenen A;, Giese B;, Fraunholz M;, May K;, Siegmund W;, Hammer E;, Völker U;, Jedlitschky, G;, Kroemer H.K.;, Grube M. Rapid Modulation of the Organic Anion Transporting Polypeptide 2B1 (OATP2B1, SLCO2B1) Function by Proteine Kinase C mediated Internalization. J. Biol. Chem. 2010, 285, 11336-11347

7) Jedlitschky, G.; Tirschmann, K.; Lubenow, L. E.; Nieuwenhuis, H. K.; Akkerman, J. W.; Greinacher, A.; Kroemer, H. K. The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. Blood 2004, 104, 3603-3610.

8) Jedlitschky, G.; Cataneo M.; Lubenow, L. E.; Rosskopf, D.; Lecchi, A.; Antoni A.; Motta G.; Niessen J.; Kroemer, H. K.; Greinacher, A. The nucle-otide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. Blood 2004, 104, 3603-3610.

S16. Regulation of Drug Transporters in Different Disease States and its Toxicological and Clinical Implications

Jose E. Manautou

Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, USA

Exposure to toxicants and genetic conditions leading to acute or chronic liver diseases are known to change the expres-sion of multiple hepatobiliary transporters. Although the regulatory features and biological consequences of some of these transporters changes are relatively well studied, much remains to be done. This presentation will concentrate on

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the regulation of liver transporters during acetaminophen (APAP)-induced hepatotoxicity. Drug-induced liver injury (DILI) is a leading cause of acute liver failure in the United States and other countries, with the widely used non-prescription medication acetaminophen (APAP) accounting for roughly 50% of all DILI cases. Our laboratory has shown that changes in the expression of hepatobiliary transporters is a compensatory response to chemical-induced liver injury with potential importance to tissue recovery and development of resistance to subsequent toxicant exposure. The multidrug resistance-associated protein 4 (Mrp4, Abcc4) is the most significantly induced transporter in response to acetaminophen (APAP) hepatotoxicity in mice and humans. Other hepatotoxicants with varied mechanisms of tox-icity similarly induce Mrp4 expression. We have also shown that the transcription factor Nrf2, Kupffer cell function and the nuclear receptor PPARαall contribute to liver Mrp4 regulation. Current studies in our laboratory are aimed at characterizing the transcriptional regulation of the MRP4 gene and its proximal promoter. Reporter gene assays in HepG2 cells using progressive promoter truncation constructs showed that the human MRP4 promoter-proximal region is constitutively active, and that the first 100 bp are required for maximal activity. Overexpression of the transcription factors NRF1, SP2, STAT1, KLF10 and TFAP2A in HepG2 cells increases MRP4 reporter gene expression, while HES1, KLF15 and ZFP161 are repressive. Among all transcription factors tested, regulation of MRP4 by NRF1 and HES1 was most prominent. Overexpression of an NRF1 dominant-negative form or site-directed mutations in putative NRF1 binding sites suppressed MRP4 reporter gene activity. Site directed-mutagenesis of a short module associated with HES1 function prevented the suppressive effect of HES1 on MRP4 reporter gene activity. Overall, these results suggest the presence of complex regulatory mechanisms for hepatic MRP4 gene expression involving transcription factors such as NRF1 and HES1. Since oxidative stress can be a common event associated with toxicant exposure and varied forms of liver diseases, it is our goal to investigate the commonality of these regulatory events as a compensatory attempt to restore liver function [This work was supported by The National Institutes of Health Grant DK069557; JEM is the Marlene L. Cohen and Jerome H. Fleisch Scholar at the University of Connecticut].

S17. Quantification of Transporter/Enzyme Protein in Human Tissue by Mass Spectrometry: A New Path to Pharmacoproteomics

Tetsuya Terasaki1 and Sumio Ohtsuki2

1Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan,2Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Membrane transporter, receptor and enzyme proteins are expressed in organs or tissues and play key roles in blood-tissue barrier function, as well as in the absorption, distribution, metabolism and elimination of drugs in vivo. Knowing the quantitative expression amount of transporter, receptor and enzyme proteins in humans is a key issue for a rational prediction of in vivo ADME in human, especially at the diseased state. We have employed a quantitative targeted absolute proteomics (QTAP) to establish the protein quantification method of enzyme, receptor and transporter. After trypsin digestion of membrane proteins or whole cell lysate, a stable isotope-labeled peptide of the protein to be quantified was added as an internal reference. The sample was analyzed by LC tandem mass spectrometry with multiplexed Multiple Reaction Monitoring (Multiplexed MRM) mode. The highly selectivity resulting from the two stages of mass selection yields a very specific and sensitive determination of the targeted peptide of the transporter protein in the sample. Moreover, we developed an in-silico design method to select an appropriate peptide exhibiting strong MS signal in the LC/MS/MS quantification. Using 300 channels of MS/MS, a simultaneous (37 proteins) and highly sensitive (>1 fmol/assay) absolute protein quantification method has been established (Pharm. Res., 25: 1469-1483, (2008)). In this symposium, we will show our recent results to answer to the fundamental questions in ADME 1) how the primary cultured human hepatocyte is close to the human liver? 2) how the liver of uPA/SCID mouse inoculated with human hepatocyte is close to the human liver ? 3) how we could clarify the interspecies difference of the blood-brain barrier (BBB) function in mouse, monkey and human? 4) how we could predict in vivo brain drug distribution both in normal and diseased states ? 5) how we could clarify the drug resistant mechanism in cancer cells ? To answer to these questions, we have determined the transport, receptor and enzyme protein expression amount and also the transport activities in the cell line. The present method enables us to clarify the differential drug absorption, distribution, metabolism, elimination, toxicity and pharmacological effect among in vitro, in vivo, species, gender, age in the various tissues, ages and diseased states. This strategy should have many potential applications for the future of the ADME research in animal and human. The established method is applicable to any important proteins such as drug target and disease marker, the method described here provides a

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means to move from pharmacogenomics to pharmacoproteomics, and should in turn lead to improved strategies of future drug discovery and development.

S18. The Regulatory Mechanisms and Physiological Functions of Multi-Functional Dioxin (Aryl Hydrocarbon) Receptor

Yoshiaki Fujii-Kuriyama

Medical Research Institute, Tokyo Medical and Dental University, Tokyo, JapanThe dioxin receptor (also named aryl hydrocarbon receptor or AhR) was originally identified as a ligand-activated transcription factor with structurally characteristic motifs of bHLH and PAS that is involved in induction of xenobiotic-metabolizing P4501A1 (CYP1A1) in response to polycyclic aromatic hydrocarbons (PAH). The ligand-activated AhR translocates from the cytoplasm to the nuclei where it forms a heterodimer with another bHLH-PAS protein, Arnt (AhR nuclear translocator) to enhance the expression of AhR-target genes through binding the XRE sequence in their promoter. For past several decades, AhR research was mainly focused on the functions related to pharmacology and toxicology. Recently, however, more research efforts have been devoted to physiological functions of AhR. The AhR signaling activity can be precisely regulated by a feedback inhibitory mechnism in which AhR repressor (AhRR), whose expression is enhanced by the activated AhR/Arnt heterodimer, represses the transcriptional activity of AhR through SUMOylation and subsequent recruitment of corepressor, ANKRA2, HDAC4 and HDAC5. Recent progress in our AhR research reveals that AhR is involved in the regulation of estrous cycle by acting as an activator in transcription of P450 aromatase (CYP19) gene and in the Treg and Th17 cell differentiations from naive T cells. With a novel finding that AhR functions as an E3 ubiquitin ligase, our knowledge on the AhR functions further extends to tumor suppression and regulation of the steroid hormone activities. I would like to summarize and discuss our recent work on the AhR research with an attention to inhibitory mechanisms of AhRR and physiological functions of AhR. [This work has been performed in collaboration with Dr. H. Sekine (Univ. of Tsukuba), Drs. F. Ohtake, S. Kato (Univ. of Tokyo), and Dr. K. Kawajiri (Saitama Cancer Center), and financially supported in part by CREST and SORST of JST].

S19. Functional Synergy Between CAR and β-catenin

David Moore

Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA

Hepatocarcinogenesis resulting from the action of phenobarbital as a tumor promoter shows strong selection for activat-ing mutations in β-catenin. This tumor promotion is completely dependent on CAR. Acute activation of either β-catenin or CAR results in hepatomegaly that is both rapid and strictly limited. We have reproduced the functional interaction between CAR and β-catenin by combining pharmacologic activation of the nuclear receptor with TCPOBOP, and genetic activation of the oncogene via targeted deletion of the inhibitory exon 3. When the activated β-catenin allele is het-erozygous, this results in the appearance of spontaneous adenomas after 8 months. When β-catenin is homozygously activated, pharmacologic activation of CAR results in unlimited growth of apparently normal liver. Doubly activated mice die when the weight of the liver reaches a quarter of their total body weight. We are exploring the mechanisms by which this combined activation overcomes the strict limits that restrain the growth responses to activation of either CAR or β-catenin alone.

S20. CAR and Gene Regulation: Mechanism and Action

Masahiko Negishi

Pharmacogenetics Section, LRDT, NIEHS, NIH, Research Triangle Park, NC, USA

Nuclear xenobiotic receptor CAR (NR1I3) is an orphan member of the nuclear hormone receptor superfamily. Since CAR was first characterized as a phenobarbital-activated transcription factor, the scope of regulations performed by CAR has expanded from drug metabolism to hepatic energy metabolism and to cell death. Accordingly, CAR-targeted genes

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have expanded from CYP2B to include genes such as G6Pase and PEPCK1 to Growth arrest DNA damage-induced 45B (GADD45B). Our recent study demonstrated that CAR both activates the GADD45B gene and directly interacts with the GADD45B protein, repressing TNFa-induced death of mouse primary hepatocytes (1). By regulating gene expressions, thus, CAR can be involved in the development of various diseases such as liver injury, diabetes and cancer.

Upon xenobiotic activation, CAR translocates from the cytoplasm into the nucleus, regulating transcription of its target genes. Many activators such as phenobarbital indirectly activate CAR to translocate it into the nucleus. Since protein phosphatase inhibitor okadaic acid prevented the nuclear translocation and activation of CAR, it has been suggested that de-phosphorylation is required for CAR activation, the underlying mechanism for which should be a cell signal-mediated regulation. The target of this de-phosphorylation, however, has not been identified at the present time. Recently we found that PKC phosphorylates a conserved threonine (Thr38 and Thr48 in human and mouse CAR DBDs, respectively), retaining CAR in the cytoplasm, and that phenobarbital de-phosphorylates this threonine to translocate CAR into the nucleus in mouse primary hepatocytes (2). The molecular mechanism by which protein phosphatase 2A (PP2A) de-phosphorylates threonine and the signal mechanism that regulates this de-phosphorylation have now begun to be understood. PP2A binds to the N-terminal region of CAR LBD to de-phosphorylate the threonine in CAR DBD, which requires Receptor for Activated C-Kinase 1 (RACK1) to act as the regulatory subunit. Growth factors facilitate binding of phospho-ERK1/2 to the C-terminal region of the CAR LBD, preventing PP2A binding and de-phosphoryla-tion, thus sequestering the phosphorylated CAR in the cytoplasm. Phenobarbital may inactivate phospho-ERK1/2, de-phosphorylating threonine to translocate CAR into the nucleus. This de-phosphorylation mechanism should provide CAR with significant flexibility for xenobiotic-cell signal cross talk to differentially regulate a diverse array of genes, such as sex-specific expression, activation versus repression and the expression in centrilobular versus periportal hepatocytes.

References

1. Yamamoto, Y., Moore, R., Flavell, R.A., Lu, B., and Negishi, M.: Nuclear receptor CAR represses TNFa-induced cell death by interacting with the anti-apoptotic GADD45B. PLoS ONE, 5(4): e10121, 2010.

2. Mutoh, S., Osabe, M., Inoue, K., Moore, R., Pedersen, L., Perera L., Rebolloso, Y., Sueyoshi, T., and Negishi, M.: Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). J. Biol. Chem. 284: 34785-34792, 2009.

S21. CAR and PXR and the Regulation of Gene Involved in the Metabolism of Anticancer Drugs

Jean-Marc Pascussi

University of Montpellier 1, INSERM U632, Montpellier, France

One of the major obstacles in predicting the treatment outcome in patients in terms of cancer treatment efficacy and toxicity is the large interindividual variability of chemotherapy response. Multiple factors are known to be responsible for these differences such as genetic polymorphisms in biotransformation and drug transport genes, disease status and environmental determinants such as co-medications. Because of the specific properties of anticancer drugs, like a narrow therapeutic index and a steep dose-toxicity curve, small changes in the pharmacokinetic profile can significantly alter the clinical response to these drugs. Indeed, a substantial part of the variability observed among patients might be caused by drug-drug or diet-drug interactions and variation in expression of DME via the xenobiotic receptors CAR and PXR. These nuclear receptors are key regulators of genes encoding drug metabolizing and transport proteins, and they have been recently implicated in anticancer drug resistance. Some reports have shown that CAR and PXR not only affect hepatic anticancer drug metabolism, but also tumoral drug metabolism and cancer cells viability. Notably, it has been observed that the expression of PXR in prostate, ovarian and colon cancer cells can modulate tumor cells metabolism and sensitivity to several cytotoxics. For instance, PXR ligands such as rifampicin and hyperforin (the active constituent of the herbal antidepressant St. John′s Wort) significantly enhance both the hepatic elimination of irinotecan in patients, and the intratumoral glucuronosylation and inactivation of the drug. In contrast, while co-administration of phenytoin (CAR activator) has been reported to decrease the level of the prodrug cyclophosphasmide and increase the plasma levels of its cytotoxic metabolite 4-hydroxycyclophosphamide through the hepatic induction of CYP2B6 and CYP3A4, CAR increases cancer cells resistance to 4-hydroxycyclophosphamide by increasing the expression of ALDH1A1, that catalyze the detoxification of this compound. In addition, it has been proposed that CAR and PXR directly may affect tumour growth by controlling the expression of genes involved in motility, apoptosis and cell-cycle progression in cancer

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cells. These data suggest that these receptors may represent novel determinants of tumor responses toward chemo-therapy, and that environmental and genetic factors affecting CAR or PXR (expression or activation levels) may affect the cytotoxic threshold of tumor cells to chemotherapy which can consequently mask or attenuate pharmacogenetic associations. This presentation will give you an overview of how CAR and PXR affect anticancer drugs metabolism and cancer cells resistance.

S22. Transporter Mediated Toxicity: The Role of Mitochondrial and Plasma Membrane Transporters

Jashvant D. Unadkat

Department of Pharmaceutics, University of Washington, Seattle, WA, USA

Transporters can constitute the rate-limiting step in the entry of drugs to their site of efficacy and/or toxicity. This site may be the cell cytoplasmic compartment or an intracellular organelle such as the mitochondria. In this case, the absence and presence of the transporter in the cell membrane or the membrane of the organelle, and its level of expression there, can significantly determine the efficacy and/or toxicity of the drug. Using nucleoside transporters as examples, I will show how the expression of such transporters in the plasma and mitochondrial membrane can significantly affect the efficacy and toxicity of nucleoside drugs such as ribavirin, gemcitabine and filauridine. Supported by NIH GM5447

S23. Safety and Efficacy of Amphiphilic Cyclodextrins for Drug Delivery

A. Atilla Hincal1, Hakan Eroglu2 and Erem Bilensoy3

1Güniz Sokak 44/3, ÝDE Pharma Information Center, Education, Consultancy Ltd Co, Ankara, Turkey,2Basic Sciences Dept, Hacettepe University Pharmacy Faculty, Ankara, Turkey,3Pharmaceutical Technology Dept, Hacettepe University Pharmacy Faculty, Ankara, Turkey

Today we know that some active substances used in the treatment or those contained in the formulation of some dos-age forms are not optimally and specifically delivered to target areas. In particular, toxicity and adverse effects of active substances used in cancer treatment, their effect on not only the target organ that needs treatment but also on the whole system in other words non-selective cytotoxicity, variability that result from several factors such as formulation aspects or pharmacokinetic properties of drugs and dissolution and stability problems lead researchers working on drug delivery systems to new horizons.

Since the drug delivery systems prepared with cyclodextrins which have a hydrophilic exterior and a lipophilic cavity, failed to produce the desired results, amphiphilic cyclodextrins developed from natural cyclodextrins, were investigated. The notion of using new cyclodextrin (CD) derivatives as carrier systems was a result of several factors including toxicity and serious adverse events that occur with administration of cancer drugs, ineffective results obtained from chemotherapy administrations, drug resistance and the deficits in orally administered drugs. As a matter of fact, one of the main reasons which lead to the use of these carriers in preparation of nanoparticles is that amphiphilic cyclodextrins make is possible to develop nanoparticles without using surfactants which have systemic toxic effects over long term or chronic use.

In pharmaceutical sciences, studies on nanoparticular delivery systems gained pace beginning from the early 1980s. On one hand, obstacles experienced in active substance administration mentioned above and on the other hand studies to investigate and develop new drug delivery systems have lead to intensive research on nanoparticulate drug car-rier systems in cancer treatment. The primary objective in cancer treatment is to overcome cellular and non-cellular mechanisms of resistance, to improve the selectivity of drugs to cancerous cells and to minimize adverse effects and toxicity on healthy cells. For this reason, nanoparticles are preferred as delivery systems in transferring anticancer active substances to target areas. Various natural and synthetic polymers are known to be used to this end. A major excipient group included in drug trials for the past two or three decades are cyclodextrins. These natural molecules have numerous advantages both in terms of functionality and activity, but they also have disadvantages, the most important of which are hemolysis and nephrotoxicity they present. Modified cyclodextrins have been developed to overcome these disadvantages of cyclodextrins. Today, we will focus on the safety and efficacy of drug delivery systems obtained in studies with amphiphilic cyclodextrins. This presentation will particularly emphasize β-CDC6, β--OCAPRO,

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6N-CAPRO, 6N-MYRISTO, Ramified CD derivatives, which are amphiphilic cyclodextrins with completed synthesis. For these carrier agents to be used for drug delivery, the attributes of nanoparticles to which drug is loaded, their hemolytic activity, cytotoxicity will be mentioned, and the new drug delivery systems obtained by loading anticancer drugs will be discussed in terms of safety and efficacy, and Drug delivery systems developed with amphiphilic cyclodextrins by different research groups will be evaluated regarding FDA′s “Guidance for industry: Nonclinical studies for safety evalu-ation of Pharmaceutical excipients” dated May 2005 and IPEC′s “IPEC, novel excipients safety evaluation procedure” and examples from nanoparticle and amphiphilic cyclodextrin studies conducted at the Department during the last two decades will be given.

S24. Application of Nanotechnology in Cancer Therapy and Imaging

Dong Moon Shin

Hematology & Medical Oncology, Emory University, Atlanta, GA, USA

Common challenges encountered by current cancer therapies include nonspecific systemic distribution of anti-cancer agents and their toxicity, vehicle-free anti-cancer drug delivery, frequent incidence of drug resistance, inadequate drug concentrations needed to reach tumor cells, and the limited ability to monitor therapeutic responses. An ideal therapeutic drug should selectively reach tumor cells with minimal damage to normal cells or organ sites. A rational approach to achieve these goals is to conjugate therapeutic drugs with monoclonal antibodies, their fragments, pep-tides, or other ligands that selectively bind to antigens or receptors that are usually abundantly or uniquely expressed on the tumor cell surface, not on the normal cell surface. Several key properties of anti-cancer nanoparticles include nanoparticle size, surface properties, targeting ligands, and different carriers. To deliver nanoparticles to specific cancer cells, the addition of targeting ligands to provide specific nanoparticle-cell surface interactions may play a critical role in the ultimate nanoparticle location. Currently ligand-targeted therapeutic strategies being developed include chemotherapeutics, molecularly targeted agents, anti-sense RNAs, immunotoxins, radio-immunotherapeutics, and drug immunoconjugates. These strategies also depend on the size and surface properties with conjugated ligands of the nanoparticles. In this presentation, we will describe the key elements of nanoparticles and how they differ from other types of cancer drugs. The selection of different ligands with tumor specific properties depending on the tumor cell types will also be discussed. For example, human transferin as a targeting ligand for binding to transferrin receptor (TFR) that typically is upregulated in several cancer cell types is an outstanding ligand for delivery of small interferring RNAs (SiRNA) targeting ribonucleotide reductase subunit M2 (RRM2). Suppression of RRM2 by transfection of SiRNA in vitro strongly inhibited cell growth in head and neck and lung cancer cells. Using xenograft (Tu212 cells) as an in vivo model, we found nanoparticles (10 mg/kg) significantly reduced RRM2 suppression and tumor growth. Further biologic studies indicated that SiRNA-mediated suppression of RRM2 increases apoptosis in a p53-independent manner. Next, the presentation will focus on preclinical development of several promising therapeutic novel nanoparticles. We will then summarize current clinical uses of earlier generations of nanoparticles therapeutics and their limitations, and those on the near horizon for cancer therapeutics. Finally, we will discuss the future directions regarding synthesis and utilization of mutifunctional and multiplexed nanoparticles, which are simultaneous synthesis and delivery of imaging and therapeutics in the same nanoparticles.

S25. Nanobiotechnology in Clinical Proteomics

Alexander I. Archakov and Yuri D. Ivanov

Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia

A certain concentration barrier exists in proteomics: the protein molecules residing in the concentration range below 10-12 Ì cannot be identified. The atomic force microscopy (AFM) nanotechnological method appears to be one of the most perspective diagnostic tools and enables not to measure protein concentration but merely to count single protein molecules and their complexes. Biospecific fishing method enables to extend the concentration limits of proteomics, as it enables to fetch the protein from the solution and therefore to increase its concentration on the surface of the biochip with immobilized partner proteins. Based on combination of AFM and biospecific fishing method we have created the AFM biochips for registration of hepatitis B and C and ovarian cancer biomarkers in patient serum. The described

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approach attains the concentrations of about 10-15 M; however, to reveal the lower concentrations (e.g. 10-18 M) it is necessary to convert the reversible complex formation into irreversible process.

Several other nanotechnological approaches can be useful for proteomic research, among them nanowire electrical detectors and optical biosensors. The optical biosensor method to register in real time the intermolecular interaction reactions (â òîì ÷èñëå Ag/Ab) without labels and calculate the kinetic rate constants and affinities for the complexes.. Nanowire electrical detector is designed as nanowire arrays modified with antibodies. These detectors show discrete conductance changes with biospecific binding of viruses to nanowire.

S26. Pharmacologically Regulated Stem Cell Therapy

C. Anthony Blau

Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA

Hematopoietic stem cells (HSC) are distinguished by their ability to stably engraft, to self-renew, and to differentiate across multiple blood lineages. Since the number of HSCs in an individual far exceeds the number contributing to blood cell production at any given moment, HSCs can be viewed as competing for the opportunity to contribute to blood cell formation. However, the rules governing this competition are poorly understood. Having the ability to regulate the competitive stance of transplanted cells would be advantageous for many applications of gene and cell therapy.

One method for increasing the frequency of genetically modified hematopoietic cells uses drugs that kill cells unless they are protected by a corresponding resistance gene. While a number of cytotoxic drug/resistance gene combinations have been evaluated, their effects are often short-lived due to the inability of most drugs to kill unmodified HSC. In these cases, achieving sustained selection through chronic drug administration is not feasible due to side effects. In contrast, the most potent among these methods uses a gene encoding a derivative of the enzyme methylguanine methyltrans-ferase (MGMT), followed by selection with O6 benzylguanine combined with an alkylating drug such as temozolomide. The potent and sustained selection observed in mice and dogs is attributable to the activity of the temozolomide/O6 benzylguanine combination against unmodified HSC. Although there are concerns regarding the potential for alkylating drugs to induce secondary leukemias, and regarding recent primate studies that question whether sustained selection can be achieved in humans, a larger question pertains to whether sustained and irreversible selection constitutes a uniformly desirable goal. Situations do exist in which a conditional growth advantage might be preferable (Miller and Blau, Gene Therapy, 2008).

We and others have developed an alternative method for selection that relies on conditionally regulatable signaling molecules containing portions of cell surface receptors or kinases fused to binding sites for drugs called chemical inducers of dimerization (CIDs). The activity of these signaling proteins is regulated through self-association that can be controlled using CIDs. Most attractive for eventual clinical use are CIDs that interact minimally with endogenous proteins, and domains for CID docking that are unlikely to evoke immune responses. We have used a system that fulfills these criteria to show that a conditionally activated derivative of the thrombopoietin receptor (F36VMpl) can support the expansion of genetically modified hematopoietic cells in vitro and in vivo.

In a previous report we described the hematological responses of two dogs transplanted with autologous CD34+ cells that had been transduced with a bicistronic gammaretrovirus vector encoding green fluorescent protein (GFP) and F36VMpl, followed by administration of AP20187, a CID. Both dogs received two 30-day intravenous courses of AP20187, and both responded with rises in GFP+ red blood cells (RBCs), platelets and white blood cells (WBCs), the latter comprised mainly of B cells. Here we report results from life-long monitoring as well as responses to additional courses of CID. In contrast to the previously described 30-day intravenous regimen we tested 5-day subcutaneous courses of AP20187 and its closely related analog, AP1903. Clonal tracking demonstrates the ability to conditionally regulate the contributions of individual HSC clones to hematopoiesis over the lifetimes of these large animals.

S27. Metabolic Interactions of Agrochemicals and Human Health

Andrew D. Wallace1 and Ernest Hodgson2

1Dept of Evironmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, USA,2North Carolina State University, Raleigh, NC, USA

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Agrochemicals can potentially impact human xenobiotic metabolism pathways and the consequences to human health are largely unknown. Unlike pharmaceutical drug testing procedures that utilize surrogate animals, human in vitro systems, and human clinical trials, the health risks of agrochemicals are determined primarily from studies using surrogate animals. Extrapolation from surrogate animals is problematic due to the uncertainties of the relevance to humans and to considerations of human variation and species-specific differences. Our research groups have investigated agrochemicals with the potential to cause metabolic inter-actions including inhibition and/or induction of xenobiotic metabolizing enzymes. Studies were undertaken using human in vitro model systems consisting of microsomes, enzymes, cell lines, and hepatocytes. Studies of the insecticide chlorpyrifos using microsomes have determined that metabolism of chlorpyrifos leads to formation of a reactive metabolite that inhibits xenobiotic metabolism of other agrochemicals and endogenous substrates. Further studies using individual human microsomes have characterized that large differences exist between individuals in terms of their ability to form the reactive metabolite. Agrochemicals can also impact the expression of xenobiotic metabolizing enzymes such as cytochrome P450s (CYPs). Increases in CYP3A4 and CYP2B6 expression were observed after human hepatocyte exposure to a number of agrochemicals. The mechanism(s) of the observed increases in CYP enzymes were investigated using human cell lines and various human receptor expression plasmids. By the use of multiple human in vitro model systems, we have deter-mined the ability of agrochemicals to cause metabolic interactions based on inhibition or induction. Some of the observed effects can occur at low concentrations and may pose unacceptable human health risks, and findings from these studies suggest that high use and new agrochemicals should be assessed for their ability to cause human metabolic interactions.

S28. Human Health Risk Assessment Associated with Cyanotoxin Exposure

Emanuela Testai and Enzo Funari

Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy

Human Health Risk Assessment Associated with Cyanotoxin Exposure In the last years the nutrient overload from agriculture and other anthropogenic activities, determining eutrophic conditions in water bodies, has increasingly favoured the growth of ubiquitous photosynthetic procariota, known as cyanobacteria, up to such an elevated density to result in blooms and scum. Many cyanobacterial species are able to produce cyanotoxins as second-ary metabolites, among which microcystins (MC) are a wide group of eptapeptides, consisting up to now of 80 different congeners; all of them are hepatotoxins, although with different potency in inducing acute effects. Long term effects are known for one congener (MC-LR) and used for all. Due to their tumour promoting activity, IARC has recently classified MC as possible human carcinogens (class 2B). In rodents and fish, MC-LR has been shown to be detoxified by glutathione S-transferases (GST) and recent results from our group evidenced that also recombinant human GSTs efficiently catalysed this reaction. Some other cyanotoxins, such as anatoxins, act as potent neurotoxicants, whereas cylindrospermopsin (CYN) is per se hepato- and nephrotoxic, due to protein-inhibition activity. On the basis of indirect observations it has been hypothesised that CYN is bioactivated by CYP to metabolites suspected to be genotoxic. The oral route is by far the most important source of human exposure, due to consumption of contaminated drinking water or food (including fish and food supplements derived from cyanobacteria) or by ingesting water during recreational activities. Dermal and inhalation exposure may also occur during recreational, sport, and professional activities. The parenteral route of exposure may also occur, when water from cyanotoxins contaminated superficial water bodies is used for hemodialysis, as it was in Brazil, where 56 patients out of 130 in hemodialysis treatment died, after receiving water contaminated by MC. Although acute intoxication episodes after contaminated drinking water consumption may occur when efficient treatments are not applied, the major health concern in developed countries is related to repeated exposures. Indeed, the death of a young woman due to liver failure has been recently associated to chronic use of MC-contaminated food supplements. Some epidemiological data have suggested a role for CTXs in the aetiology of neurodegenera-tive diseases, including Amyotrophic Lateral Sclerosis. In spite of the emergent health problems, the available toxicological information are limited, data on humans are very scant, and risk assessment is possible only in few cases, with huge degree of uncertainties. The available data on the more representative cyanotoxins have been critically reviewed in order to evidence the major human health risks. Some specific examples of risk assessment and research needs will be presented.

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S29. Persistent Toxic Substances: Individual Exposures, Population Distributions, and Health Effects

Miquel Porta

IMIM and Universitat Autonoma de Barcelona, Barcelona, Catalonia, Spain

The virtually universal presence of persistent toxic substances (PTS) in the environment, food chains and human popu-lations is a direct result of the way we live; the consequence of multiple private and public policies –food, agricultural, industrial, environmental policies–, we inherited PTS exposure from the most widely accepted socioeconomic models of the 20th. century. We need collective mechanisms of protection against PTS; as individuals there is little we can do to decrease our personal accumulation of PTS. Prevention of human PTS contamination cannot be treated as an individual problem: more energetic policies and population strategies are required.

Systems that monitor human contamination by PTS in a representative sample of the general population are scarce worldwide. Few efforts have been devoted to the representation and analysis of the full population distribution of PTS concentrations in humans. “PTS Geoffrey Rose curves” help visualize that such distributions stem from and belong to a population, and emphasize the importance of shifting the whole distribution of PTS concentrations through public and private policies.

Findings on PTS concentrations in the general population stress the need to develop appropriate surveillance sys-tems. The path to follow is not to perform individual measurements of PTS, but population surveillance and control of PTS. Indeed, “Geoffrey Roses′s big idea” (i.e., changing the population distribution of a risk factor prevents more burden of disease than targeting people at high risk) is even more relevant to PTS than to classic risk factors for chronic diseases. The only way to improve matters is to shift the population distribution of PTS.

Hence, there are strong reasons to conduct population surveys on PTS concentrations. Main results of the recent Report “Distribution of serum concentrations of Persistent Organic Pollutants (POPs) in a representative sample of the general population of Catalonia” will be presented (available at: http://www.imim.es/media/upload//arxius/porta/COPs%2004.pdf)

From mechanistic and epidemiological perspectives it is also interesting that there currently is a re-emergence of knowledge on the influence of environmental agents on gene expression; e.g., the carcinogenicity of compounds like Nickel, Cadmium or Arsenic involves DNA hypermethylation and histone deacetylation, both of which contrib-ute to heterochromatin condensation and the epigenetic silencing of some genes. The adverse health effects of PTS through indirect, non-genotoxic -perhaps epigenetic- mechanisms is also highly relevant. Accumulation of genetic and epigenetic alterations is often a fundamental etiopathogenic process in diseases of complex etiology. It often has environmental causes, as PTS exposure. It is a scientific mistake to disregard or underestimate the environmental causes of the accumulation of genetic and epigenetic alterations in diseases of complex etiology.

PTS offer both old and new challenges to health sciences. Not least among them is the need to explain findings on PTS exposure and effects in a way that is culturally acceptable to wide sectors of our societies. Otherwise PTS will only cause more fear. Because we aim at societies as free from fear as possible, we should also develop a scientific pedagogy that is as culturally sustainable as possible.

S30. Dialkylanilines are Ubiquitous and Potently Toxic and Mutagenic via Multiple Pathways

Steven R. Tannenbaum, Paul L. Skipper and Gerald N. Wogan

Dept of Biological Engineering, MIT, Cambridge, MA, USA

Aromatic amines are typically metabolized in the liver by CYP450s to form the N-hydroxy derivative which is sufficiently stable to enable diffusion out of the hepatocyte into a red blood cell where it reacts with hemoglobin to form a stable cysteine sulfinamide. Hydrolysis of the adduct releases the original aromatic amine which can be characterized by GC-MS. In an epidemiological study on bladder cancer patients we discovered that 3 alkylanilines (2,6-dimethyl-; 3,5-dimethyl-; and 3-ethylaniline) had a higher association with bladder cancer risk than the well-known human bladder carcinogen 4-aminobiphenyl (J Natl Cancer Inst. 2004, 96:1425-31). This was a surprising result because the methyl-anilines are weak or non-carcinogens. However, the result was fortified by a study that showed that 2,6-dimethylaniline (2,6-DMA) was a rat nasopharyngeal carcinogen. Subsequent research in our laboratory showed that the principal enzyme responsible for 2,6-DMA metabolism (CYP 2A6) was absent in rat liver, but present in mouse and human liver, and that all 3 compounds formed DNA adducts in several tissues in the mouse (Chem. Res. Toxicol. 2006, 19:1086-

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1090), further suggesting that these compounds receive serious consideration as human toxicants. In addition, a recent analysis of this class of chemicals by NTP/NIEHS revealed that at least 19 alkyl substituted anilines were prevalent in the environment and had never been evaluated for toxicity. It is important to be able to understand the mechanisms of toxicity of these 3 compounds in order to be able to make predictions on the remainder of the class. Therefore we have undertaken to develop cell culture based systems for analysis of toxicity and mutagenicity, and to prepare 14C-labeled compounds for both in vitro and in vivo experiments on DNA adduct formation. The N-hydroxy and N-acetoxy amines, and the corresponding p-aminophenols and their corresponding quinoneamines have all been prepared and tested in vitro, and the parent amines and the aminophenols have been tested in vivo. The results show that both N-hydroxylation and subsequent aminophenol formation are important for toxicity, DNA adduct formation, and mutagenicity, but the rules for prediction have yet to be established.

S31. CYP51: A Target for Drug Design

Michael R. Waterman and Galina I. Lepesheva

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 (USA)

Design of novel drugs which have a profound effect on the function of essential biochemical processes in infectious organisms but little effect in the host is the general approach for development of treatment of biological infections. Sterol biosynthesis is essential in maintenance of membrane integrity and therefore survival of many eukaryotes including animals, plants, fish, yeast, fungi, protozoa and chordates. Of these organisms yeast, fungi and protozoa are well known to cause infectious diseases in humans.A particularly effective drug target for these infectious organisms is sterol 14α-demethylase (CYP51) which catalyzes an essential step in sterol biosynthesis. Inhibition of this reaction and consequently synthesis membrane sterols can be lethal for infectious organisms. CYP51, a microsomal P450, is the most widely distributed of all P450 enzymes. In addi-tion to its broad distribution in eukaryotes, it is found in a limited number of prokaryotes. The CYP51 primary sequence between different biological kingdoms ranges between 20-30%. Yet all of these enzymes can use only 5 closely related sterols as substrates. CYP51 3-dimensional structures of human and protozoa are similar even though the signature sequence consists of only 36 amino acids across all biological kingdoms.Azole compounds have been used for many years as treatment for external yeast/fungal infections such as athlete’s foot and ringworm. Many of these azoles can also efficiently inhibit human (host) CYP51 and therefore are safe only for external treatment. Examples of internal infections where CYP51 inhibition is effective are Candida and Aspergillus especially in HIV patients, trypanosomes causing sleeping sickness and Chagas disease and fungal infections of plants. In a few cases azole compounds specific for CYP51 in the infectious organism and not the host have been developed. High throughput screening is one approach that has been used to identify additional types of CYP51 inhibitors by our laboratory and others. Recently we have identified highly specific and potent, stoichiometric inhibitors and deter-mined the 3-D structures of their complexes with trypanosomal CYP51s. These molecules serve as scaffolds for further development of inhibitors and describe a new approach for drug design for treatment of CYP51-dependent infectious diseases.

S32. Germline Pharmacogenomics in the Treatment of Breast Cancer

David A. Flockhart

Division of Clinical Pharmacology, Indiana University, Indianapolis, IN, USA

Both the risk of breast cancer and mortality due to breast cancer are increasing when measured globally. The treatment of breast cancer has improved markedly over the last 10 years with the advent of increasingly individualized approaches that have paved the way towards a more personalized treatment of many other malignancies. These approaches have exploited advances in tumor pathology, and include the targeting of endocrine therapy towards tumors that have estrogen or progesterone receptors, of the ant-HER2 antibody trastuzamab (Herceptin™) to tumors that overexpress the Her2 growth factor receptor, and the targeting of chemotherapy to patients with specific multigene RNA expression profiles such as the Oncotype Dx™ and Mammaprint tests. While an increasing body of literature supports the contribution of germline variation to breast cancer risk and therapeutic toxicity, germline approaches have not been used to study efficacy until

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recently, when it became clear that active metabolites of tamoxifen are generated through the action of cytochrome P450 2D6. Concentrations of the active metabolite of endoxifen clearly associate with CYP2D6 genotype and some. But not all studies conducted in the prevention, adjuvant and metastatic settings suggest that patients with CYP2D6 poor metabolizer genotypes respond less well to tamoxifen. This is important because there is a clear alternative to tamoxifen for these patients: the aromatase inhibitor class of drugs. Despite the fact that aromatase inhibitors appear slightly but definitively superior to tamoxifen as adjuvant therapy in postmenopausal women, the relatively low cost of tamoxifen make it the only viable monotherapy in many countries, and the aromatase inhibitors are ineffective, and therefore inappropriate mono-therapy in premenopausal women. In the treatment of breast cancer with biologic agents, it has been demonstrated that the combination of the anti-VEGF antibody bevacizumab with paclitaxel showed greater reductions in disease free survival than paclitaxel alone. Our group has recently demonstrated that germline genetic variability in the VEGF receptor was associated with outcomes, and can identify 2 populations: a group that experienced overall survival benefits not different from placebo, and a group that survived on average a year longer. It is of note that variants in the same gene are also associ-ated with risk for the most common toxicity of bevacizumab therapy: clinically significant hypertension. Collectively these data suggest that germline genomic variability in candidate genes, but also in pharmacologic and physiologic pathways may be valuable approaches to further refining the targeting of patients with breast cancer to maximize efficacy, but also to reduce toxicity and optimize the overall risk:benefit ratio of therapy for breast cancer.

S33. Pharmacogenomics of Thiopurines in Treatment of Acute Childhood Leukemia

Matthias Schwab

Dr.Margarete Fischer Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, Stuttgart, Germany

The individual reaction toward a given drug varies between individuals, and may depend on many factors such as age, sex, weight, body fat, concomitant drugs, nutritional status, liver and renal function, etc. Moreover pharmacogenetic polymorphisms are known affecting biotransformation and clinical outcome. Treatment results in childhood acute lymphoblastic leukemia (ALL) are one of the true success stories of modern clinical oncology, with overall cure rates of >80% achieved by application of intensive multiagent chemotherapeutic regimens. Modern regimens consist of at least four elements: 1) an induction phase using multiple cancer chemotherapeutic agents; 2) a consolidation segment to eradicate residual leukemic blasts in patients who are in remission; 3) extra-compartment therapy such as central nervous system-directed therapy, and 4) a maintenance period to further stabilize remission. Adjustment of therapy according to the risk of treatment failure has become a common feature in the clinical management of ALL. Continuing research on the clinical and biological aspects of leukemias has identified numerous features with prognostic potential. These factors include clinical (e.g., initial leukocyte count, age at diagnosis), immunological (e.g., immunophenotype) and genetic characteristics (e.g., non-random recurrent chromosomal aberrations). The thiopurines 6-mercaptopu-rine (6-MP) and 6-thioguanine (6-TG) are essential components of treatment protocols for ALL. In the past 25 years, insights into thiopurine pharmacology have been gained and led to the development of strategies for improving efficacy and reducing toxicity. One important route of metabolism for thiopurines is methylation by the enzyme thiopurine S-methyltransferase (TPMT). The gene coding for TPMT is subject to phenotypically relevant genetic variation, but the validation of this information and its potential translation into clinical practice is still an ongoing process. However, it is unlikely that one single gene will affect exclusively disease and/or treatment outcome, and therefore, a more compre-hensive approach will be to consider genetic polymorphisms in entire biological/pharmacological pathways. Moreover, recently developed ‘-omics appoaches′ (e.g., genomics, transcriptomics, proteomics) will be helpful to identify further putative targets for better prediction of drug response and will complement each other. Microarray technologies (e.g. cDNA arrays, GWA) have shown to be helpful for identifying novel susceptibility genes in ALL therapy, redefining disease diagnosis and predicting therapy response to specific drugs.

Supported by the Robert-Bosch Foundation, Stuttgart, Germany.

S34. Translation of Irinotecan Pharmacogenetics into Clinical Practice in the Treatment of Colorectal Cancer

Giuseppe Toffoli

Experimental and Clinical Pharmacology Unit, CRO- National Cancer Institute, Aviano, Italy

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Recent developments in a number of molecular profiling technologies, including genomic/genetic testing, proteomic profiling and metabolomic analysis have allowed the development of ‘personalized medicine′. Irinotecan is one of the models for personalized medicine based on pharmacogenetics, and a number of clinical studies have revealed significant associations between UGT1A1*28 and irinotecan toxicity. Based on this cumulative evidence, the US FDA and pharmaceutical companies revised the irinotecan label in June 2005. However, a recommended strategy for irinotecan-dose adjustments based on individual genetic factors has not yet been fully established. Irinotecan is activated in vivo by systemic carboxylesterases (hCEs) to 7-ethyl-10-hy droxycamptotecin (SN-38), active as a topoisomerase I inhibitor. Subsequently, SN-38 is glucuronidated by uridine disphosphate glucuronosyltransfer ases (UGTs) to form an inactive metabolite, SN-38 glucuronide (SN-38G). UGT1A1 is the main UGT isozyme that inactivates irinotecan to SN-38, whereas UGT1A7 and UGT1A9 play a minor role in the inactivation of SN-38. UGT1A9 is highly expressed in human liver and UGT1A7 is expressed in extrahepatic tissues and is potentially relevant to the enterohepatic circulation of SN-38. In addition to conversion to SN-38, irinotecan undergoes oxidation to metabolites, 7-ethyl-10-(4-N-[5-aminopentanoic acid]-1-piperidino)-carbonyloxy-camptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino)-carbonyloxycamp-tothecin (NPC), by CYP3A4/5 enzymes. The membrane transporter responsible for the uptake of SN-38 from plasma into hepatocytes is OATP1B1, whereas the elimination of irinotecan and its metabolites into bile is mediated by ABCB1, ABCC2 and ABCG2. The ABCC1 transporter is responsible for the efflux of SN-38 from hepatocytes into the interstitial space.

UGT1A1*28 polymorphism, characterized by an extra TA repeat in the promoter region of the gene, is associated with a reduced glucuronidation of SN-38, with effects on the drug pharmacokinetics and in the toxicity profiles. In a prospective study, we had analyzed the effect of UGT1A1*28 polymorphism on irinotecan pharmacodynamics and pharmacokinetics, in 250 mCRC patients first-line treated with FOLFIRI regimen. We noticed an increased hematologic toxicity in *28/*28 patients after the first cycle of chemotherapy, that was associated with a trend for improved tumor response. We concluded that reducing the dose in *28/*28 patients could be questionable due to the link between toxicity and response. On the contrary we pointed out that *1/*1 and *1/*28 patients could be under-dosed for optimal tumor response. Based on this suggestion, we recently conducted a phase I study and demonstrated the clinical util-ity of an innovative pharmacogenetic approach in a genotype-based (UGT1A1*28 polymorphism) phase Ib study in metastatic colorectal cancer (mCRC) patients, treated with FOLFIRI (irinotecan, 5-fluorouracil and leucovorin) regimen. We pointed out that the absence of the homozygous variant *28/*28 genotype was associated with a higher irinotecan MTD than that previously defined with a traditional FOLFIRI phase I study. Even if the response to treatment is not the aim of phase I trials, patients treated at higher doses seemed to respond better, as suggested by our previous data. Prospective FOLFIRI studies in a large population of metastatic CRC patients should be performed to test whether higher irinotecan doses can increase the therapeutic index of FOLFIRI as compared to the standard dosing and whether other polymorphisms may be clinically important.

S35. Pharmacogenomics of Colorectal Cancer Including Prognostic and Predictive Biomarkers

Sabine Tejpar

Digestive Oncology Unit, University Hospital Gasthuisberg, Leuven, Belgium

The number of agents which are potentially effective in the treatment of resectable and metastatic colon cancer is increasing. Consequently it is important to ascertain which subgroups of patients will benefit from a specific treatment. Despite more than two decades of research into the molecular genetics of colon cancer there is a lack of prognostic and predictive molecular biomarkers with proven utility in this setting. We will discuss our work on prognostic markers in stage II and III colon cancer, and how we approach the identification of prognostic subgroups in this disease in this post genomic era. We will also discuss the development of predictive biomarkers, taking as example the recent developments to predict sensitivity to EGFR inhibitors in colorectal cancer.

S36. Engineering of Glutathione Transferases for Biocatalysis

Bengt Mannervik, Arna Runarsdottir and Abeer Shokeer

Department of Biochemistry and Organic Chemistry, Uppsala University, Uppsala, Sweden

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A major paradigm in natural enzyme evolution is gene duplication accompanied by mutations that result in redesign of the preexisting molecule. The mutagenesis can involve point mutations as well as recombination of segments of primary structure. In the engineering of enzymes to obtain novel functional properties the same principle of redesign can be used. Glutathione transferases (GSTs) are particularly well suited to demonstrate acquisition of altered catalytic proper-ties, since they have low activity with many alternative substrates, and enhanced activity with the particular substrate can often be obtained by minor structural alterations of an enzyme (Josephy and Mannervik, 2006). It would appear that the evolutionary trajectory from an already specialized enzyme to an alternative selective and efficient biocatalyst generally involves intermediate states of promiscuous low-activity functions. Some of the naturally occurring GSTs appear to represent such generalist enzymes. We will present examples of GSTs that have undergone structure-based rational redesign or been subjected to stochastic mutations followed by screening for novel activities with alternative substrates. (The work has been supported by the Swedish Research Council and the Swedish Cancer Society)

Reference

Josephy, P.D. and Mannervik, B. Molecular Toxicology, 2nd Ed., Oxford University Press, New York, 2006

S37. Engineering P450s by Rational Design

Luet Lok Wong1, Christopher J. C. Whitehouse1, Wen Yang2, Jake A. Yorke1, Christopher F. Blan-ford1, Stephen G. Bell1, Mark Bartlam2, Zihe Rao2,3

1Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, Oxford, United Kingdom,2Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, China,3Laboratory of Structural Biology, Tsinghua University, Beijing, China

The cytochrome P450 (CYP) superfamily of heme monooxygenases plays important roles in the biosynthesis of endogenous compounds as well as xenobiotic metabolism and detoxification. The C–H bond activation activity of P450 enzymes enables microorganisms to grow on otherwise chemically inert substrates such as alkanes and aromatic hydrocarbons, while oxidation typically to the alcohol derivative renders hydrophobic organic compounds more soluble and reactive and hence more readily degraded. Human P450 enzymes are involved in the metabolism of a large pro-portion of prescription medicines, but also the activation of procarcinogen molecules to express their carcinogenicity. The remarkable C–H bond oxidation activity of P450 enzymes has significant potential in many areas of biotechnology including the synthesis of fine chemicals and metabolites of candidate drug molecules under benign conditions and environmental remediation.

We have studied the engineering of stable, soluble bacterial P450 enzymes for the oxidation of non-natural substrates. Our efforts have been focused on the extensively characterised class I P450cam (CYP101A1) system from Pseudomonas putida and the self-sufficient class I enzyme P450BM3 (CYP102A1) from Bacillus megaterium.1 New, fully reconstituted P450 systems have also been isolated from metabolically diverse bacteria.2,3 Rational design of the P450cam system has generated variants capable of oxidising alkanes down to ethane, polyaromatic hydrocarbons, polychlorinated benzenes and terpenoids. The P450BM3 system is a particularly attractive target for rational and directed protein evolution studies because it is self-sufficient and highly active. We will report on the rational engineering and directed evolution of these two P450 enzymes for the oxidation of non-natural substrates and our efforts to gain insights into the effect of mutations on enzyme structure, substrate binding and reaction pathways leading to product selectivity as well as the electronic and biophysical properties of the heme.

References

1) Bell, S.G., Hoskins, N., Whitehouse, C.J.C. & Wong, L.L. (2007) Met Ions Life Sci 3, 437-476.2) Bell, S.G., Hoskins, N., Xu, F., Caprotti, D., Rao, Z. & Wong, L.L. (2006). Biochem Biophys Res Commun 342, 191-196.3) Bell, S.G. & Wong, L.L. (2007) Biochem Biophys Res Commun 360, 666-672.4) Whitehouse, C.J.C., Bell, S.G., Tufton, H.G., Kenny, R.J., Ogilvie, L.C. & Wong, L.L. (2008) Chem Commun 966-968.5) Whitehouse, C.J.C., Bell, S.G., Yang, W., Yorke, J.A., Blanford, C.F., Strong, A.J.F., Morse, E.J., Bartlam, M., Rao, Z. & Wong, L.L. (2009)

Chembiochem 10, 1654-1656.6) Whitehouse, C.J.C., Bell, S.G. & Wong, L.L. (2008) Chem Eur J 14, 10905-10908.

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S38. Optimization of CYP102A1 for Biocatalysis

Vlada B. Urlacher

Institute of Biochemistry, Heinrich-Heine University of Düsseldorf, Düsseldorf, Germany

The diverse chemical reaction catalyzed by members of the cytochrome P450 family opens up new potentials for prac-tical application of these protein systems. Many interesting oxygenase-catalyzed reactions have been described in literature, but examples for process implementation and scale-up to pilot or industrial scales are comparatively rare. Obviously, it needs a long time and excellent knowledge of the corresponding P450 systems to develop industrial proc-esses due to the complexity of these enzymes.

The focus of our research was to elaborate the concepts of biocatalysis with bacterial P450 monooxygenases in pre-parative scale with respect to feasibility of such systems and to exploiting these enzymes for the production of valuable oxyfunctionalized compounds. To reduce the complexity of the P450 systems, we have been focusing on self-sufficient bacterial monooxygenases such as CYP102A1 from Bacillus megaterium (also referred to as P450 BM3), which do not depend on exogeneous electron transfer proteins. The enzyme can now be produced in gram scale using the fed-batch fermentation of E. coli. The use of expensive NADPH could be circumvented using mutants of CYP102A1, which were engineered to accept the much cheaper NADH. Further an efficient and cheap cofactor regeneration system that is stable in vitro, has been established and successfully applied in different reaction systems. Furthermore, our studies have shown that methods of protein design as well as enzyme immobilization can lead to improved thermo-, and storage stability of P450 enzymes.

The problem of low substrate solubility in water was solved by using a two-phase system or by adding cyclodextrines. In both systems, the P450 enzyme remains active over a long period, resulting in significant total turnovers and space-time-yields. Highly valuable products such as hydroxylated highly-branched fatty alcohols or oxidized monoterpenes or sesquiterpenes, such as (-)-cis-verbenol or (+)-nootkatone, were synthesized on a preparative scale. The developed systems can be applied for other P450 monooxygenases with new activities or altered selectivities.

An iterative process of site-directed mutagenesis, subsequent characterization of the mutants, and computer modeling was used to gain structural information on CYP102A1. Our results have demonstrated that mutagenesis of residues in the active site can lead to altered regio- and stereoselectivity. Nevertheless, the effects of a substrate on the regiochemistry of CYP102A1 should be taken into account during protein design processes. This novel insight in catalytic processes involving CYP102A1 can be used in future research in order to support the effective development of new mutants with interesting properties.

S39. The Explosive-Degrading Cytochrome P450 System XplA/B: From Enzyme Discovery to Engineering Transgenic Plants

Neil Bruce

Department of Biology, University of York, York, United Kingdom

Explosives are examples of toxic, synthetic compounds that pollute large areas of land and ground water on a global scale. Environmental contamination by explosives such as RDX has arisen mainly as a result of the manufacture and military use of munitions. Our recent studies on the biodegradation of energetic compounds by bacteria resulted in the isolation of a strain of Rhodococcus rhodochrous from explosive-contaminated land that is capable of degrading RDX when provided as the sole source of nitrogen for growth. The gene, xplA, responsible for RDX degradation in R. rhodochrous strain 11Y was cloned and identified as encoding a novel cytochrome P450 (CYP177A1). We have demonstrated for phytoremediation applications that expression of xplA along with its partnering reductase xplB in the model plant system Arabidopsis confers the ability to tolerate and degrade high concentrations of RDX. XplA is of unique structural organization in that it was found to possess a flavodoxin domain fused to the N-terminus of the P450 domain. Recombinant XplA, with XplB, was shown to efficiently degrade RDX both aerobically and anaerobically by a reductive denitration mechanism. Interestingly, the presence of oxygen determines the final products of RDX degrada-tion, demonstrating that the degradation chemistry is flexible, but both pathways result in ring cleavage of the RDX molecule and release of nitrite. A reductive denitration mechanism is unusual for P450s and details of which currently remain unclear. The structure of the heme domain of XplA has been solved in two crystal forms. The structural features and rational engineering of XplA and its potential use for biotechnological applications will also be discussed.

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S40. Oxidative DNA Damage, DNA Repair Enzyme NEIL1, Polymorphisms and Cancer

Miral Dizdaroglu

Biochemical Science Division, National Institute of Standards and technology, Gaithersburg, MD, USA

Oxidatively induced damage generates a multiplicity of base lesions in DNA. This type of damage is implicated to play a role in disease processes such as cancer and aging. Most of DNA base lesions are repaired by base excision repair, which is initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1 and others with different substrate specificities. Human and mouse NEIL1 proteins specifically remove 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA containing multiple lesions. However, NEIL1 possesses no significant activity for another major lesion, 8-hydroxyguanine. We characterized four known polymorphic variants of human NEIL1. Two of them showed near wild-type enzyme specificity and kinetics, whereas the others were devoid of glycosylase activity. Loss of NEIL1 was shown to result in significant biological consequences. Thus, inactivating mutations in neil1 correlated with some diseases including cancer. Knockdown of neil1 hypersensitized murine cells to killing effects of ionizing radiation. Downregulation of NEIL1 increased spontaneous DNA damage and mutations in cultured mammalian cells. In the absence of exogenous oxidative stress, several types of cancer and symptoms of metabolic syndrome were observed in neil1–/– mice. Significant accumulation of FapyAde and FapyGua were shown in several organs of neil1–/– mice, proving these lesions as in vivo substrates of NEIL1. Furthermore, (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines accumulated in neil1–/– mice, suggesting the involvement of NEIL1 in nucleotide excision repair of these lesions. Extrapolation of accumulated data suggests that individuals, who are heterozygous for inactive variant neil1 alleles, may be at increased risk for carcinogenesis and diseases associated with the Metabolic syndrome.

S41. Role of Transcription Coupled DNA Repair in Human Disease

Philip C. Hanawalt and Graciela Spivak

Department of Biology, Stanford University, Stanford, CA, USA

Replication and maintenance of the genome are absolute requirements for life. A proliferating cell must duplicate its DNA with astonishing precision in the face of a barrage of endogenous and environmental genotoxic insults. A com-pounded threat may arise when an advancing replication fork encounters an arrested transcription complex.

Expressed genes are scanned by translocating RNA polymerases, which sensitively detect DNA damage and initiate transcription-coupled repair (TCR), a sub-pathway of nucleotide excision repair targeted to lesions in the transcribed strands of expressed genes. Human hereditary diseases that present only a deficiency in TCR are characterized by sunlight sensitivity but no cancer. Multiple gene products are implicated in this pathway but we lack knowledge of the precise signals for TCR.

UV-sensitive syndrome (UVSS) is a human hereditary disease with mild clinical manifestations. No neurological or developmental abnormalities or predisposition to skin cancer have been reported. UVSS is genetically heterogeneous, in that it appears in individuals with mutations in CSB, in CSA, or in a still unidentified gene.

Cells from UVSS and Cockayne syndrome (CS) exhibit similar hypersensitivity to UV, resulting from defective TCR of photoproducts in expressed genes.

The severity of the developmental and neurological symptoms in CS patients may be caused by a defect in process-ing base damage generated by endogenous reactive oxygen species, with consequent premature cell death in tissues undergoing intense metabolic activity, while UVSS patients may be proficient in such pathways. In support of this idea, we have shown that CS cells (but not UVSS cells) are hypersensitive to the oxidants hydrogen peroxide, potassium bromate and menadione.

Using a host cell reactivation (HCR) assay with UV-irradiated plasmids, we found that expression of the plasmid-encoded lacZ gene is reduced in the TCR-deficient CS-B and UVSS cells. When the plasmids contained the oxidative base lesion thymine glycol, CS-B cells were defective in recovery of expression, whereas UVSS cells exhibited levels of expression similar to those in wild type cells. Similarly, plasmids containing 8-oxoG resulted in defective HCR in CS-A and CS-B cells. However, repair of thymine glycols in the lacZ gene was efficient and without strand bias in all cell lines tested. Evidence from in vitro transcription of substrates containing uniquely positioned oxidized bases suggests that transcription past oxidative lesions in DNA is defective in CS cells.

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References

Hanawalt & Spivak, Nat.Rev.Mol.Cell Biol 9:958-970 (2008);Spivak & Hanawalt, DNA Repair, 5:13-22 (2006);Spivak, PNAS 43:15273-15274 (2004);Spivak, Mutat. Res. 577:162-169 (2005);Nardo T, et al. PNAS 106:6209-6214 (2009).Support from National Cancer Institute, NIH.

S42. Base Excision Repair in Cancer and its Modulation by Oxidative Stress

Barbara Tudek

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

Oxidative stress is involved in the pathogenesis of colon cancer. It appears at premalignant phase of the disease devel-opment and is manifested as depletion of antioxidant vitamins level, aberrant oxidative DNA damage, as well as aber-rant transcription and activity of base excision repair enzymes. Analyzing three groups, healthy individuals (controls), patients with benign adenoma (AD), and colorectal carcinoma patients (CRC) we observed that the level of vitamins A, C and E in blood plasma decreased gradually. 8-OxodG level in leukocyte DNA and urine increased in CRC and AD patients. Transcription of DNA repair genes, namely OGG1 glycosylase and APE1 endonuclease was induced in leukocytes of AD individuals. Similar increase in mRNA level of OGG1, APE1, MTH1, ANPG and TDG repair enzymes was also observed in blood leukocytes of CRC patients. Repair rate of different DNA lesions measured by the nicking assay also changed in CRC patients in relation to controls. 8-OxoGua excision rate was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. However, excision rate of lipid peroxidation (LPO) derived etheno DNA adducts, 1,N6-ethenoadenine (ϵA) and 3,N4-ethenocytosine (ϵC) was lower in leukocytes of CRC patients than in controls. Model studies performed on purified BER enzymes and in cell cultures showed that LPO product 4-hydroxynonenal (HNE) can modify BER efficiency. HNE decreases the excision rate of ϵA and ϵC, but not 8-oxoG, increases AP sites incision and blocks the religation step after gap filling the by DNA polymerase. Thus, during onset of carcinogenesis, repair of different DNA damages in the colon seems to be under complex transcriptional and post-transcriptional control, whereby deregulation may act as a driving force to malignancy.

S43. Oxidative Stress and Antioxidants, Beneficial or Pathological?

Barry Halliwell

Department of Biochemistry, National University of Singapore, Singapore, Singapore

Free radicals and other reactive species are generated continually in the human body, both for useful purposes and by “accidents of chemistry”. They are significant contributors to age-related diseases and perhaps to the ageing process itself. In particular, they contribute to cancer development [1]. Nevertheless, reactive species also have beneficial effects in killing invading organisms and coordinating the inflammatory response. Indeed, many epidemiological studies have failed to demonstrate protective effects of antioxidants against the development of diseases such as cancer. By contrast, fruits, vegetables and products derived from them appear to play a key role in the human diet, in health maintenance and disease prevention, for at least some forms of cancer and of other diseases. Many constituents could contribute to such effects, including antioxidants and (seemingly paradoxically) pro-oxidants. These agents can affect physiology and pathology both within the gastrointestinal tract and also systemically after absorption. The care that is needed to establish the true effects of antioxidants and pro-oxidants in cell culture studies [2] and in vivo [3] will be illustrated by data from our recent studies.

References

Halliwell B (2007) Oxidative stress and cancer: have we moved forward? Biochem J. 401, 1-11.

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Halliwell B (2008) Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch. Biochem. Biophys. 476, 107-112.

Lee CY, Isaac HB, Wang H, Huang SH, Long LH, Jenner AM, Kelly RP, Halliwell B (2006) Cautions in the use of biomarkers of oxidative damage; the vascular and antioxidant effects of dark soy sauce in humans. Biochem Biophys Res Commun. 344, 906-911.

S44. Soy Isoflavones in Prostate Cancer Treatment

Ömer Küçük

Winship Cancer Institute, Emory University, Atlanta, GA, USA

Dietary intake of soy products has been associated with a decreased risk of prostate cancer. Genistein, the principal isoflavone in soy, has shown anti-cancer effects in cell culture, animal and human studies. Genistein is a selective estrogen receptor modulator (SERM) with greater preference for estrogen receptor (ER)-beta compared to ER-alpha. Potential mechanisms of action of genistein may also include its anti-oxidant and anti-inflammatory effects as well as inhibition and reversal of DNA methylation. Genistein has also been reported to lower serum VEGF levels and have ant-angiogenic activity. We have recently observed anti-RANKL activity as well. Genistein increases osteoprotegerin expression and decreases RANKL activation and MMP-9 activation, thereby preventing osteoporosis and bone metas-tases in a mouse model of prostate cancer bone metastasis (Li et al. Cancer Research, 2006). We have also observed genistein induced inhibition of NF-kB and sensitization of prostate cancer cells to radiation and chemotherapy both in vitro and in vivo. Some of the genistein effects on prostate cancer may be due to down-regulation of AR and up-regulation of Cx43. Tumor suppressor genes that are downregulated in prostate cancer may also be upregulated by genistein through demethylation of promoter regions of these genes. Potential benefits of genistein include prevention of prostate cancer, stabilization of advanced disease in patients with prostate cancer, and increasing the efficacy and decreasing the toxicities of androgen deprivation therapy, chemotherapy and radiation therapy.

S45. Chemopreventive and Chemotherapeutic Effects of Curcumin

Marc Diederich

Hopital Kirchberg, LBMCC, Luxembourg, Luxembourg

Prostate cancer is the most commun cancer in men in the western world. Lifestyle, diet and environmental as well as genetic factors (e.g. disregulations of the Wingless (Wnt) signaling pathway) promote the malignant transformation of healthy prostatic epithelium. Due to the high prevalence and the slow progressive development of prostate cancer, pri-mary prevention appears as an attractive strategy to eradicate prostate cancer. During the last decades, chemoprevention using non-toxic natural or synthetic compounds appeared to play an important role in cancer prevention and treatment, alone or in conjugation with usual drugs. Curcumin (diferuloylmethane), a major component of turmeric (Curcuma Longa), is one of the most well studied natural compounds characterized for its anti-inflammatory, anti-carcinogenic, anti-proliferative, anti-angiogenic and anti-oxidant properties. With this in mind, we studied the anti-proliferative potential of curcumin in prostate cancer. We demonstrate here that curcumin has the same intracytoplasmic localization in all prostate cancer cells tested. Interestingly, the androgen sensitive cells appear more sensitive to curcumin treatment than the androgen independent ones. We report that curcumin has an impact on the proliferation of androgen sensi-tive prostate cancer cells through the induction of cell cycle arrest in G2/M. By the same way, curcumin was shown to affect the Wnt signaling pathway highly implicated in prostate cancer cell proliferation. We observe a modulation of the expression of proteins implicated in this pathway, that leads to a decrease of the Wnt (beta-catenin/Tcf-4) transcriptional activity resulting in the decrease of Wnt target gene expression. Altogether our results suggest that curcumin could be considered as an interesting chemopreventive agent for early but not for late, metastatic prostate cancer.

S46. Tocopherols and Cancer Prevention

Chung S. Yang, Guang-Xun Li, Gang Lu, Mao-Jung Lee and Nanjoo Suh

Chemical Biology, Rutgers, The State University of New Jersey, Piscataway, NJ, USA

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Tocopherols, which exist in alpha, beta, gamma, and delta forms, are antioxidative nutrients also known as vitamin E. Although gamma-tocopherol (gamma-T) is the most abundant form of vitamin E in our diet, alpha-T is the major form found in the blood and tissues. Low vitamin E nutritional status has been suspected to increase cancer risk. However, a robust cancer preventive activity of these tocopherols has not been demonstrated. The disappointing results from recent large-scale human trials with high doses of alpha-T point out the need for a better understanding of the biological activities of different forms of tocopherols. Using a tocopherol mixture that is rich in gamma-T (gamma-TmT, which contains 57% gamma-T, 24% delta-T and 13% alpha-T), we have recently demonstrated the inhibition of cancer formation and growth in different animal models. When given in the diet at 0.17 and 0.3%, gamma-TmT inhibited colon tumorigenesis in mice treated with azoxymethane (AOM) and dextran sulfate sodium. Inhibition of colon carcinogenesis (reduction of adenocarcinoma and adenoma formation by ∼80%) was observed when dietary treatment with 0.3% gamma-TmT was initiated either before or after AOM/DSS treatment. The levels of 8-isoprostane, nitrotyrosine, prostaglandin E2 and leukotriene B4 in the plasma and/or colon were decreased by the gamma-TmT treatment. The anti-inflammatory activity of pure gamma-T and delta-T was also demonstrated in this model. In another set of experiments, dietary 0.3% gamma-TmT inhibited lung tumorigenesis in the A/J mice, induced by the tobacco carcinogen NNK or NNK plus benzo[a]pyrene, as well as inhibited the growth of human lung cancer H1299 cells in xenograft tumors in NCr-nu/nu mice. In both experimental systems, gamma-TmT decreased the levels of 8-hydroxydeoxyguanosine (a DNA oxidation product), gamma-H2AX (an indicator of DNA repair caused by DNA double-strand breakage), and nitrotyrosine in tumors. Dietary gamma-TmT also inhibited the growth of subcutane-ous tumors derived from CL13 murine lung cancer cells in A/J mice. In addition, dietary gamma-TmT was shown to prevent N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats by Suh et al.; the inhibition was associated with increased tumor cell apoptosis, activation of PPAR- gamma and down regulation of estrogen receptor-alpha. When different tocopherols were compared, delta-T was found to be more active than gamma-T in the inhibition of cancer cell growth (and induction of apoptosis) in cell culture and in H1299 lung cancer xenograft tumors and in the inhibition of AOM-induce aberrant crypt foci formation in rats, whereas alpha-T was relatively inactive. In order to understand the biological fates and activities of different tocopherols, the levels of these vitamin E forms and their metabolites were systematically analyzed after supplementation with different doses of tocopherols to mice and rats. These studies demonstrate the broad cancer preventive activities of gamma-TmT and higher activity of delta-T. We suggest the possible use of these agents, rather than pure alpha-T, for cancer prevention. (Supported by NIH grants CA120915, CA122474, CA141756, and the John Colaizzi Chair Endowment Fund).

S47. Regulation of Antioxidant Gene Expression by Selected Dietary Phytonutrients

Young-Joon Surh

College of Pharmacy, Seoul National University, Seoul, South Korea

Regulation of antioxidant gene expression by selected dietary phytonutrients Young-Joon Surh WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea The low risk of chronic diseases, such as coronary heart disease, neurodegenerative disorders and certain cancers, observed in some population groups results from consumption of relatively large amounts of fruits and vegetables. Health benefits associated with functional foods and beverages containing a great array of bioactive phytochemicals or phytonutrients have been extensively investigated and well-documented. As oxidative stress is implicated in a wide array of human disorders, antioxidant phytochemicals have also been frequently included as active ingredients of many functional foods. The most important botanicals for use as nutracuetical formulations include tea, soy, pomegranate, grape seeds, pycnogenol, turmeric, ginkgo biloba, aloe, ginseng, etc. The majority of bioactive chemical compounds contained in the dietary supplements/nutraceuticals have antioxidant activities. Nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) plays a crucial role in regulating induction of antioxidant or cytoprotective gene expression. Many bioactive substances derived from edible plants have been found to activate this particular redox-sensitive transcription factor, thereby potentiating cellular antioxidant or detoxification capacity. Nutraceutical and functional food markets are rapidly growing, and there are ample outstanding opportunities of research for the development of botanicals as dietary supplements or nutraceu-ticals. References 1. Surh, Y.-J. (2003) Cancer chemoprevention with dietary phytochemicals. Nature Reviews Cancer, 3: 768-780 2. Surh, Y.-J., Kundu, J.-K., and Na, H.-K. (2008) Nrf2 as a master redox switch in turning on the cellular signaling Involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med.,

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74: 1526-1539. 3. Surh, Y.-J., Dong, Z., Cadenas, E., and Packer, L. (2008) Dietary Modulation of Cell Signaling Pathways, CRC Press-Taylor & Francis

S48. Pharmacoepigenetics of Drug Pharmacokinetics

Magnus Ingelman-Sundberg

Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, SwedenPharmacoepigenetics of Drug Pharmacokinetics

The interindividual differences in drug metabolism are extensive. At present we do understand a major part of the true genetic reasons to such variability as copy number variations, in/dels and SNPs. Still the bases for a large extent of interindividual differences in enzyme expression or activity as revealed to be inherited from in vivo studies is extensive and remains to be elucidated. Such differences can be explained by epigenetic factors such as DNA methylation, post-translational modification of histones and expression of ncRNAs such as microRNAs and RNAas. Among the P450s such regulation is to be expected to be of importance for e.g. the variation in CYP1A2 and CYP3A4 expression, where no functional genetic polymorphism has been found. Today indeed, epigenetic regulation of the expression of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, CYP3A5, and CYP3A7 has been described. Among the transporters tissue specific expression has been found to be caused by specific epigenetic control. In addition, microRNA regulated expression of several CYPgenes and nuclear receptors have been described that is of importance for the regulation of drug disposition.

The lecture will give an update in the field of genetic polymorphism and epigenetics of genes of important for predic-tion of drug metabolism and ADRs focusing on clinically relevant examples.

S49. MicroRNA Regulation of Nuclear Receptors and Drug Metabolizing Cytochrome P450s

Miki Nakajima and Tsuyoshi Yokoi

Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

MicroRNAs (miRNAs) are endogenous ∼22-nucleotide non-coding RNAs that regulate gene expression through the translational repression or degradation of target mRNAs. The human genome may contain up to 1000 miRNAs and over 30% of human mRNAs are predicted to be targets of miRNAs. We investigated the role of miRNAs in the regulation of nuclear receptors and drug metabolizing cytochrome P450s. To evaluate the involvement of potential miRNAs on the regulation of targets, we examined the following: 1) the relationship between the mRNA and protein levels of the concerned target and the miRNA levels in a panel of human livers, 2) the effects of overexpression or inhibition on the expression level of the target and its outcome, 3) the results of a luciferase assay to identify the functional miRNA recognition element (MRE) in the target mRNA. We found that human pregnane X receptor (PXR) is regulated by miR-148a1). The contribution was supported by the fact that the PXR protein level in a panel of human livers was not correlated with the PXR mRNA level, but the translational efficiency of PXR was inversely correlated with the miR-148a level. The miR-148a-dependent repression of PXR protein attenuated the induction CYP3A4 mRNA. Notably, a potential miR-148a recognition element was also identified in the 3′-UTR of CYP3A4. However, the luciferase assay revealed that miR-148 could not recognize the MRE on the CYP3A4. Since the CYP3A4 protein level in human livers was significantly correlated with the CYP3A4 mRNA level, the post-transcriptional regulation would be minor for CYP3A4. We found that human hepatocyte nuclear factor (HNF) 4α is regulated by miR-24 and miR-34a2). Interestingly, miR-24 recognized the MRE in the coding region causing mRNA degradation, whereas miR-34a recognized the MRE in the 3′-untranslated region causing translational repression. The down-regulation of HNF4α by these miRNAs resulted in the decrease of its targets such as CYP7A1 and CYP8B1. We also have evidence that human proliferators-activated receptor α (PPARα), aryl hydrocarbon receptor nuclear translocator (ARNT), vitamin D receptor (VDR)3), CYP244), CYP1B15), and CYP2E16) are directly regulated by miRNAs. Collectively, miRNA plays an important role in the regulation of metabolism and disposition of endo/exobiotics. Although the regulation of miRNAs themselves is largely unknown, but the expression of miRNAs is readily altered. Therefore, dysregulation of specific miRNAs might lead to pharmacokinetic or pathophysi-ologic changes.

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References

1) Takagi et al., J. Biol. Chem., 283: 9674-80, 2008.2) Takagi et al., J. Biol. Chem., 285: 4415-22, 2010.3) Mohri et al., Int. J. Cancer, 125: 1328-33, 2009.4) Komagata et al., Mol. Pharmacol., 76: 702-9, 2009.5) Tsuchiya et al., Cancer Res., 66: 9090-8, 2006.6) Mohri et al., Biochem. Pharmacol., 79: 1045-52, 2010.

S50. RNA-seq Reveals Alternative Transcripts and Splicing Variants of P450 Genes in Human Hepatoma HepaRG Cells in Response to Xenobiotics

Xiao-bo Zhong, Dan Li, Steven N. Hart, Lai Peng and Hans Tregear

Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA

Change of cytochrome P450 gene expression is a major phenomenon in human liver cells in response to xenobiot-ics. About 90% human genes transcribe multiple transcripts or splicing variants. However, it is unclear how many isoforms each P450 gene can transcribe and how their transcription can be influenced by xenobiotics. Therefore, we used RNA-seq to examine changes of transcription in CYP1, 2, 3, and 4 gene families in the human hepatoma HepaRG cells treated with rifampicin, phenobarbital, or dexamethasone in comparison to an untreated control. RNA-seq was done by 36-base pair-end reads on an Illumina Genome Analyzer. The RNA-seq reads were aligned to human reference genome by TopHat. Transcripts were assembled by Cufflinks and differential expression of the P450 genes was tested by Cuffdiff. Of the 41 annotated genes in the CYP1, 2, 3, and 4 families, 36 genes were detectable in the control cells. Seven genes (CYP2C8, 2C9, 2C18, 2C19, 3A4, 3A5, and 3A7) significantly increased, but three (CYP2E1, 4B1, and 4F22) significantly decreased expression levels in at least one treatment. Twenty of the 36 detected P450 genes expressed multiple (2-9) isoforms, including several different types of alternative transcript events, such as alternative promoters, alternative poly(A), and exon skipping. Influence of transcription by xenobiotics was different among the isoforms with bigger changes in some isoforms than in other isoforms. In conclusion, by using RNA-seq, we have revealed alternative transcripts and splicing variants in the major P450 genes and found changes of their expression levels are different in response to xenobiotics in the hepatoma HepaRG cells.

S51. Lung P450 Expression, Bioactivation, and Mechanism-Based Inactivation by Glucocorticoids and Mutagens

Garold S. Yost

Dept of Pharmacol & Toxicol, Univ of Utah, Salt Lake City, UT, USA

The selective expression of cytochrome P450 enzymes in the respiratory tract is often a major factor in the metabolism and/or toxicity of numerous xenobiotics. In addition, the metabolism of xenobiotics by cytochrome P450 enzymes to produce reactive intermediates is surprisingly selective, because most P450s accommodate a wide variety of sub-strates. Many of the reactive intermediates are dehydrogenation products that are toxic and/or are mechanism-based inactivators. 3-Methylinole (3MI) is a highly potent mutagen, it was dehydrogenated by the lung-selective 1A1, 2F1, and 2A13 enzymes – the 2A13 and 2F1 enzymes were inactivated. Other enzymes tested included 1B1, 2E1, 3A4, 3A5, 2A6, 2B6, 2D6, 2C19 but none of them was inactivated by 3MI. Conversely, only CYP3A4, the hepatic enzyme, inactivated two other indoles, zafirlukast and SPD-304, a TNF-α inhibitor. Thus, remarkable selectivity for lung or liver enzyme-mediated bioactivation or mechanism-based inactivation by 3-substituted indoles was demonstrated. Glucocorticoids (GC) are primarily metabolized in respiratory tissues by the three cytochrome P450 3A enzymes (CYP3A4/5/7). Selective expression and/or inhibition of one or more CYP3A enzymes could substantially modify target lung cell or systemic concentrations of GCs, and lead to therapeutic resistance or adrenal suppression-induced adverse effects. CYP3A4 is the major hepatic 3A enzyme, but we and many others have confirmed it is not expressed in respiratory tissues. Conversely, CYP3A5 is a predominant lung P450, and surprisingly CYP3A7 mRNA was highly transcribed in amounts 10- to 300-fold higher than 3A5 from isolated pediatric human lung cells. We evaluated the mechanism-based inactivation of CYP3A4, 3A5 and 3A7 enzymes by beclomethasone dipropionate, triamcinolone

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acetonide, flunisolide, budesonide, and fluticasone propionate (FLT). FLT was the only GC that inactivated any CYP3A enzyme, and it potently inactivated CYP3A5 with KI, kinact and partition ratio of 16 μM, 0.027 min-1 and 3, respectively. In contrast, FLT minimally inactivated CYP3A4, and did not inactivate 3A7, even with a concentration of 100 mM. Thus, selective transcription of CYP3A4/5/7 genes and/or inactivation of the enzymes in the most relevant airway cells of pediatric patients may profoundly regulate the metabolism of inhaled GCs. Supported by NIH Grants GM074249 and HD060559.

S52. The Arachidonic Acid Epoxygenase: Functional Roles and relevance to the Pathophysiology of hypertension and Tumor Angiogenesis

Jorge H. Capdevila, Ambra Pozzi and Nataliya Pidkovka

Medicine, Vanderbilt University Medical School, Nashville, TN, USA

In addition to roles in xenobiotic metabolism, cytochrome P450 (P450) bio-activates arachidonic acid (AA) to 5,6-, 8,9-,11,12-, and/or 14,15-epoxyeicosatrienoic acid (EET)(AA epoxygenase); with human CYP2C8 and CYP2C9, mouse Cyp2c44, and rat CYP2C23 identified as predominant epoxygenases in the liver, kidney, and endothelium (1.2). The characterization of the AA epoxygenase as a formal metabolic pathway, suggested functional roles for its EET metabo-lites, and led to the identification of their roles in cell proliferation, vascular reactivity, and Ca++, K+, and Na+ ion channel regulation (1,2).

Experimentally (xenobiotic) induced or genetically determined changes in the expression and/or the activity of the Cyp2c44 epoxygenase alters renal and vascular function with important physiological and/or pathophysiological consequences (1,2). Thus, disruption of the Cyp2c44 epoxygenase gene leads to alterations in: a) in sodium transport in the distal nephron, and the development of dietary salt sensitive hypertension, and b) in tumor angiogenesis and growth. On the other hand, PPAR(alpha) ligands down-regulate endothelial Cyp2c44 expression, and reduce tumor angiogenesis and growth. The demonstration of a tumor selective expression of human CYP2C9, a functional homologue of murine Cyp2c44, and of its regulatory control by PPARα ligands suggest a role for this enzyme in human cancer, and for human PPARα as a target for the development of novel and better tolerated anti-tumor strategies.

These and other studies introduce a paradigm shift that could influence our views of the roles played by the P450 enzyme system in disease and toxicology, from that of vehicles for drug disposition and/or activation, to that of active participants in the pathophysiology of hypertension and cancer. It is expected that they will stimulate efforts to: a) develop novel, CYP2C based, inhibitors of tumor angiogenesis, and b) to a consideration during drug evaluation of the physiological and/or pathophysiological consequences of interfering with the activity and/or expression of P450s involved in endogenous metabolic pathways. (Supported by NIDK 38226 and GM 37922).

References

Capdevila, JH. (2007) Curr, Op. Nephr. & Hypertens. 16:465-470, and cited references.Pozzi A. et al. (2010) J. Biol. Chem. Feb 23 [Epub ahead of print].

S53. Function and Regulation of CYP2S1

Oliver Hankinson

Pathology, and Molecular Toxicology, University of California, Los Angeles, USA, Los Angeles, CA, USA

CYP2S1 is a recently described dioxin-inducible cytochrome P450 that is expressed in several extrahepatic organs, including the lung, skin, and urinary and gasterointestinal tracts. Several investigators have set out to identify sub-strates for CYP2S1, but no substrates were identified unambiguously. We expressed human CYP2S1 in bacteria and found that CYP2S1 is not reduced by NADPH-cytochrome P450 reductase and thus no activity was found with NADPH. However, we found that CYP2S1′s oxidative activity is supported by certain lipid peroxides such that the enzyme can oxidize various carcinogens via the peroxide shunt. We then investigated whether human CYP2S1 can metabolize

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various cyclooxygenase (COX) and lipoxygenase (LOX) derived lipid peroxides in a NADPH-independent fashion. Human CYP2S1 metabolizes prostaglandin G2 (PGG2) into several products including 15-keto-PGG2, 12-oxo-5-Z,8E,10E-heptadecatrienoic acid (12-oxoHT) and 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT). It also metabolizes prostaglandin H2 (PGH2) into malondialdehyde (MDA), 12-HHT, and thromboxane A2 (TXA2). The turnover of 12-HHT by human CYP2S1 (1.59±0.04 min-1) is 40-fold higher than that of TXA2 (0.04 min-1). Other P450s such as CYP1A1, 1A2, 1B1, and 3A4 also metabolize PGH2 into MDA and 12-HHT. In addition to prostaglan-din metabolism, human CYP2S1 efficiently metabolizes the hydroperoxyeicosatetraenoic acids (HpETE), 5S-, 12S-, 15S- HpETEs and 13S-hydroperoxyoctadecadienoic acid (13S-HpODE) into 5-oxo-eicosatetraenoic acid (5-oxoETE) (turnover = 16.7 ± 0.3 min-1), 12-oxo-eicosatetraenoic acid 1(2-oxoETE) (11.5 ± 0.9 min-1), 15-oxo-eicosatetraenoic acid (15-oxoETE) (16.9 ± 0.8 min-1), and 13-octadecadienoic acid (13-oxoODE) (20.2±0.9 min-1), respectively. Human CYPs 1A1, 1A2, and 1B1 also carried out similar conversions, but at slower rates. The hydroperoxy fatty acids were also converted by human CYP2S1 to several epoxy alcohol and trihydroxy derivatives. Our data indicate that PGG2, PGH2 and hydroperoxy fatty acids represent endogenous substrates of CYP2S1 and suggest that the enzyme may play an important role in the inflammatory process, since some of the products that CYP2S1 produces have been shown to participate in inflammation.

S54. Essential Role of Cytochromes P450 in Vitamin D Signaling

Inge Schuster

Institute of Theoretical Chemistry, University Vienna, Vienna, Austria

Vitamin D - produced in the skin from 7–dehydrocholesterol upon exposure to sunlight or supplied from the diet – is converted in the mammalian body to its major biologically active form 1alpha,25(OH)2D that acts in the way of steroid hormones. Bound to its nuclear receptor (VDR), 1alpha,25(OH)2D regulates the transcription of a wide variety of target genes that play key roles in numerous physiological processes such as mineral and bone homeostasis, cellular growth and differentiation, inflammation, innate immunity, and cellular metabolism. Meta-analyses of wide epidemiological studies are in line with these functions as they show vitamin D deficiency causally underlying a plethora of severe health problems, including neoplasms that might be prevented/improved on adequate vitamin D supplementation.

Levels, life-time, and consequently function of hormonally active vitamin D are controlled by cytochromes P450 (“vitamin D hydroxylases”) that catalyze all steps in the activation process of vitamin D to 1alpha,25(OH)2D as well as in its subsequent metabolism and eventual inactivation. Activation is achieved in two sequential steps, starting with a hydroxylation at the C-25 site that is mainly catalyzed by CYP2R1, but other CYPs (CYP27A1, CYP3A4, and CYP2J2) might also work as vitamin D 25-hydroxylase. Thereafter, a single enzyme - CYP27B1 – inserts a OH-group in the C-1alpha-position of 25(OH)D leading to the active hormone that may enter the circulation and become distributed to target sites expressing the VDR. While the VDR is expressed in most cell types and many of them are capable of 1alpha,25(OH)2D synthesis, the hormone can also function in an autocrine/paracrine way (as illustrated for several tissues/cell types). An important autocrine function of the hormone is the VDR-mediated rapid upregulation of its own metabolism via CYP24A1. This enzyme (termed vitamin D 24–hydroxylase) repeatedly attacks the side chain of 1alpha,25(OH)2D leading to a broad cascade of metabolites and the eventual loss of activity. In addition to the crucial role of vitamin D-hydroxylases, recent studies demonstrate that CYP11A1 recognizes vitamin D (and its precursor 7-dehydrocholesterol) as substrate(s) generating new cascades of products with potentially important biological activities.

The cascade of vitamin D metabolites shows considerable resemblance to the steroid hormone cascade: In generating wide arrays of transient lipophilic products that could activate signaling through nuclear receptors, Nature might have repeated the same strategy on either the rigid 4-ring system of cholesterol or the more flex-ible B-ring-cleaved secosterol derived from 7-dehydrocholesterol. Co-expressed in various tissues, both cascades show significant cross-talk with respect to their co-factors, reductases, CYPs, substrates, and nuclear receptors. Still little is known about intrinsic biological activities of individual vitamin D metabolites - all observed activi-ties are commonly attributed to 1alpha,25(OH)2D. As selective inhibitors of vitamin D hydroxylases increase the lifetime of otherwise highly transient metabolites they can serve as valuable research tools to analyze mechanistic details of the reactions and identify intrinsic activities of the metabolites without major contributions from their sequential products.

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Poster Awards Competition

A1. Abstract Withdrawn.

A2. Diclofenac Causes Inhibition of TNFα-induced NF-κB Nuclear Translocation Leading To Enhanced Death Receptor Signaling-dependent Hepatocyte Apoptosis

Lisa Fredriksson, Bram Herpers and Bob van de Water

Toxicology, LACDR, Leiden, Netherlands

Drug-induced liver injuries (DILIs) are the major cause of drug failures and are often idiosyncratic in nature. We hypothesize that idiosyncratic DILI occurs due to crosstalk between drug reactive metabolite and cytokine stress signaling. To study this hypothesis, human hepatoma HepG2 cells were exposed to diclofenac, which causes idi-osyncratic DILI in humans, in the presence of the pro-inflammatory cytokine TNFα. Diclofenac itself induced a mild concentration-dependent apoptosis of HepG2 cells. While TNFα itself was not cytotoxic, it strongly enhanced the diclofenac-induced apoptosis. This apoptosis was associated with the onset of caspase-8, -9 and -3 activation and inhibition of caspase-8 and -3 activity prevented apoptosis onset. Systematic siRNA-based knock-down of all individual apoptotic machinery components indicated that diclofenac/TNFα-induced apoptosis is mediated through a caspase-8/Bid/Apaf1/caspase-3 pathway. TNFα alone caused a rapid activation of the IKK-dependent phosphorylation of IκBα reflected by an oscillating nuclear translocation of NF-κB. Diclofenac inhibited IκBα phosphorylation, and dampened the nuclear NF-κB translocation oscillation in association with reduced NF-κB transcriptional activity. Both inhibition of IKK as well as stable lentiviral shRNA-based knock down of RelA sensi-tized hepatocytes towards diclofenac/TNFα-induced cytotoxicity. Finally, while diclofenac itself induced sustained activation of JNK, inhibition and siRNA-based knock down of JNK inhibited both diclofenac and diclofenac/TNFα-induced apoptosis. Together these data suggest a model whereby diclofenac-mediated stress signaling, possibly through JNK-dependent pathways, suppresses TNFα survival signaling routes and sensitizes cells to apoptosis. Currently, we apply both transcriptomics as well as siRNA-based functional genomics strategies to further unravel the signaling pathways that underlie the diclofenac/TNFα-induced hepatotoxicity. We anticipate that our work will enable us to identify mechanism-based biomarkers that can predict idiosyncratic DILI in a pre-clinical drug development setting.

A3. Humanized CYP3A Mice: (2) Functional Expression of Human CYP3A Isoforms in CYP3A-HAC Mice and Inhibition of CYP3A via Mechanism-Based Inactivation

Mio Watanabe1, Sasitorn Aueviriyavit1, Kaoru Kobayashi1, Nahoko Iuchi1, Yasuhiro Kazuki2, Mitsuo Oshimura2 and Kan Chiba1

1Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 2Graduate School of Medical Science, Tottori University, Tottori, Japan

Cytochrome P450 3As (CYP3As)-humanized mice were constructed by human artificial chromosome (HAC) vector system. The CYP3A-HAC mice possess the human CYP3A gene cluster which contains CYP3A4, CYP3A5, CYP3A7, CYP3A43 genes and their essential regulatory regions. In this study, we analyzed the functional expression of CYP3A enzymes in liver and intestine of CYP3A-HAC mice and studied inhibitory effects of erythromycin on CYP3A enzymes via mechanism-based inactivation (MBI). The quantitative real-time PCR analysis and western blot analysis showed that CYP3A4 mRNA and CYP3As protein were expressed in both of liver and intestine of adult CYP3A-HAC mice. To analyze the metabolic function of CYP3A enzymes expressed in CYP3A-HAC mice, we determined the formation of α-hydroxytriazolam (α-OH TRZ) and 4-hydroxytriazolam (4-OH TRZ) as the markers of CYP3A activity. The formation of α-OH TRZ and 4-OH TRZ in both liver and intestinal microsomes of Cyp3a-knockout mice were remarkably lower than those in wild-type (WT) mice. On the other hand, the formation rates of α-OH TRZ and 4-OH TRZ in liver and intestinal microsomes of CYP3A-HAC mice were comparable with those in human liver and intestinal microsomes, respectively. The formation rates of α-OH TRZ and 4-OH TRZ in liver and intestinal microsomes were observed in the

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4-week- to 2-year-old male CYP3A-HAC mice. Next, we investigated whether CYP3A inhibitor erythromycin showed similar inhibitory effects on the formation of 4-OH TRZ in liver microsomes between CYP3A-HAC mice and human. Erythromycin exhibited markedly weaker MBI effect on CYP3A activity in liver microsomes of WT mice compared to human, suggesting species differences in MBI of CYP3A by erythromycin. On the other hand, the potent MBI effect of CYP3A by erythromycin observed in human was well reproduced in CYP3A-HAC mice. In conclusion, the results of the present study demonstrated that the functional CYP3A enzymes were expressed in both of liver and small intestine of CYP3A-HAC mice and CYP3A inhibition caused by MBI in human liver microsomes was well reproduced in CYP3A-HAC mice. In the development of new drugs, CYP3A-HAC mice would be useful in vivo model for the estimation of CYP3A-mediated metabolism and prediction of drug-drug interaction risk caused by MBI in humans.

A4. Activation of Epac-Rap Signaling Protects against Cisplatin-induced Apoptosis of Mouse Renal Proximal Tubular Cells

Yu Qin1, Geurt Stokman1, Kuan Yan2, Sreenivasa Ramaiahgari1, Fons Verbeek2, Bob van de Water1 and Leo Price1

1Division of Toxicology, Leiden-Amsterdam Center for Drug Research, Leiden University, Leiden, Netherlands, 2Section Imaging and Bioinformatics, Leiden Institute of Advanced Computer Science, Leiden University, Leiden, Netherlands

Background: Cisplatin is a widely used chemotherapy drug for the treatment of several solid tumors. However, severe side effects, especially nephrotoxicity, which occurs after consecutive cisplatin administration, limit the clinical use of cisplatin for more efficient treatment with a higher dose. Nephrotoxicity is primarily associated with pathological alterations of renal proximal tubule epithelial cells, including reorganization of the actin cytoskeleton, disruption of cell-cell and cell-matrix adhesions, and cell death—mostly by apoptosis. The small GTPase Rap is a pivotal regulator of cell adhesions. Endogenous Rap can be activated by Epac (exchange protein directly activated by cAMP), and functions in adhesion-associated processes including cell survival. Of all organs, Epac is most highly expressed in the kidney, being particularly enriched in tubule epithelium, suggesting a functional role here for Epac-Rap signaling. The recent discovery of an Epac-specific activator, 8-pCPT-2′-O-Me-cAMP (007) provides a unique means of manipulating this pathway. In this study, we have investigated the effect of activated Epac-Rap signaling on cisplatin-induced loss of cell-cell adhesion and cell apoptosis, and its potential to protect against these injuries. Methods: Immortalized mouse proximal tubule epithelial cells (IM-PTEC) were treated with cisplatin, in the presence or absence of 007. The Epac-Rap activity was monitored by Rap1-GTP pull down assay and western blotting. Cell injury was scored microscopically, by caspase-3 activity assay and cell cycle analysis. Cells were stained for the adherens junction protein beta-catenin and the tight junction protein ZO-1, and followed by fluorescence microscopy. RNA interference was performed with siRNAs that specifically target Epac1 and Rap1. Results: Activation of Epac-Rap signaling by 007 profoundly inhibited the elevation of caspase-3 activity and reduced the percentage of cells in sub-G1 phase after cisplatin treatment, indicating a protective effect on IM-PTEC cells against cisplatin-induced apoptosis. Microscopic examination demonstrated that activation of Epac-Rap signaling using 007 also preserved cell-cell junctions against disruption during cisplatin treatment. Both protections were blocked in either Epac1 or Rap1 knockdown cells, confirming that the effects of 007 on survival and cell-cell junction stability were mediated by Epac-Rap signaling. Conclusion: Pharmacological activation of Epac-Rap signaling using the Epac-specific cyclic AMP analogue 007 preserves cell-cell junctions and protects against cell apoptosis of renal proximal tubular cells during cisplatin treatment. Therefore activating Epac-Rap signaling by 007 or analogous compounds is a potential strategy to reduce the nephrotoxicity and consequent renal insufficiency in clinical cancer treatment with cisplatin.

A5. Ribavirin Uptake Systems are Required for the Anti-hepatitis C Virus Activity of Ribavirin

Minami Iikura1, Tomomi Furihata1, Masanori Ikeda2, Nobuyuki Kato2 and Kan Chiba1

1Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Siences, Chiba University, Chiba-shi, Japan,2Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Department of Tumor Virology, Okayama, Japan

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Ribavirin is a key component in the recommended treatment method for hepatitis C virus (HCV) infection. Since effectiveness of the therapy is currently limited to about 55% of patients, the efficacy of anti-HCV therapy must be improved.We have recently identified a nucleoside transporter as a ribavirin uptake transporter in human hepato-cytes (1). Nucleoside transporters can be classified into two fomilies: equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs). ENTs facilitate the bidirectional diffusion of nucleosides across the cell membrane in a substrate concentration-dependent manner and their activities are specifically inhibited by nitrobenzylmercaptpurine riboside (NBMPR). CNTs utilize the sodium concentration gradient as a driving force to import nucleosides into the cells against a substrate concentration gradient. Considering that at least four of the five proposed anti-HCV mechanisms require ribavirin import into the cell, we hypothesized that ribavirin uptake by hepatocytes plays an essential role in the anti-HCV action of ribavirin. In this study, we attempted to clarify the role of nucleoside transporters in the anti-HCV action of ribavirin.The genome-length HCV RNA (strain O of genotype 1b) replication system (OR6 cells) with luciferase as a reporter, which has been recently established (2), was used for evaluation of anti-HCV efficacy of ribavirin.Expression of nucleoside transporter mRNAs in OR6 cells was examined by reverse transcription-PCR. The results showed that ENT1 and ENT2 mRNAs were abundantly expressed, whereas CNT2 and CNT3 mRNAs were expressed at only trace levels. Ribavirin uptake by OR6 cells was measured by an oil-filtration assay. All ribavirin uptake activities observed in OR6 cells were sodium-independent and they were mostly abolished by addition of NBMPR. These results suggest that ENTs are exclusively involved in ribavirin uptake in OR6 cells. The following experiments were performed to examine the role of ENTs in antiviral activity of ribavirin. When OR6 cells were treated with ribavirin alone, the level of antiviral activity was increased in a concentration-dependent manner. The level of anti-HCV activity of ribavirin was decreased by NBMPR in a concentration-dependent manner, which was associated with concentration-dependent inhibition of ribavirin uptake by NBMPR. In the above experiments, cytotoxicity of ribavirin with/whithout NBMPR was evaluated by lactase dehydrogenase release assay, the results of which showed that they exhibited no cytotoxic effect. In conclu-sion, these results suggest that ENTs determine the level of anti-HCV activity of ribavirin through ribavirin uptake into the cells. Therefore, cellular ribavirin uptake by nucleoside transporters is thought to play a critical role in the anti-HCV efficacy of ribavirin.

References

1. Fukuchi Y., Furihata T., Hashizume M., Iikura M., Chiba K. Characterization of ribavirin uptake systems in human hepatocytes. J Hepatol. 2010;52:486-92.

2. Ikeda M., Abe K., Dansako H., Nakamura T., Naka K., Kato N. Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system. Biochem Biophys Res Commun. 2005;329:1350-9.

A6. A Novel mRNA Splicing Isoform Is a Real Cancer-type Organic Anion Transporting Polypeptide 1B3 mRNA

Miki Nagai1, Tomomi Furihata2, Sayaka Matsumoto1, Seiya Ishii3 and Kan Chiba2

1Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 2Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 3Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Organic anion transporting polypeptide 1B3 (OATP1B3, encoded by SLCO1B3) is specifically expressed in the liver under normal physiological condition and mediates the cellular transport of a wide range of endogenous and exog-enous amphipathic compounds including clinically important drugs, such as HMG-CoA reductase inhibitors, cardiac glycosides and anti-cancer drugs. In addition to the liver, it has been reported that OATP1B3 is expressed in some solid tumors such as colon, breast and prostate cancers. To date, studies aimed at clarification of the functions of OATP1B3 in the cancer tissues have been performed on the basis of consideration that OATP1B3 expressed in cancer cells is the same molecular as that in the liver. However, the results of our preliminary experiments revealed that a novel splice variant was expressed in cancer cells. Therefore, we aimed to identify and characterize this novel OATP1B3 variant,

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termed cancer-type OATP1B3 (ct-OATP1B3), in the present study. RNA ligase mediated-rapid amplification of 5′ cDNA ends with RNA obtained from colorectal tumor, LS180 (colon cancer) and PK45p cells (pancreatic cancer) and cDNA cloning were performed to address the nature of OATP1B3 expressed in cancer cells. The results showed that the transctiption start site (TSS) was located in the intron 2 of the SLCO1B3 gene and generated a novel exon (exon 1′). Accordingly, the promoter activity was observed in the upstream region of the novel TSS in LS180 and PK45p cells. The result of cDNA cloning showed that cDNA of ct-OATP1B3 consisted of exon 1′ and exon 3 to 15 of known OATP1B3. To analyze relative expression levels of novel OATP1B3 and known OATP1B3 mRNA, quantitative real-time PCR was performed. The results showed that the mRNA expression level of ct-OATP1B3 was 61.0-, 29.3-, 2.3-, 3.7- and 176.0- fold higher than that of known OATP1B3 in two samples of human colorectal tumor, LS180, HCT116 (colon cancer) and PK45p cells, respectively. On the other hand, in normal liver, known OATP1B3 mRNA was substantially expressed while ct-OATP1B3 mRNA was hardly expressed. The effect of epigenetic modification on expression of ct- OATP1B3 mRNA in cancer cells was also examined. Treatment of Caco2 cells (colon cancer) and Caki-1 cells (kidney cancer) with DNA methylation inhibitor, 5-aza-2′-deoxycytidine (5 μM), significantly increased the level of ct-OATP1B3 mRNA expression. In conclusion, our results suggest that the novel isoform of OATP1B3 mRNA, cancer-type OATP1B3, is predominantly expressed in cancer and its expression is controlled by epigenetic mechanisms in cancer-specific manner.

A7. Inhibition of Breast Cancer Resistance Protein (BCRP/ABCG2) by Purine Cyclin-dependent Kinase Inhibitors In Vitro and In Situ

Jakub Hofman, Davoud Ahmadimoghaddam, Lenka Hahnova, Martina Ceckova and Frantisek Staud

Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic

Inhibition of cyclin-dependent kinases (CDKs) is a modern approach to stop proliferation of tumor cells. While the mechanism of CDKs inhibition by purine CDK inhibitors (CDKi) is relatively well understood, interactions of these compounds with other biological structures remain unknown. In this study we investigate possible interactions between five purine CDKi (purvalanol A, olomoucine I and II, roscovitine and bohemine) and breast cancer resist-ance protein (BCRP/ABCG2), an efflux transporter which plays important role in the pharmacokinetics, drug-drug interactions and multidrug resistance of many routinely used medicaments. In vitro experiments were carried out in parallel on parent MDCKII cell line and its genetically modified counterpart, MDCKII-ABCG2, expressing human ABCG2. Using accumulation studies with ABCG2 substrates, glyburide and Hoechst 33342, we observed inhibition of BCRP by all CDKi tested except for olomoucine I. Purvalanol A and olomoucine II reached the potency of a model BCRP inhibitor, fumitremorgin C. Furthermore, we used in situ method of dually perfused rat placenta to confirm our in vitro results on the organ level. Similarly to in vitro data, with the exception of olomoucine I we observed significant effect of all tested compounds on the transplacental passage of glyburide. Interestingly, all CDKi except for olomoucine I were found to be stronger inhibitors of placental BCRP in comparison with fumitremorgin C. Regarding their potency to inhibit BCRP, the tested compounds can be ranked as follows: purvalanol A > olomoucine II > roscovitine = bohemine > olomoucine I, with slight differences among methods used. In conclusion, our data suggest substantial impact of the studied CDKi on the pharmacokinetic behaviour of ABCG2 substrates. Apart from inhibiting CDKs, we expect these compounds might as well help overcome multidrug resistance of tumor cells to concomitantly administered cytotoxic agents that are substrates of BCRP. In addition, purvalanol A and olomoucin II could also be used as alternative BCRP inhibitors in biomedical research. This work was supported by grants GAUK 114909/C/2010 and SVV-2010-261-003.

A8. Improved Transfer of Zidovudine by Dehydroepiandrosterone Sulfate in CD4(+) T Lymphosyte, Molt-4 Cells

Tomohiro Nishimura, Masatoshi Tomi, Yoshimichi Sai and Emi Nakashima

Faculty of Pharmacy, Keio University, Tokyo, Japan

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Zidovudine (3′-azidothymidine, AZT) is a well-known nucleoside reverse transcriptase inhibitor and widely used for antiretroviral therapy. We previously showed that the uptake of AZT in TR-TBT 18d-1 cells, a placental cell line, was transporter-mediated and that it was enhanced by the addition of a certain type of estrogen sulfate such as dehydroe-piandrosterone sulfate (DHEAS). The aim of present study was to clarify 1) the cellular selectivity of the enhancement of AZT uptake by DHEAS including CD4(+) T lymphocyte, a pharmacological target of AZT, and 2) the effect of DHEAS on cellular uptake of thymidine. Molt-4 cells was used as a model for CD4(+) T lymphocyte. Radiolabelled agent uptake in Molt-4 cells was performed by silicone layer centrifugation method. The uptake was measured in the presence and absence of a specific enhancer. Contrary to TR-TBT 18d-1 cells, the uptake of [3H]AZT by the addition of 1 mM DHEAS was not enhanced in TR-BBB cells, rat blood-brain barrier model cell line and Cos-7 cells, indicating that the enhancement effect of DHEAS has cellular specificity. Molt-4 cells showed saturable uptake both of [3H]AZT and [3H]thymidine. By the addition of 1 mM DHEAS, [3H]AZT and [3H]thymidine uptake was respectively increased and decreased in Molt-4 cells. Michaelis-Menten equation revealed Km values of 2.6 µM and 1.2 µM for [3H]AZT uptake in the absence and presence of DHEAS, respectively. These results indicate that [3H]AZT uptake by Molt-4 cells is enhanced in the presence of DHEAS by changing transport affinity. The increased uptake of AZT and the decreased uptake of thymidine may provide implications towards antiretroviral therapies. Travel grant for this study was sup-ported by The Nagai Foundation Tokyo.

A9. Clathrin Adaptor Protein Complex 2 (AP2) Regulates The Internalization Of Bile Salt Export Pump (BSEP/ABCB11)

Hisamitsu Hayashi, Kaori Inamura, Kensuke Aida and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Bile salt export pump (BSEP/ABCB11) mediates the biliary excretion of bile acids in an ATP-dependent manner. Reduced BSEP expression at the canalicular membrane caused by various factors like pregnancy and sepsis can lead to dysfunction of BSEP, ultimately inducing or deteriorating intrahepatic cholestasis. Although the rapid internaliza-tion and subsequent degradation of BSEP has been suggested to be responsible for the reduced BSEP expression under cholestatic conditions, the underlying molecular mechanism has not yet been clarified. Our current study explored the molecular mechanism of BSEP internalization. We specifically focused on clathrin adaptor protein complex 2 (AP2), which helps clathrin-mediated internalization by recruiting targeted protein into clathrin coated pits, since BSEP has several potential recognition sites for AP2 in its cytosolic region. Studies using immunostaining and co-immunoprecipitation showed the colocalization of BSEP with α-subunit of AP2 (AP2α) on the plasma mem-brane in 3×FLAG BSEP-expressing HeLa cells and the canalicular membrane in rat hepatocytes as well as interaction between BSEP and AP2α, suggesting the plasma membrane as a potential site of interaction. Biotinylation study demonstrated that 3×FLAG-BSEP expression at the cell surface was markedly enhanced in clathrin heavy chain knockdown HeLa cells and AP2 function-deficient HeLa cells, which were constructed by siRNA-mediated depletion of AP2α. In addition, the internalization rate of 3×FLAG-BSEP was significantly slower in AP2 function-deficient HeLa cells than in control cells by almost 85%. In conclusion, AP2 mediates clathrin-mediated internalization of BSEP through the direct interaction with BSEP, and thereby negatively modulates BSEP expression at the cell surface.

A10. Effect of Drug Structure on Glucuronidation Clearance in Multiple Species: Utility of In Silico/In Vitro Parameters and Bile-Duct Cannulated Monkey/Dog

Yoshimasa Kobayashi1, Tsuneo Deguchi1, Masaru Iwasaki1, Yumi Nishiya1, Tomoyo Honda1, Yoshitake Furuta1, Katsuhiro Igeta1, Nobuaki Watanabe1, Keiichi Fusegawa2, Hidenori Ikenaga2, Atsushi Kurihara1, Osamu Okazaki1 and Takashi Izumi1

1Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo, Japan, 2Biological Research Laboratories, Daiichi Sankyo Co., Ltd, Tokyo, Japan

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[Objective] Human glucuronidation clearance has been predicted from animal data and from in vitro data. But the prediction accuracy is sometimes very low. In addition, effect of drug structure on glucuronidation clearance has been less clarified than that about oxidation clearances. Here we investigated how drug structures/physicochemi-cal properties affect the glucuronidation clearance and its species differences. [Methods] Animal in vivo intrinsic glucuronidation clearance (CLglc,vivo) values for 20 compounds (marketed:12, discovery stage:8) were estimated by the data obtained from bile-duct cannulated animals (mouse, rat, dog, and monkey). Biliary tract homeostasis in the cannulated monkey and dog was maintained enough for repeated experiments. Plasma and microsome free fraction values for intrinsic clearance calculation were predicted with SimCYP™ (SimCYP) or experimentally measured by the ultracentrifugation method. In vitro intrinsic glucuronidation clearance (CLglc,vitro) for 1274 compounds were estimated with the liver microsome (human and animal) reaction system in the presence of alamethicin. Octanol-water distribution ratio (Log D) values at pH 7.4 were predicted with Pallas™ (CompuDrug) or experimentally measured using the flask-shaking method. [Results] The CLglc,vivo/CLglc,vitro ratio of mon-key was well correlated with that of human, for any type of compound. For some aliphatic carboxylic acids, the CLglc,vitro of rat tended to be lower than the CLglc,vitro of human and higher than the CLglc,vivo of rat. From a viewpoint of physicochemical properties, CLglc,vitro values were well correlated with log D values for any animal species as well as human. When log D values were in the same range, CLglc,vitro values of aromatic carboxylic acids tended to be higher than that of aliphatic ones, and CLglc,vitro values of phenols tended to be higher than that of alcohols. [Conclusion] Human clinical CLglc of compounds is successfully predicted by the CLglc,vitro value after correction with the monkey in vitro-in vivo correlation data. Some carboxylic acids give low prediction accuracy even using this method. At a drug discovery stage, in order to decrease CLglc of a candidate compound, it is preferable to design new compounds with lower lipophilicity and to transform the substructure near the glucuronidation site.

A11. Uptake and Efflux of Abacavir and Tenofovir by In Vitro Renal Transporter Systems

Zoe Riches1, Helen C. Pearce2 and Gabrielle M. Hawksworth3

1Division of Applied Medicine, University of Aberdeen, Aberdeen, United Kingdom, 2GSK, Uxbridge, United Kingdom, 3University of Aberdeen, Aberdeen, United Kingdom

Nucleoside reverse transcriptase inhibitors (NRTIs) are a class of drug used in combination therapy for treatment of HIV. However, case studies have shown an association of some NRTIs with nephrotoxicity, including renal tubular damage and Fanconi′s syndrome. It has been suggested that the uptake and efflux of these drugs in renal cells may be involved in their side-effects and that the efflux by multi-drug resistant proteins (MRP) is rate limiting (Reid et al., 2003). To investigate this, in vitro transporter systems were used, including cell lines stably transfected with human organic cation transporters (OCTs), transduced cell lines using baculovirus and BacMam technology (Ames et al., 2007) for human organic anion transporters (OATs), and finally inside-out insect membrane vesicles for human MRPs. Radiolabelled tenofovir and abacavir were incubated with the transporter-expressing cells or membranes and quanti-fied by scintillation counting. Known probes, tetraethylammonium bromide for OCTs, p-aminohippuric acid (PAH)

Table 1 Uptake and efflux by human OAT, OCT and MRP transporters. Substrate concentrations for the OAT and OCT assay were 100 μM, except PAH (50 μM), and incubation times were 5 min. For the MRP assay substrate concentrations were 10 μM, with a 5 min. incubation time and 10 μg vesicles (n≥4).

Control OAT1 OAT3 Control OCT1 OCT2 Control MRP2 MRP4

pmol/min/104 cells (fold difference from control)

pmol/min/104 cells (fold difference from control)

pmol/min/mg (fold difference from control)

Tetraethylammonium bromide

1.0 3.3(3.3) 2.5(2.5)

p-Aminohippuric acid 0.9 5(5.6) 1(1.1)

Estradiol-β-glucuronide 5 20(4) 96(19)

Tenofovir 0.2 19(95) 2.1(11) 7 6(0) 7(0) 0.8 0.8(0) 4.3(5)

Abacavir 0.1 0(0) 0.3(3) 4 6(1.5) 5(1.3) 1 0.4(0) 0.5(0)

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for OATs, and estradiol-β-glucuronide for the MRPs were used as positive controls (Table 1). Tenofovir was transported by OAT1 and OAT3 95- and 11-fold times higher than into control cells, respectively (Table 1). A 5-fold increase in uptake occurred with MRP4-expressing vesicles, but no significant transport was seen with MRP2, OCT1 or OCT2 at the substrate concentrations used. Abacavir had a 3-fold greater uptake into OAT3-expressing cells than controls, and a 1.5- and 1.3-fold increased uptake with OCT1 and OCT2 cells. However abacavir was not transported by OAT1, MRP2 or MRP4-expressing cells or vesicles. Determination of kinetics will allow prediction of relative proximal tubular cell concentrations. In conclusion, tenofovir is actively transported by OATs and effluxed by MRP4 in vitro, while abacavir is a weak substrate for OAT3. Thus there is potential for accumulation of tenofovir in proximal tubular cells and for drug-drug interactions due to competitive uptake.

References

Ames, R. S., Kost, T. A., & Condreay, J. P. (2007). BacMam technology and its application to drug discovery. Expert Opinion on Drug Discovery, 2(12), 1669-1681.

Reid, G., Wielinga, P., Zelcer, N., De Haas, M., Van Deemter, L., Wijnholds, J., et al. (2003). Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Molecular Pharmacology, 63(5), 1094-1103.

A12. Microscale Engineered Human Liver Model for Investigative Toxicology, Drug Metabolism and Efficacy Assessment

Salman Khetani1, Stacy Krzyzewski1, Amanda Moore1, Jeannemarie Gaffney2, Jack McGeehan2, Wei Wei Wang3, David Duignan3 and R. Scott Obach3

1Research and Development, Hepregen Corporation, Medford, MA, USA, 2Process Development and Manufacturing, Hepregen Corporation, Medford, MA, USA, 3Pfizer, Groton, CT, USA

Primary hepatocytes are notoriously difficult to maintain in conventional (extracellular-based) models as their phenotypic functions display a precipitous decline within a few hours after isolation from the native microenviron-ment of the liver. Indeed, unstable hepatocytes in these models have been shown to be poor predictors of clinical outcomes. Here, we have utilized microfabrication technologies and tissue engineering techniques to develop a human liver model with precise microscale cytoarchitecture and optimal stromal interactions that displays pheno-typic stability for several weeks in vitro (HepatoPac™) as compared to only a few hours in conventional cultures. HepatoPac™ has been coupled with miniaturization strategies (i.e. 24- and 96-well format) and assay technologies for high content and predictive applications in investigative toxicology, drug metabolism, and efficacy assessment. Drug-induced liver injury (DILI) is the leading cause of acute liver failures and the high attrition rate of pharmaceu-ticals; therefore, HepatoPac™ has been optimized specifically for the in vitro screening of genotype-specific and clinically- relevant drug disposition and coupled DILI using standard end-points (i.e. ATP depletion, mitochondrial activity) as well as state-of-the-art technologies such as high content imaging. We have subsequently investigated the toxicity of several known clinical hepatotoxins (i.e. Troglitazone, Fialuridine, Diclofenac) in HepatoPac™ under both acute (hours to days) and chronic (weeks) dosing regimens. Since metabolism is an important determinant of the overall disposition of drugs and the profile of metabolites can have an impact on efficacy and safety, the utility of HepatoPac™ for generating human in vivo metabolites was evaluated. Twenty-seven compounds of diverse chemical structure and subject to a range of drug biotransformation reactions were assessed for metabolite profiles in HepatoPac™ using pooled cryopreserved human hepatocytes. The ability of HepatoPac™ to generate metabolites that are >10% of dose in excreta or >10% of total drug-related material in circulation was assessed and compared to data obtained in human hepatocyte suspensions, liver S-9 fraction, and liver microsomes. HepatoPac™ generated 82% of the excretory metabolites that exceeded 10% of dose and 75% of the circulating metabolites that exceeded 10% of total circulating drug-related material. This exceeds the performance of hepatocyte suspension incubations and other in vitro systems. Phase 1 and phase 2 metabolites were generated, as well as metabolites that arise via two or more sequential reactions. Finally, applications of HepatoPac™ for discovery and optimization of compounds targeting the liver and its diseases are also under development. In conclusion, microscale engineered liver plat-forms may find broad utility in the development of several classes of therapeutic compounds (drugs, biologics), in

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evaluating the disposition and injury potential of environmental toxicants, in fundamental investigations of liver physiology and disease, and in personalized medicine for liver disease. In the future, continued combination of microtechnology with tissue engineering may spur the development of other tissue models and their integration into the so-called ‘human-on-a-chip′.

Poster Presentations

P1. Genotoxic effect of inorganic arsenics in human hepatoma cells (HepG2 cells) using single cell gel electrophoresis

Ghazalla M. Benhusein1, Elaine Mutch2, Mabruka Elashheb3 and Faith M Williams2

1Al-fateh University, Faculty of Pharmacy, Department of Pharmacology and Clinical Pharmacy, P.O. BOX 13645 Tripoli, Libya, 2Medical Toxicology Centre and Institute for Research in Environment and Sustainability, Devonshire Building, Newcastle University, UK, 3Department of Pharmacology, Faculty of Medicine, Al-fateh University for Medical Sciences, Tripoli-Libya

Arsenic is an environmental chemical of toxicological concern today. It has been shown to be a human genotoxic and carcinogen. This project investigate three forms of inorganic arsenic compounds are arsenate (As+5), arsenite (As+3) and dimethylarsinic acid (DMA).The aim of this study was to determine the toxic effect of arsenate, arsenite, and DMA in HepG2 cells by measuring i) DNA damage with and without buthionine sulfoximine (BSO, GSH synthesis inhibitor) ii) depletion of glutathione level also with and without BSO. HepG2 cells were treated with arsenate, arsenite and DMA (10 µM) for 24hr with and without BSO (10 µM). The cells were then analysed for GSH concentration by reverse phase HPLC with fluorescence detection. Harvested cells were assayed using the Comet technique to quantify DNA damage. DNA damage was significantly different in cells dosed with arsenate p<0.05, arsenite p<0.01 but not DMA all compared to the control. There was increase in DNA damage in cells treated with arsenate, arsenite with BSO but not DMA p<0.05, p<0.01 and P=ns respectively compared to the control.There was significantly increase in reduced GSH level (nmol/mg protein) in cells treated with arsenate p<0.05, arsenite p<0.001 and significantly not different in DMA without BSO compared to the control. There was increase in reduced GSH in cells treated with arsenate p<0.01, arsenite p<0.001 but not DMA plus BSO all compared to the control. Our results indicate that, both arsenite and arsenate induce oxidative DNA damage in HepG2 cells. This study showed an increase in the GSH levels in low doses. Oxidative DNA damage was induced also in the presence of BSO.

P2. Polyphenolic acetates are superior to polyphenols in the prevention of benzene induced genoto-xicity in bone marrow and lung cells

Ajit Kumar1, Vishwajeet Rohil1, Hanumantharao G. Raj1, Ashok K. Prasad2 and Virinder S. Parmar2

1Department of Biochemistry, VP Chest Institute, University of Delhi, Delhi, India, 2Department of Chemistry, Bioorganic Laboratory, University of Delhi, Delhi, India

R6 R6

R5 R5

R4 R4

R3 R3 R2

R2 R1

R1 O O

O

O

I II 1:R1=R3=R4=H, R2=CH3, R5=R6=OCOCH3

4:R1=R3=R4=R6=H, R2=CH3, R5=OCOCH3

2:R1=R3=R4=H, R2=Ph, R5=R6=OCOCH3

3:R1=H, R2=Ph, R3=R4=H, R5=R6=OCOCH3

6:R1=Ph, R2=H,R3=R4=R6=H, R5=OCOCH3

5:R1=CH3, R2=COCH3,R4=R6=H, R5=OCOCH3

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Polyphenols have attracted immense interest because of their diverse pharmacological activities1. Not much is docu-mented about the biological activities of acetyl derivative of polyphenols. In our previous publications, we have reported the inhibition of AFB

1/benzene induced genotoxicity possibly due to acetylation of cytochrome P-450 apoprotein by 7,8-

diacetoxy-4-methylcoumarin (1) catalyzed by acetoxy drug: protein transacetylase (TAase)2,. Later studies confirmed this TAase as Calreticulin, a calcium binding protein in the lumen of endoplasmic reticulum and it was appropriately termed as Calreticulin Transacetylase (CRTAase)3,4. In this report, we have focused on agents that interfere with P-450 linked MFO such as acetoxy derivative of coumarin, chromone, flavones and xanthones, which could offer protection against benzene-induced genotoxicity. For this purpose, rats were separately pretreated with various polyphenolic acetates, followed by the administration of benzene either intratracheally (IT) or intraperitoneally (IP), and sacrificed 26 h after the injection of benzene. The incidence of micronuclei in bone marrow and lung were assessed by light and fluorescent microscopy, respectively. A dose-dependent induction of micronuclei in bone marrow and lung cells was observed in rats administered with benzene. A significant reduction in benzene-induced micronuclei in bone marrow and lung cells was observed as a result of polyphenolic acetates administration to rats; a higher dose of polyphenolic acetates resulted in greater inhibition of clastogenic action of benzene as revealed by micronuclei incidence. Polyphenols, the deacetylated product of polyphenolic acetates, demonstrated relatively lesser potency in inhibiting the clastogenic action of benzene. This observation is consistent with the ability of polyphenolic acetates to inhibit the formation of benzene oxide during the course of benzene metabolism. It was pertinent to note that differential specificities of several polyphenolic acetates in the inhibition of benzene induced micronuclei incidence was in tune with their specificities to CRTAase in following order 7,8-diacetoxy-4-methylcoumarin(1)>7,8-diacetoxy-4-phenylcoumarin(2)=7,8-diacetoxyisoflavone(3)=7- acetoxy-4-methylcoumarin(4)=7-acetoxy-3-acetyl-2-methylchromone(5)>7 acetoxyflavone(6).These observations further sub-stantiated our hypothesis that the biological effects of polyphenolic acetates are mediated by the action of calreticulin transacetylase that catalyzes the acetylation of concerned proteins.

References

1. Elliot- Middleton, J. R., Kandaswami, C., Theoharides, T. C. The effect of plant flavonoides on mammalian cells: Implications for inflammation, heart disease and cancer. Pharmacol. Rev. (2000) 673-751.

2. Raj, H. G., Kohli, E., Rohil, V., Dwarkanath, B. S., Parmar, V. S., Malik, S., Adhikari, J. S., Goel, S., Gupta, K., Bose, M., Olsen C. E. Acetoxy of 4-methylcoumarins confer differential protection from aflatoxin B

1 - induced micronuclei and apoptosis in lung and bone marrow cells.

Mutat. Res. 494 (2001) 31-403. Raj, H. G., Kumari, R., Seema, Muralidhar, K. M., Dwarkanath, B. S., Rastogi, R. C., Prasad, A. K., Watterson, A. C., Parmar, V. S. Novel function

of calreticulin: Characterization of calreticulin as transacetylase-mediating protein acetylation independent of acetyl CoA using polyphenolic acetates. Pure & Appl. Chem. 78 (2006) 985-992.

4. M. Michalak, E.F., Corbett, N., Mesaeli, K., Nakamura, M. Opas, Calreticulin: one protein, one gene, many functions, Biochem. J. 344 (1999) 281-292.

P3. Method Validation in Pharmaceutical Analysis

Marika Kamberi

Analytical Chemistry, Abbott Vascular, Inc, Santa Clara, CA, USA

Analytical methods used in pharmaceutical analysis must be sufficiently accurate, specific, sensitive and precise to ensure quality and reliability of the results, which in turn are crucial for ensuring quality, safety and efficiency of pharmaceuticals. Method validation is the process of proving that an analytical method is acceptable for its intended purpose. Validation is primarily concerned with the identification of the sources of the potential errors in the method and their quantification. It describes in mathematical and quantifiable terms the performance characteristics of a method. Performance is strongly connected with both the requirements and the design of the individual analytical procedure. Consequently, the analyst has to identify relevant parameters which reflect the routine performance of the given analytical procedure, to design the experimental studies accordingly and to define acceptance criteria for the results generated. The extent and Departmenth of the validation studies, as well as acceptance criteria, should be defined in relation to the required performance and the stages of product development. The acceptance criteria can be defined from regulatory requirements, statistical considerations, type of the individual test procedure and experience. However, statistical significance test should be applied with cautions because they do not take into consideration the practical relevance. Validation must be not regarded as a singular event. The analyst is responsible for the continued maintenance of the validated status of an analytical procedure.

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P4. How to isolation and identification, of New Thiophene Derivatives in postmortem rat tissues

T.M. Ibrahim1 and Mohamed Kreet2

1Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Egypt, 2Department Of Toxic and Narcotic drug ,Forensic Medicine, Cairo, Egypt

Thiophene derivatives are metabolized primarily by S-oxidation, followed by conjugation with glutathione; however, other routes of metabolism, involving ring cleavage, are also possible. The resulting mercapturic acid derivative is eliminated in the urine.Ring-substituted thiophenes also undergo S-oxidation and glutathione conjugation. In a study in which radiolabelled 3-benzoylthiophene was injected intraperitoneally into Sprague-Dawley rats, 15% of the urinary radiolabel was accounted for by a mixture of two diastereomers of the mercapturic acid conjugate of 4,5-dihydro-3-benzoylthiophene. The N-acetylcysteine residue (mercapturate) is bonded to the 4-position of the thiophene ring. Experiments with rat liver microsomes confirmed that thiophene sulfoxide is a reactive interme-diate in the conversion of 3-benzothiophene to the dihydromercapturic acid metabolite. The desired compound was extracted from viscera by different method which Used in forensic medicine { Arab Republic of Egypt in the laboratories of poisons and drugs} and examined via coloring tests and new chromatographic experiments such as U.V spectra, I.R spectra, G.C Mass and NMR spectra. Samples were used in all cases: Stomach content, Blood sample, Part of liver, half of both kidneys, and urine. Results: Different doses were administrated orally to rats to detect LD

50 of Thiophene Derivatives. LD

50 was found to be 1000 mg of Thiophene Derivatives per each Kg of rat

weight. it is clear that Marqui′s reagent gives a orange colour which changed to brown then to olive green with Thiophene Derivatives indicating the presence of ring sulphur and aromatic compound which consist of C, H, N. PDA reagent gives deep pink colour indicating the presence of active methylene group. On the other hand, the remainder reagents gave negative reactions with Thiophene Derivatives FeCl

3 and Beam reagents due to the

absence of phenolic hydroxyl group. While Zwikker reagent gives no response to Thiophene Derivatives because of presence of other substituents on the nitrogen atom and FPN reagent gives no color due to the absence of tricyclic structure of Thiophene Derivatives.

P5. Abstract Withdrawn.

P6. Abstract Withdrawn.

P7. Split calibration curve: An innovative approach to avoid repeat analysis of the samples exceeding upper limit of quantitation of a conventional calibration curve approach

Sudipta Basu, Rajanikanta S, Alpana T and Subrahmanyam V

DMPK & Toxicology, Sai Advantium Pharma Ltd, Pune, India

The objective of this research was to overcome the limitations associated with the conventional calibration curve approach in the analysis of the concentrations exceeding the upper limit of smaller range calibration curves using a split calibration curve approach. Dapsone was used as a model compound in this study over a concentration range of 1.04-5001.32 ng/ml in plasma matrix. Protein precipitation with acetonitrile was used to clean the plasma samples. Samples were analyzed for dapsone using LC-MS/MS. Chromatographic separation of dapsone and metadapsone (internal standard) was accomplished on a C-18 column interfaced with a triple quadrupole mass spectrometer. The mobile phase consisting of acetonitrile, methanol and 5 mM Ammonium acetate in water (20: 20: 60 v/v/v) was delivered at a flow rate of 0.8 ml/min. Analytes were determined by electro spray ionization tandem mass spectrometry in the positive ion mode using multiple reactions monitoring (MRM). Dapsone was monitored by scanning MRM transitions m/z 249.0 –>155.8, and metadapsone (IS) 249.0–> 92.0. Concentrations-response relation of Dapsone from 1.04-468.87 ng/ml fit with conventional calibration curve approach had shown a linear relationship with a regression co-efficient more than 0.9974. However, concentration-response relationships from 468.87-5001.32 ng/ml were best fit to a quadratic equation with regression coefficient more than 0.9979. Thus a calibration curve that can fit a wide dynamic range of concentrations v.i.z. ∼1 to 5000 ng/ml was introduced and validated accurately and precisely under a single run using a split approach.

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P8. Development and Validation of the Analysis Methods of Midodrine HCl Tablets and Trazodone HCl Capsules Using HPLC System For the Setting of Dissolution Standards

Jin-Young Lee1, Jae-Hee Auem1, Ji-Young Lee1, Sheen-Hee Kim1, Woo-Seong Kim1, Chan-Soon Kang1, In-Kyu Kim2, Dong-Sup Kim2 and Kab-Ryong Chae1

1Center for Food & Drug Analysis, Busan Regional Korea Food and Drug Administration, Busan, South Korea, 2Pharmaceuticals and Medical Devices Research Department, National Institute of Food and Drug Safety Evaluation, Seoul, South Korea

Drug absorption from a solid dosage form after oral administration depends on the release of the drug substance from the medicine, which can be expected by the dissolution of the drug under physiological conditions. Therefore, in vitro dissolution may be relevant to the prediction of in vivo performance. Based on this general consideration, in vitro dissolution tests for immediate release solid oral dosage forms, such as tablets and capsules, are used to manage the batch-to-batch quality of a drug product. As the dissolution profile can provide useful informa-tion for controlling the quality of drug, we have conducted research to set up dissolution specifications of drugs preparation which have no existing dissolution specifications. Accordingly, we selected midodrine hydrochloride (HCl) tablets and trazodone HCl capsules among drugs preparation in Korean Pharmaceutical Codex (KPC) to perform dissolution test on a single active ingredient of oral dosage forms. Midodrine hydrochloride (HCl) tablet is a vasopressor agent. Trazodone HCl capsule is an antidepressant drug. These two solid oral drugs are prescrip-tion medicines which are frequently used. They are defined to be analyzed by UV spectrophotometer and to be examined by disintegration test in Korean Pharmaceutical Codex (KPC). The aim of this study is to develop and validate the analysis methods for midodrine HCl tablets and trazodone HCl capsules using HPLC system instead of UV measurement for the setting of dissolution standards. Using a C18 column, the best result of midodrine HCl was obtained at 290 nm and the mobile phase composition of methanol and 0.1M monobasic sodium phosphate buffer. And as for trazodone HCl, the best result was gained at 246 nm and the mobile phase composition of methanol and 0.1M phosphate buffer. The methods showed accuracy, precision, linearity and specificity within the acceptable range. Moreover, the dissolution profiles of these drugs at the various dissolution solutions(pH 1.2, 4.0, 6.8 buffer and distilled water) were executed for setting of optimal conditions and dissolution standards to carry out the dissolution test instead of the disintegration test. The in vitro dissolution samples were analyzed using validated HPLC methods in this study. Taken together, the outcome of this study is going to be applied to revised version of Korean Pharmaceutical Codex.

P9. Study on the decreasing curve of nitrite residue in sausages conteins 40, 60 and 90 percent meat in sorage time

Hamid Mirzaei

Food Hygiene Department, Islamic Azad University, Tabriz branch, Tabriz, Iran

The use of sodium and potassium nitrite as preservative and colour stabilizer in meat products like sausages is common. High concentrations of these Salts in meat products increases the likelihood of cancer, malignancy and teratogenesis amongst consumers, whilst low concentrations increases the risk of c.botolinum growth during stor-age and the production of lethal toxin of botulism. Therefore, the use of these salts shuld be cotroled during the production and storage of meat products. The purpose of this study, was to plot the decreasing curve of nitrite in sausages containing 40, 60 and 90 percent meat during storage and then to compare them in order to evaluate the effect of meat concentration on nitrite decrease. In this study, 10 kg of each type of sausages containing 40, 60 and 90 percent meat were produced from the same meat source and 120 PPM of nitrite was added to all of them. Three samples were taken from each sausage type daily and, cosecutively, on the first week, every other day during the second week and in 2 day interval during the third and fourth weeks and the nitrite levels were measured using the spectrophotometric technique and the mean of three samples recorded as the nitrite residue level on that day. The results indicated that nitrite residues decreases during storage and the pearson correlation coefficient between storage time and nitrite residues in three sausage types containing 40,60 and 90 percent meat, respectively, was r = -0.91, r = -0.89 and r = -0.86 which is significant (p<0.01). The results olso indicated that nitrite residues in sausage

41

containing 40 percent meat was significantly higher than sausages containing 60 and 90 percent meat on different time intervals and also these residues were significantly higher in sausage containing 60 percent meat than sausage containing 90 percent meat (P<0.05).

P10. Accurate mass and nominal mass MSMS comparison for the simultaneous acquisition of quali-tative and quantitative data in DMPK studies

Joanne Mather, Marian Twohig, Ignatious Kass and Robert Plumb

Pharmaceutical Business Operations, Waters Corporation, Milford, MA, USA

A crucial phase of the drug discovery process is the quantification and qualitative analysis of candidate pharmaceuti-cals and their metabolites in biological fluids. The information generated is used to determine key pharmacokinetics parameters such as clearance, half-life, Tmax, and bioavailability. Traditionally this would involve the use of a triple quadrupole mass spectrometer for the quantitative analysis of low exposure compounds, qualitative data is derived from either hybrid quadrupole time of flight (QToF) mass spectrometer (MS) or ion trap instrumentation. This need for multiple instruments and analytical runs results in reduced productivity and increased instrument capital costs. The two approaches considered for metabolite detection were accurate mass full scan LC/MS(MS) which offers the best full scan data sensitivity and Tandem Quadrupole LC/MS/MS which offers greater detection sensitivity (MRM). The QToFs MSE functionality uses the LC/MS data from the low collision energy (CE) MS scan and high CE MS scan to be obtained during a single UPLC injection therefore allowing the simultaneous acquisition of precursor and product ion data with the high stringency of accurate mass. The use of new detector electronics allowed for stable mass measurements and a wide dynamic range. New tandem quadrupole MS instrumentation allow for simultaneous MRM and full scan data collection with product ion confirmation, permitting the detection, quantification and metabolite confirmation in a single injection. We also present the use of an automated approach to method development in the form of an MRM method generating tool that predicts possible bio-transformations and writes MRM methods for them. thus combining the sensitivity of MRMs without the need to develop methods for all possible metabolites. In this poster we present a comparison of the benefits and drawbacks for the simultaneous acquisition of qualitative and quantitative LC/MS/MS data using either a QToF MS instrument or a triple quadrupole instrument. The throughput, sensitivity and spectral quality were compared using a set of structurally diverse compounds prepared in rat plasma. Metabolites of one of the compounds, nefazodone, were prepared by incubation with rat liver microsomes in order to assess two different approaches to metabolite detection.

P11. Quantitation of Ursolic Acid in Human Plasma By Ultra-performance Liquid Chromatography Tandem Mass Spectrometry and Its Pharmacokinetic Study

Guangli Wei1, Yuanyuan Xia2, Duanyun Si2 and Changxiao Liu2

1Tianjin Institute of Pharmaceutical Research, Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin, China, 2Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin, China

Ursolic acid is a pentacyclic triterpene acid, which exists wildly in plants. As for its anti-cancer activity, ursolic acid is reported capable of inhibiting several types of cancer cells by disturbing the STAT3 activation pathway. The ability to measure ursolic acid quantitatively in biological matrixes is crucial for evaluating the pharmacokinetic/pharmacody-namic (PK/PD) relationship in support of their preclinical and clinical development. Hence, a reliable bioanalytical method with high selectivity, sensitivity, accuracy, precision, and throughput is necessary for further study of ursolic acid. Till now, no LC-MS/MS method has been rigorously validated for various biosamples. Therefore, we developed a simple, rapid and sensitive assay for the quantitation of ursolic acid in human plasma. The method used a small sample volume (100µL) with a simple LLE for sample preparation, a runtime of 6 min, and 10 ng/mL of quantitation limit. Plasma samples were pretreated by liquid - liquid extraction with ethyl acetate and were chromatographed by an ACQUITY UPLC BEH C

8 column (length of 100mm and 2.1mm, i.d) with the mobile phase consisting of acetonitrile

and 10mM ammonium formate (90:10, v/v) at a flow rate of 0.2 mL/min. The multiple reaction monitoring (MRM)

42

transitions were performed at m/z 455.1 to 455.0 for ursolic acid and m/z 469.3 to 425.2 for glycyrrhetinic acid (IS) in the negative ion mode with electrospray ionization (ESI) source. The present method showed good linearity over the concentration range of 10 to 5000 ng/mL for ursolic acid in human plasma with a lower limit of quantification of 10 ng/mL. The mean extraction recovery was 73.2 ± 4.5% and the matrix effect ranged from -11.4% to -5.6%. The intra- and inter-day precisions were less than 7.0% and 7.2%, respectively, and the accuracy was within ±2.0%. Ursolic acid was stable during the analysis and the storage period. The validated method has been successfully applied to a pharmacokinetic study of ursolic acid after intravenous infusion of a ursolic acid nano-liposome formulation to 24 healthy volunteers.

P12. Analysis of tetrodotoxin in puffer fish by liquid chromatography tandem mass spectrometry

YoungWoon Kang, Yoonsook Lee, SungKug Park, OckJin Paek, Hoon Choi, CheonHo Jo and MeeHye Kim

Food Contaminants Division, Korea Food and Drug Administration, Seoul, South Korea

Tetrodotoxin (TTX) found in a number of organisms including puffer fish is an important xenobiotic that causes neurotoxicity in humans and animals. The mechanism of toxicity is known as its high affinity to block voltage-gated sodium channels which transport Na+ between the exterior and interior of cells. Tetrodotoxin poisoning is sporadically reported in Korea, Taiwan, and Japan. Thus, the testing TTX to ensure the safety of seafood consumed is important in these countries. The current standard for testing TTX in foodstuffs is the mouse bioassay (MBA) in Korea as in many other countries. However, this test suffers from potential ethical concerns over the use of live animals. In addition, the mouse bioassay does not test for a specific toxin thus a sample resulting in mouse incapacitation would need further confirmatory testing to determine the exact source toxin (e.g., TTX, STX, brevotoxin, etc.). Furthermore, though the time of death is proportional to toxicity in this assay, the dynamic range for this proportional relationship is small thus many samples must be diluted and new mice be injected to yield a result that falls within the quantitative dynamic range. Therefore, in recent years, there have been many efforts in this field to develop alternative assays. High performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) has been emerged as one of the most promis-ing options. To adopt LC-MS-MS method as alternative standard for testing TTX, we performed a validation study. A LC-MS-MS method involves solid-phase extraction (SPE) and followed by analysis using an electrospray in the positive ionization mode and multiple reactions monitoring (MRM). This method has been validated against the quantification of TTX in puffer fish according to AOAC method validation guideline. The preliminary results showed the linearity within concentration ranged of 10-1,000 ng/g and limit of detection was 1 ng/g. The full analysis for correlation between the results of mouse bioassay and LC-MS-MS method is underway. We will present results comparing the mouse bioassay and LC-MS-MS method.

P13. Abstract Withdrawn.

P14. A validated enantioselective assay for the determination of ibuprofen in human plasma using ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS)

András Szeitz1, Andrea N. Edginton2, Henry T. Peng3, Bob Cheung3 and K. Wayne Riggs1

1Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC, Canada, V6T 1Z3, 2School of Pharmacy, University of Waterloo, Waterloo, ON, Canada, N2L 3G1, 3Defence Research and Development Canada, Toronto, ON, Canada, M3M 3B9

A modified ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of ibuprofen enantiomers in human plasma. Ibuprofen and flur-biprofen (internal standard) were extracted from human plasma at acidic pH, using a single-step liquid-liquid extraction with methyl-tert-butyl ether. The enantiomers of ibuprofen and flurbiprofen were derivatized to yield the corresponding diastereomers. Chromatographic separation was achieved using a phenyl column with a run

43

time of 20 minutes. (R)- and (S)-ibuprofen were quantitated at the multiple reaction monitoring (MRM) transition of m/z 360.2 → 232.1, and (R)- and (S)-flurbiprofen were monitored at the MRM transition of m/z 398.3 → 270.1. The method was validated for accuracy, precision, linearity, range, limit of quantitation (LOQ), limit of detection (LOD), selectivity, absolute recovery, matrix effect, dilution integrity, and evaluation of carry-over. Accuracy for (R)-ibuprofen ranged between -11.8% and 11.2%, and for (S)-ibuprofen between -8.62% and -0.335%. Precision for (R)-ibuprofen was ≤10.3%, and for (S)-ibuprofen ≤6.98%. The calibration curves were weighted (1/X2, n=7) and were linear with r2 for (R)-ibuprofen ≥0.988 and for (S)-ibuprofen ≥0.990. The range of the method was 50 to 5000 ng/mL with the LOQ of 50 ng/mL, and LOD of 1 ng/mL, for (R)- and (S)-ibuprofen requiring 100 µL of sample. The method was applied successfully to a pharmacokinetic study with the administration of a single oral dose of ibuprofen capsules to human subjects.

P15. Analysis of Factors Determining Incomplete Intestinal Drug Absorption as Observed in Cyclosporine Formulations by Use of Tube Model Incorporated Nernst Equation

Katsumi Iga, Akiko Kiriyama and Akino Honbo

Pharmaceutical Sciences, Doshisha Women′s College of Liberal Arts, Kyotanabe, Japan

Purpose: Intestinal absorption rate after oral administration of cyclosporine is known to be variable depending on formulation and administration conditions (fasted or fed). In the present study, we analyzed factors determining absorp-tion rate of cyclosporine by model calculation.Methods: For model calculation, we developed a tube model incorporated Nernst equation and used the PK data of Sandimmune (emulsion formulation) and Neoral (micro-emulsion formulation).Results: Caco-2 cell permeability of cyclosporine in early reports suggested that Ka is around 5/hr, with possible reduction due to efflux by Pgp.Solubility behaviors in bile acid (NaTC) in an early report, suggested that Cs-increase in administration of Sandimmune (emulsion) in fed conditions is about 3 times.Rate profiles of dissolution, absorp-tion and saturation obtained by the present model calculation, assuming a drug like cyclosporine, gave the following general information of drug absorption, (i) absorption rate is highly dependent on dose (D), aqueous solubility (Cs), and absorption rate constant (Ka), while it′s not dependent on particle size (a0) or intensity of stirring conditions of dissolution (Df/h), (ii) dissolution rate process is not rate limiting to absorption rate on a time scale of 0-5 hr, (iii) incomplete drug absorption is attributed solely to the saturation in drug dissolution where D is high enough, Cs is low enough, and Ka is small enough.In absorption-rate matrix derived by a simple tube model neglecting dissolution rate process, absorption rate of Sandimmune was layout in a low solubility region where absorption rate is sensitive to change in Cs, while absorption rate of Neoral (micro-emulsion) was layout in a high solubility region where absorption rate is not sensitive to change in Cs. However, even in the region for Neoral, the absorption rate is still sensitive to the change in Ka. High dose in cyclosporine was shown to be disadvantageous to complete absorption.

Enalaprilat300

250

200

150

100

50

00 5 10 15 20

pmol

/ml

hours

human (n=30)

dog (n=6)pig (n=6)

Enalapril400

300

200

100

00 5 10 15 20

pmol

/ml

hours

human (n=30)

dog (n=6)pig (n=6)

Figure 1 Average concentration time profile (aritmetical means) of enalaprilat and enalapril in serum after administration of 20 mg preparation in pig, beagle and human.

44

Conclusion: The present model calculation of drug absorption rate gave the following general information of drug absorption: (i) a simple tube model neglecting dissolution rate process is useful to predict drug absorption rate, (ii) incomplete drug absorption is attributed solely to the saturation in drug dissolution where D is high enough, Cs is low enough, and Ka is small enough.

P16. Preparation of controlled release tablets of Rheum palmatum and evaluation in vivo

Ruizhi Zhao1, Shaojun Liu1 and Liling Zhou2

1Second Affiliated Clinical college, Guangzhou university of triditional Chinese medicine, Guangzhou, China, 2Department of Pharmacy, Guangzhou University of Traditional Chinese Medicine, Guangzhou, China

Release rate of pharmaceuticals is very important to preparations, it directly influence on the pharmacodynamics and side effect of drugs. People realized it for many years and developed many methods for controlling the release rate of preparations, the study of the release rates has been the hotspot of pharmaceutical research, such as sus-tained release preparations, controlled release preparations, immediate release preparation especially for poor dis-solution drugs and so on, in modern times, We could use different form of preparation or different matrix to control the release rate almost as we like. But this is not for the Traditional Chinese Medicine (TCM). In ancient times of China, people usually use decoction and powder to realize immediate release of drugs. But it is very inconvenience, and the taste is terrible. So in modern times, people try to make it to injection and oral liquid pharmaceutical, and Dropping Pill, some of it is very successful, such as injection of Qingkailing and Bingduling oral liquid, and composite Salviae Dropping Pill, etc, but most drugs are not suited to prepare to liquid pharmaceutical, because liquid pharmaceuticals are easily changed, the drugs property limited its pharmaceuticals. And Droppling Pill can carry very little drug, to most of the TCM drugs it is not suit. So how to achieve a rapid and prolonged release is an important problem in the pharmaceutical study of TCM. However, little research has been found in the literature relating to in vivo performance of preparation of TCM especially for immediate release. The aim of this study was to prepare controlled release tablets of Chinese Traditional Medicine. The Rheum palmatum was chosen as the model drug, and the Anthraquinone free derivatives being the index of Rheum palmatum, concentrations of Anthraquinone free derivatives in serum were determined by HPLC. The tablets were prepared by directly compressed method, and the release behavior in vivo was evaluated by the serum concentration in dogs. Drug release from tablets containing HPMC and lactose is rapidly and prolonged for a long time than capsules, and the AUC of tablets was 4.6 times compared to capsules. It indicates that the mixture of HPMC and lactose could be used as the matrix of the rapid and prolonged release of Traditional Chinese Medicine. Key wards: Rheum palmatum, controlled release tablets,in vivo evaluation

P17. The Influence of Albumin to Brain Tissue Binding

Hinnerk Boriss1 and Lydia Helmdach2

1Sovicell GmbH, Leipzig, Germany, 2Pharmaceutical Technology Group, University of Halle, Halle, Germany

The Influence of Albumin to Brain Tissue Binding The binding of drugs to brain tissue components, such as lipids and albumin, is a major determinant in the disposition of drugs. The brain tissue binding of test compounds was studied in-vitro using equilibrium dialysis and a test kit (TRANSIL Brain Absorption Kit) for the determination of membrane affinity. Sixteen compounds from different compound classes covering a wide range of brain tissue binding (fu(brain) between 0.07 – 40.65 %) were selected. We analyzed the contribution of albumin to brain tis-sue binding. Since the brain dry weight consists mainly of lipid, the brain tissue binding is primarily influenced by binding of the test compounds to brain lipids. To estimate how the protein binding contributes to brain tissue binding albumin and lipid content of brains were quantified and the affinity of drugs to both albumin and lipids determined. The albumin content of the brain was several orders of magnitude lower than the lipid content, while the affinity of the tested drugs was higher to lipids than to albumin. Hence, albumin binding contributed only to a minor extent to brain tissue binding. Consequently, there experimental systems that predict brain tissue binding

45

need not account for albumin binding. However, dialysis with brain homogenates may be biased by albumin from the brain capillaries as an increase in binding by up to 15% was observed in non-perfused versus perfused rat brains. Thus, albumin binding may play a significant role in biasing brain tissue binding when using non-perfused brains for dialysis experiments.

P18. Poor Membrane Permeability Ability of SANT75 across Caco-2 Cell Monolayer Due to High Cellular Accumulation

Na Li1, Ying Hu1, Yang Yu1, Song Li2, Chao Che3 and Ling Yang4

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking University, Shenzhen, China, 3Shenzhen Biosynergo Bio Technology Co.,Ltd,Shenzhen, China, 4Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Background: SANT75(Smoothened antagonist 75) has been identified and characterized as Smo inhibitor by screening from a small molecule library designed based on the scaffold of Smo agonist SAG[1]. It is a structural analog of SAG with the methyl group substituted by propyl group. As part of effort to address pharmacological effects of SANT75, we investigate the permeability of SANT75 using an in vitro cell culture model, Caco-2 cell monolayer.

Method: The viability of Caco-2 cells in the presence of SANT75 was measured using Sulforhodamine B(SRB) cytotoxic-ity experiment. The Caco-2 cells were cultured in Transwell-clear inserts with 1.12cm2 permeable polyester membrane in 12-well plates for 21days. The integrity of the cell monolayers was evaluated by measuring transepithelial electri-cal resistance (TEER) using a Millicell-ERS voltohmmeter and paracellular leakage of Lucifer Yellow. Furthermore, permeability coefficients (P

app) of dexamethasone and digoxin were measured as additional markers. Transport study

was initiated by replacing the transport medium on the apical side with an equal volume of HBSS/HEPES buffer con-taining 10 μM SANT75 and fresh HBSS/HEPES buffer on the basolateral side. 100 μl of the medium from the receiver compartment was taken at every 30 min up to 2 h, and the sample was quantified by ultra fast liquid chromatograph (UFLC) method. To determine whether cellular accumulation occured in Caco-2 cells or not, the methanol extract of cell collection was analyzed by UFLC method mentioned above.

Results: Based on the cytotoxicity assay results, the concentration of 10μM was selected for transport experiment. The P

app(A-B) values of dexamethasone and digoxin were (5.1 ± 0.5)×10−6 cm·s−1 and (2.0 ± 0.04) ×10−6 cm·s−1 respectively,

which were in accord with the reported values previously for permeability assay with the same tested compounds[2,3]. No celluar accumulation of dexamethasone and digoxin was found during the transport experiment. However, using SANT75 as tested compound, the amounts permeated to the basolateral side was too low to be detected, while a high cellular accumulation was detected in the cell extracts. The cellular accumulation rate reached to (75.2 ± 0.1)% and the amount of SANT75 retained in the apical side was only (4.7 ± 0.1)%, thus the total recovery percentage during the transport experiment was (79.9 ± 0.1)%.

Conclusion: SANT75 showed poor permeability ability across Caco-2 cell monolayer due to high cellular accumulation. The phenomenon is very uncommon and might be due to its high cellular protein binding, which is worthy of further research. The results suggested that it was necessary to optimize SANT75 structurally further to gain good membrane permeability keeping its activity.

References

[1] Yang HB, Xiang J, Wang ND, et al. Converse Conformational Control of Smoothened Activity by Structurally Related Small Molecules[J]. Journal of Biological Chemistry. 2009, 284(31): 20876-20884.

[2] Zambito Y, Fogli S, Zaino C, et al. Synthesis, Characterization and Evaluation of Thiolated Quaternary Ammonium-chitosan Conjugates for Enhanced Intestinal Drug Permeation[J]. European Journal of Pharmaceutical Sciences, 2009, 38(2): 112-120.

[3] Han Y, Tan TMC, Lim LY. In Vitro and in Vivo Evaluation of the Effects of Piperine on P-gp Function and Expression[J]. Toxicology and Applied Pharmacology, 2008, 230(3): 283-289.

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P19. hCYP3A4 Over-Expressing IEC-18 Cell Lines as an In Vitro Model to Assess Gut Permeability and CYP3A4 Metabolism

Swathi Vangala1, Jae H. Chang2, Ning Liu3, William K. Chan4 and Miki S. Park5

1University of the Pacific, Stockton, CA, USA, 2Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA, USA, 3Department of Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA, USA, 4Department of Pharmaceut & Med Chem, Univ of the Pacific, Stockton, CA, 5Department of Pharmaceutics & Med Chem, Univ of Pacific, Stockton, CA

Purpose: The oral route offers many advantages over other forms of drug administration. Therefore, many drugs are developed to ensure adequate exposure after an oral administration. The fate of an orally administered drug is dependent on many parameters before it can reach the systemic circulation, including drug absorption and first-pass metabolism in the gut and the liver. Immortal mammalian cells lines such as Caco-2 and MDCK are commonly employed to assess drug permeability but they lack physiologically relevant drug metabolism enzyme expression such as CYP3A4 which is responsible for metabolizing a significant fraction of marketed drugs. Consequently, these cell lines are not sufficient to integrate metabolism when assessing drug absorption. Here, we introduce IEC-18 cells over-expressing CYP3A4. Method. A recombinant replication-deficient adenovirus carrying the hCYP3A4 full length cDNA was constructed according to Stratagene′s AdEasy XL Adenoviral system. This construct was used to transiently transfect hCYP3A4 into IEC-18 cells, an intestinal cell line that forms polarized monolayers. Two days after transfection, Western blot was performed to assess the level of hCYP3A4 expression in the wild-type and CYP3A4 over-expressing IEC-18 (CYP3A4-IEC) cells. In situ metabolism of midazolam was performed in the six-well plates containing either wild-type or CYP3A4-IEC cells. In addition, apical to basal permeability of midazolam and its 3-hydroxyl midazolam (3-OH-M) were examined in the wild-type and CYP3A4-IEC cells. The parent and the metabolite levels were quantified using a LC-MS/MS.

Results: Abundant expression of CYP3A4 was found in CYP3A4-IEC cells but not in the wild-type cells, sug-gesting that the the CYP3A4 gene was successfully transfected into the IEC-18 cells. The activity of the trans-fected CYP3A4 activity was assessed using midazolam as a probe. Compared to the wild-type cells, there was 10-fold higher formation of 3-OH-M. In addition, apical to basal transport studies of midazolam in CYP3A4-IEC cells showed 4-fold increased appearance of 3-OH-M in the receiver compartment compared to the wild-type cells.

Conclusions: Transient transfection of CYP3A4 was successfully conducted in IEC-18 cells. The newly constructed CYP3A4-IEC cells expressed CYP3A4 proteins and showed higher CYP3A4 activity when compared with the wild-type cells. Because CYP3A4-IEC cells can capture both the metabolism and permeability component of drug disposition in the intestine, it may be a useful model to assess drug absorption and first-pass metabolism.

P20. A Novel model for Mucin-type O-glycosylation sites Prediction

Kun Zhou, Chunzhi Ai and Ling Yang

Lab of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Glycosylation, one of the most common protein post-translational modifications, is involved in a variety of important biological processes, including protein stability, solubility, secretion of signal, extracellular recognition, regulation of interactions, etc. The identification of glycosylation sites in a query protein will help to understand its biological function, while till now only a little work has done for the recognition sequence for the O-glycosylation in experi-ment due to the difficulties in isolation and analysis, therefore, it is necessary to develop computational method for identifying the glycosylation sites in protein to bridge the gap between the large number of known protein sequences and the small munber of proteins experimentally identified glycosylation sites. To accurately predict O-glycosylation sites, a new model has been developed with genetic algorithm to select crucial features among the large amount of amino acid properties encoded feature vectors[1]. 328 nonapeptides of O-glycosylation and 2855 of non-O-glycosylation sites, were retrieved from Swiss-Prot database. The peptides represented by 526 amino acid

47

properties are transformed into a numeric vector with 4737 features. Based on the selected top 50 features with genetic algorithm, a predictive model was established with neural network method. The model exhibited a good predictability with a cross-validation accuracy of 80.5% for the whole set, and the accuracies were 81.7% and 68.0% for non-glycosylation and glycosylation sites respectively, which is comparative to the NetOGlyc3.1,one of useful O-glycosylation site predictors at present[2]. According to the features selected, we can conclude that the propeties of the 4th and the 8th amino acid of the nonapeptides contribute more to the glycosylation, and the accessibility of the 8th amino acid impact most greatly. This model can not only be used as fast and accurate screener to identify the glycosylation sites of large protein darasets, but can determine the crucial amino acid properties contributing more to glycosylation.

References

1. Kawashima, S., et al., AAindex: amino acid index database, progress report 2008. Nucleic Acids Research, 2008. 36: p. D202-D205.2. Julenius, K., et al., Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology,

2005. 15(2): p. 153-164.

P21. Biomarkers releated to endothelial dysfunction in the “peripheral arterial disease” (PAD)

Suzan E. Usanmaz1, Cuneyt Koksoy2 and Emine Demirel-Yilmaz3

1Department of Medical Pharmacology, Ankara University, Faculty of Medicine, Ankara, Turkey, 2General Surgery, Ankara University, Faculty of Medicine, Ankara, Turkey, 3Medical Pharmacology, Ankara University, Faculty of Medicine, Ankara, Turkey

It is well known that endothelial dysfunction appears in the PAD. Several “biomarkers” have already been used in monitoring the progress of endothelial dysfunction. However, there have not been any “biomarkers” established and widely accepted for endothelial dysfunction in the PAD yet. If some “biomarkers” would be shown to associate with PAD development, therapeutic/preventive approaches might be beneficial in slowing-down the disease progress. In addition to diagnosis, “biomarkers” for endothelial dysfunction might also help for revealing the stage of pathology and the appropriate treatment strategy. Many substances are synthesized or released by vascular endothelium. “Nitric oxide” (NO) released from the endothelial cell, is accepted as the marker for endothelial function, and the endothelial dysfunction is attributed to the problems in NO synthesis/release. It has also known that, the oxidative stress increases in cardiovascular diseases and causes endothelial dysfunction. At the oxidative stress status, the amount of superoxide radicals increases and they combine with NO to produce peroxynitrite radicals. On the other hand, in the inflammation, high release of NO from inflammatory cell could cause “nitrosative stress” together with increased superoxide radical. Nitrosative stress impairs endothelial function by either decreasing NO bioavailability or causing to the cell damage, because of the more reactive free radicals generated. “Asymmetric dimethylarginine” (ADMA), which is increased in oxidative stress, is the endogenous inhibitor of NOS (NO Synthase). ADMA has claimed to be, the one of the factors responsible for, the decreased NO release causing endothelial dysfunction. “Hydrogen sulfide” (H

2S) is a new NO-like

acting molecule and its synthesis is increased by oxidative stress. However, the role of H2S in endothelial dysfunction

has not been established yet. In this study, plasma levels of NO, ADMA, H2S, “Total Antioxidant Capacity” (TAC, as a

Trolox® equivalent) were determined as putative “biomarkers” for endothelial dysfunction in PAD patients. NO, H2S

and TAC were measured spectrophotometrically, ADMA was measured by ELISA technique. In PAD patients (n=63), TAC and H

2S plasma levels did not change, while NO and ADMA plasma levels increased significantly, against healthy

controls (n=20) (Table 2).This study indicates that, high NO and ADMA levels of plasma may represents “nitrosative stress” that causes

endothelial dysfunction.

Table 2

Plasma levels uM NO uM Trolox® uM ADMA uM H2S

Healthy Controls 23.6 ± 2.3 705,5 ± 35.4 0.131 ± 0.23 57.4 ± 7.0

PAD patients 69.7 ± 4.2 708,8 ± 18.5 0.846 ± 0.093 51.4 ± 4.4

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P22. Comet Assay and Cytokinesis-Blocked Micronucleus Test for Monitoring The Genotoxic Effects of Narrowband UVB Treatment in Psoriasis Patients

Diren Beyoglu1, Zehra Asiran Serdar2, Ilkin Zindanci3, Gulden Z. Omurtag1 and Semra Sardas1

1Toxicology Department, Marmara University, Faculty of Pharmacy, Istanbul, Turkey, 2Dermatology Department, Haydarpasa Numune Training and Research Hospital, Istanbul, Turkey, 3Dermatology, Goztepe Training and Research Hospital, Istanbul, Turkey

Psoriasis is a common, chronic, multifactorial and inflammatory disease characterized by T-cell mediated hyperpro-liferation of keratinocytes. Epidemiological studies from around the world have estimated the prevalence of psoriasis to be anywhere from 0.6-4.8% [1]. Topical agents, appropriate wavelengths of ultraviolet radiation and systemic medications can be used alone or in combination as the treatment therapeutic modality of psoriasis. When topical treatment are inadequate or more than 5% of the body surface area is involved, phototherapy constitutes are preferred as treatment before considering other systemic treatments in psoriasis. The combination of oral 8-methoxypsoralen followed by UVA (PUVA) used as an effective therapy against psoriasis has been reported to cause mutagenic and carcinogenic effects [2, 3]. Therefore, narrowband UVB (N-UVB) phototherapy alone is widely used as efficient thera-peutic option to moderate-severe psoriasis. Recent reports about safety and efficacy of N-UVB and other treatment options (wide-spectrum UVB and PUVA) have been widely discussed [3, 4]. However, the number of studies using more than one end point genotoxicity test in psoriasis patients under N-UVB treatment are rare. The present study aims to evaluate the possible DNA damage in peripheral lymphocyte cells of psoriasis patients (n=13) before and after 20 sessions of N-UVB treatment by using alkaline comet assay and cytokinesis block micronucleus assay. Mean tail %DNA for each cell was calculated as 100-Head %DNA. The number of micronuclei (MN) in 1000 bicnucleate (BN) cell was scored and the frequency of MN/1000 BN was calculated. The mean tail %DNA basal damage in pre-treated patients (8.62 ± 1.11) highly increased as (13.59 ± 1.57) after 20 sessions of N-UVB treatment and similar increase were observed for MN frequencies (‰) with initial value 1.00 ± 0.88 raised as 1.92 ± 1.14. The study is still continuing, however the present observations indicate that the benefit/risk ratios of the available treatments should be considered and cautious interpretation is required. Ongoing risk assessment is therefore essential in the treatment of psoriasis.

References

[1]. Neimann AL, Porter SB, Gelfand JM, The epidemiology of psoriasis. Expert Rev. Dermatol., 1(1): 63-72, 2006.[2]. Menter A, Current and future management of psoriasis. Lancet, 370: 272-284, 2007.[3].Sardas S, Karahalil B, Karakaya AE, Sasmaz R, Mutagenic risk in psoriatic patients before and after 8-methoxypsoralen and long-wave ultraviolet

radiation. Mutat. Res. 312: 79-83, 1994.[4]. Hamurcu Z, Demirtas H, Ascioglu O, Donmez-Altuntas H, Aktas E, Micronucleus evaluation in mitogen-stimulated lymphocytes of PUVA

treated patients. Thoku J. Exp. Med. 198: 11-21, 2002.

P23. MALDI -Imaging of Endogenous Lipids in Tumour Xenographs

Philip Bond1, Andrew B. McEwen1, Stuart G. Wood2 and Malcolm Clench3

1Metabolic Chemistry, Quotient Bioresearch (Rushden) Limited, Rushden, United Kingdom, 2Metabolic Chemistry, Quotient Bioresearch (Rushden) Limited, Rushden, United Kingdom, NN10 6ER, 3Biomedical Research Centre, Sheffield Hallam University, Sheffield, United Kingdom, S1 1WB

Purpose: There has been a lot of recent interest in the application of matrix assisted laser desorption mass spectrometry (MALDI-MS) to investigate distribution of molecules within tissues. The aim of the current work is to develop MALDI-MS imaging methods to investigate changes in the distribution of endogenous lipids, and xenobiotic compounds in dosed and non-dosed tumours.Methods: Tumour xenografts (human colon carcinoma, HCT116) were established in mice. Xenographs obtained from non-dosed, 2hr and 6hr post dose (Vinblastine 6mg/kg IV) animals, were sectioned at 12µm and mounted onto aluminium plates. Sections were prepared for positive and negative ion imaging using an Imageprep (Bruker Daltonics, Germany). The matrix was αCHCA in 50% ethanol, 0.1%TFA was included for positive ion analysis. The coated sections

49

were analysed using a Q-star pulsar-i, and a MALDI Synapt HDMS hybrid mass spectrometer. Principal Component Analysis Discriminant Analysis (PCA DA) was performed on the spectra obtained and ion maps produced using BioMap software.Results: The data obtained indicated several lipid species which were notably elevated in the tumour region compared to the surrounding tissue. In addition several species appeared in the surrounding tissues that were not observed in the tumour region. MALDI images were compared with stained sections, and ions were assigned to different regions of the tumour.Conclusions: PCA DA separated all six groups (Tumour, necrotic centre and connective tissue from both the dosed and non-dosed tumour sections) and ions of interest were identified using MS/MS techniques. Ion mobility separation (IMS) was highly beneficial in MALDI-MS experiments. IMS reduced the complexity of signals within the mass region and enhanced imaging by separating the ions of interest based on their relative cross sectional area thus removing interferences from matrix ions.

P24. Urinary excretion of the acrylonitrile metabolite 2-cyanoethylmercapturic acid is correlated with tobacco smoke exposure

Emmanuel Minet1, Francis Cheung2, Graham Errington2, Katharina Sterz3 and Gerhard Scherer3

1Risk Characterization, British American Tobacco, Southampton, United Kingdom, 2Risk Characterization, British American Tobacco, Southampton, AL, United Kingdom, 3Analytisch-Biologisches Forschungslabor GmbH, Munich, Germany

Introduction

Acrylonitrile is an IARC class 2B carcinogen, with the general population exposure to acrylonitrile limited to tobacco smoke, accidental fires, and residual acrylonitrile in commercial polymers. Tobacco smoke is by far the major non-occupational contributor for acrylonitrile exposure. Biomarkers of exposure are the best indicators of the absorbed dose of a given toxicant. Urinary 2-cyanoethylmercapturic acid (CEMA) is an acrylonitrile metabolite and a potential biomarker for acrylonitrile exposure. So far, the dose response relationship between acrylonitrile exposure and CEMA excretion has only been partially investigated. Objective - The objective of this work was to study the dose response of CEMA in urine of non-smokers and smokers of different ISO tar yield cigarettes and its correlation with other known biomarkers of tobacco smoke exposure.

Method: 24hrs urine samples were collected from non-smokers (n=50) and smokers of 1mg (n=48), 4mg (n=45), and 10mg (n=47) ISO tar yield cigarettes. CEMA, total nicotine equivalent (Tneq), 3-HPMA (a marker of acrolein), and NNAL (a marker of NNK) were quantified by LC-MS/MS. Nicotine mouth level exposure (NMLE) was quantified by GC-FID. All statistical analyses were carried out with MINITAB v15.1. Results - CEMA levels in urine of non-smokers were significantly lower than in urine of smokers, and increased with ISO tar yield of cigarette smoked and NMLE. Urinary CEMA levels were significantly correlated (p>0.001) with biomarkers of tobacco smoke exposure (Tneq, NMLE), and with biomarkers of smoke toxicants exposure (3-HPMA, NNAL). The corresponding Pearson correlation coefficients (r) were > 0.7.

Conclusion: Urinary CEMA levels show a clear dose-response relationship to markers of tobacco smoking exposure such as NMLE and urinary Tneq and are well correlated to other biomarkers of smoke toxicants exposure.

P25. Characterization of bilirubin binding site in human serum albumin via construction and bi-lirubin binding screening of a phage library

Ai Minomo1, Yu Ishima1, Makiyo Uchida1, Yoshiaki Suwa1, Toru Maruyama1, Hiroshi Morioka1 and Masaki Otagiri2

1Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan, 2Pharmaceutical Sciences, Sojo University, Kumamoto, Japan

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Bilirubin (BR), an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in indi-viduals with impaired or immature liver function. Human serum albumin(HSA) binds BR(Kd = 10-7∼10-8 M) at a high affinity site and acts as a buffer preventing the transfer of BR from blood to the tissues, thus playing a critical role in impair-ing the development of BR encephalopathy. However, their precise binding locations on HSA have yet to be determined using crystal structure analysis and site-directed mutagenesis. Thus, this study was undertaken to identify key amino acid residues of HSA involved in BR binding using phage display as a tool of evolutionary engineering. Firstly, we revealed that domain II (187Asp-385Gln) of HSA in domain I∼III was a major binding site of BR. Therefore, a phage library displaying domain II was constructed which a total of twelve Lys and Arg residues were randomly mutated to Lys, Arg, Glu or Gly. Phage clones with higher BR binding affinity were selected using BR-coupled EAH Sepharose 4B gel. Selected 111 phage clones were evaluated for BR binding activity by peroxidase assay. 8 clones possessed higher binding affinity compared with HSA domainII. Interestingly, sequencing analysis of these 8 clones showed that they contained the basic amino residues at position 195 and 199. These results suggest that both 195Lys and 199Lys may be involved in BR binding. To provide evidence for precise details, we investigated the characteristics of K195A and K199A mutants of HSA. As a result of fluorescence binding analysis, both mutants possessed five folds lower affinity than wt-HSA. We conclude 195Lys and 199Lys play an important role in BR binding. Based on this finding and X-ray crystal structure analysis of HSA, we found that hydrophobic pocket formed from four of the alpha-helices around 195Lys and 199Lys. We speculated BR binding site was formed from these helices. To test this hypothesis, another region 116Val-293Val possessed the four helices displaying phage were evaluated for BR binding activity. 116Val-293Val displaying phage possessed equal binding affinity compared with HSA domainII, thus it was suggested that 187Asp-293Val may be BR binding site.

P26. Influence of particle size and surface charge on the cytotoxicity of nanodispersions

Phillip Martin1, Tom McDonald2, Darren Smith3, Marco Giardiello2, Steve Rannard2 and Andrew Owen3

1Pharmacology and Therapeutics, University of Liverpool, Liverpool, United Kingdom, 2Department of Chemistry, University of Liverpool, Liverpool, United Kingdom, 3Pharmacology and Therapeutics, University of Liverpool, Liverpool, United Kingdom

Nanoparticle dispersions are being developed in an attempt to improve the utility of poorly soluble drug candidates and may have additional benefits such as improved oral bioavailability or cellular targeting. However, there is currently a lack of information regarding the interaction of nanodispersions with biological systems. The purpose of this study was to investigate the influence of nanoparticle properties on the cytotoxicty of nanodispersions using ritonavir as a paradigm. A library of 68 nanodispersions with varying combinations of 23 excipients (10 polymers and 13 surfactants) was generated using previously published methodology (Zhang et al., 2008). Particle size (z average) ranged from 212 – 3682 nm and surface charge (zeta potential) ranged from -92 – +36 mV. The cytotoxicity of each nanodispersion was assessed in hepatic (HepG2), intestinal (Caco-2), lymphocyte (CEM), monocyte (THP-1) and macrophage (A-THP-1) cell systems. Cells were separately seeded at a density of 2.5 x 104 / 100 µl into each well of a 96 well plate and incubated for 24 hours at 37°C and 5% CO

2. Media was then aspirated and replaced with media containing 0.1, 1, 10, 100, 500 or 1000

µM of each dispersion and incubated for a further 24 hours. Subsequently, 100 µl of CellTiter-Glo® Reagent (Promega, USA) was added to each well and incubated at room temperature for 10 minutes. Luminescence was then measured using a Tecan Genios plate reader. Data were used to calculate IC

50 values by non-linear regression in Graphpad prism.

SPSS was then used to assess the impact of either particle properties (z average, zeta potential and polydispersity) or excipients on the resultant IC

50s in univariate and multivariate models. A wide variability in cytotoxicity was observed in

all cell systems with IC50

values ranging from 0.3 - 734 mM in A-THP-1 cells (IC50

of an aqueous solution in this cell system was 8.1 mM). Particle size and surface charge but not polydispersity were related to the cytotoxcity of the dispersions. For example, a linear relationship between z average and logIC

50 (R2 = 0.09) and a quadratic relationship between zeta

potential and logIC50

(R2 = 0.09) was observed in A-THP1 cells. Furthermore, in a multiple regression model, z average and zeta potential accounted for 25% of the variability in A-THP1 cytotoxicity. A number of polymers (F127, hydrolysed gelatine) and surfactants (chremaphor, tween 80, brij58, hyamine, CTAB) were also associated with cytotoxicity and this was partially explained by their impact upon particle properties. Importantly, none of the excipients were cytotoxic at any of the concentrations used. In conclusion, these data indicate that nanoparticle properties directly influence cytoxicity of the resulting dispersions. Further work is now required to investigate the mechanisms for these observa-tions and to establish the source of the unexplained variability in cytotoxicity.

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Zhang H, Wang D, Butler R, Campbell NL, Long J, Tan B, Duncalf DJ, Foster AJ, Hopkinson A, Taylor D, Angus D, Cooper AI and Rannard SP (2008) Formation and enhanced biocidal activity of water-dispersable organic nanoparticles. Nat Nanotechnol 3:506-511.

P27. As, Cd and Pb synergistically promote apoptosis in astrocytes and damage blood brain barrier in developing rat brain and alter neurobehavior

Sanghamitra Bandyopadhyay, Asit Rai, Shailendra Maurya and Priyanka Khare

Developmental Toxicology, Indian Institute of Toxicology Research, Lucknow, India

P28. Baclofen brain targeting: High CNS levels via baclofen methyl carbamate

Salwa Mosad1, Raimund Nieß1, Khaled Abou-Zeid1, Rasha Hanafi1, Reham Abdel-Kader1 and Hilde Spahn-Langguth2

1Faculty of Pharmacy & Biotechnology, German University in Cairo - GUC, New Cairo City, Egypt, 2Institute of Pharmaceutical Sciences, Karl-Franzens-University, Graz, Austria

Baclofen, a specific GABAB-receptor agonist, has successfully been used for decades for the treatment of spastic dis-

orders. However, the fraction of an oral or intravenous dose that reaches the CNS is negligible, while intrathecal dos-ing provides direct biophase access and yields effective CNS-levels, as indicated by the respective cerebrospinal fluid concentrations. This implies that baclofen does not sufficiently permeate through the blood-brain-barrier. Lipophilic ester prodrugs exhibit a considerably higher membrane permeability than parent baclofen. Yet, since ester prodrugs of baclofen are not readily cleaved in vivo and were found to be subject to exsorptive transport, alternative prodrug concepts were evaluated. E.g., methyl 4-(tert-butoxycarbonyl amino) 3-(4chlorophenyl) butanoate (baclofen methyl carbamate) was synthesized and its solubility and log P were evaluated as bio-relevant parameters. Baclofen methyl carbamate was then tested vs. baclofen with respect to the obtainable target organ levels. Baclofen hydrochloride as well as the methyl carbamate were dosed intraperitoneally to rats (baclofen dose: 1.0 mg/kg, carbamate dose, equimolar for direct comparability). Blood and brain samples were collected and the concentrations of the analytes determined via RP-HPLC on an octadecylsilane stationary phase (gradient elution with pH2.6 0.1mM phosphate buffer/methanol mix-tures; UV-eluateIt was found that the additional carbamate group in the baclofen methyl ester molecule had a significant influence, since the structural change was considerably decreasing the solubility in water and increasing the partitioning into lipophilic matrices. Maximum baclofen concentrations detected in brain tissue were almost 5-times higher after carbamate dosage than after administration of baclofen itself. As opposed to parent baclofen the carbamate showed a rapid distribution into tissues after i.p. dosage. In the blood compartment formation of baclofen was negligible. In the brain the prodrug was hydrolyzed to a remarkable extent, yet showing a delayed release profile for baclofen and reaching highest brain concentrations at a t

max of 3 hr. The possibility to simultaneously detect the respective ester via HPLC gave

evidence that the methyl ester cleavage represents the first of two sequential metabolic steps regenerating baclofen in vivo. Hence, carbamate derivatives appear as very promising biolabile prodrugs for brain delivery of baclofen.

P29. Benzene hexachlorides induce TOP2A expression in human primary renal cells

Keiko Hosohata, Hitoshi Ando, Akira Sasaki, Yasuo Oshima and Akio Fujimura

Division of Clinical Pharmacology, Department of Pharmacology, School of Medicine, Jichi Medical University, Shimotsuke, Japan

Benzene hexachloride (BHC), which is widely used as a drug for scabies and an agricultural chemical, has been shown to cause cancers. However, the underlying molecular mechanism remains to be elucidated. To address this issue, we investigated the effects of BHCs (alpha, beta, gamma, and delta) on gene expression profiles in primary human renal tubular cells and human renal proximal tubule epithelial HK-2 cells. Microarray analyses revealed that each BHC time-

52

dependently increased TOP2A (topoisomerase II alpha gene) expression in primary cells. The mRNA level of TOP2A was also increased in HK-2 cells after the 6 h exposure to each BHC. Moreover, TOP2A protein was significantly increased after 24 h. These results clearly demonstrated that BHCs induce TOP2A expression. Because topoisomerase II alpha is responsible for unlinking DNA during replication and a target of anticancer anthracycline, the increased TOP2A expression might be involved in BHC-associated carcinogenesis.

P30. CYP2E1*5B Polymorphism, Response to Chemotherapy and Survival in Non Small Cell Lung Cancer Patients

Volkan Karacaoglan1, Ahmet Oguz Ada1, Semih C. Kunak2, Sibel Alpar3, Meral Gulhan3, Bahar Kurt3 and Mumtaz Iscan1

1Department of Toxicology, Ankara University, Faculty of Pharmacy, Ankara, Turkey, 2Pharmacology, Giresun University, Medical Faculty, Giresun, Turkey, 3Pulmonary Diseases Clinic, Ataturk Pulmonary Diseases and Thoracic Surgery Hospital, Ankara, Turkey

Lung cancer is an increasing worldwide public health problem particularly in men. In recent years the mutations in CYP2E1 genes have been suggested to be responsible for altering the survival rates of patients with non-small cell lung cancer (NSCLC) by altering sensitivity to chemotherapy. However, results are contradictory and did not provide data with respect to their relation to response to therapy. In this study, the association between CYP2E1*5B (RsaI) polymor-phism and response to chemotherapy or survival in 137 NSCLC patients who were treated with platinum based drugs have been investigated. Genetic polymorphism analyses were determined by using the PCR/RFLP method by using lymphocyte DNA. The polymorphism did not significantly influence the responses to platinum based chemotherapy. No significant associations were noted between the responses of genotypes to chemotherapy and age, sex, smoking status, chemotherapy regimen, tumor stage or histology. Mutant carriers of CYP2E1 gene did not significantly survive shorter or longer than the wild type carriers of the gene (median survival of 18.2 months for wild type (c1/c1) genotype and 16.1 months for heterozygous mutant (c1/c2) genotype; p=0.830). Multivariate analysis also revealed no significant hazard ratio (HR) of death associated with CYP2E1 genotypes. (HR, 1.25; 95 % CI, 0.26-6.04, p=0.74). These results show that the CYP2E1*5B (RsaI) polymorphism is not associated with survival in patients with advanced NSCLC (Supported by the Research Fund of Ankara University No: 10A3336002).

P31. Cytogenetical and Cytological EFFECTS of the Diclofenac IN PLANTS Test Systems

Rena Agabeyli

Institute of Botany, National Academy of Sciences, Baku, Azerbaijan

Azerbaijan Republic Influence of the diclofenac (D) – non-steroidal anti-inflammatory drag that is widely mobilized for treatment of the different bone and muscle deceases, including rheumatoid arthritis, acute muscle pain has been studied on plants objects. The purpose of this study is to reveal the possibilities to mobilize the plants test system as more suitable from bioethics point of view for initial assessment of the influence of different new potential drugs and food additives on the cells morphology and cells proliferation. The following species have been mobilized for experiments as test objects: Triticum durum (I), Hordeum sativum (II, both monocotyledons) and Vicia faba (III, dicotyledons). The effect of the commercial D (manufactured by “Synthesis”, Russia) has been studied after growing of the seeds in the different solutions of the drag: 250, 25 and 2.5 mkg/ml. The influence of the treatment in these doses has been studied with analyses of the frequency and spectrum of the aberrations of chromosomes and proliferate activities of the cells. The growing of the seeds also has been studied. The experiments shown that influence of the D in doze 250 mkg/ml completely stopped the proliferation of the cells and decreasing growing processes of the plants roots. The influence of the other studied doses (25 and 2.5 mkg/ml) resulted in the increasing of the different types of abnormalities in the both groups of plants. These toxicological processes induced with D in plants cells cover such pathologies as depressions of the cells proliferation, increasing of the levels of the aberrations of the chromosomes, increasing of the poly-nucleus cells percentage. Early has been shown that D induced different pathological processes in the animal′s cells (Ware J.A., Pohl L. R. Immunochemical detection of diclofenac protein adducts in the small intestine of rat: possible role in allergic

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reactions - ISSX Proceedings, v. 11, 1997, p.103). The results of the experiments with plants test systems has been shown that this objects can be used in screening processes.. The effect of the diclofenac (D) - non steroid anti-inflammatory drug that is widely used for the treatment of the different bones and muscles deceases has been studied on different plant test

P32. Effect of DDT on the target genes of hormonal carcinogenesis

Vladimir Pustylnyak1, Mariya Liyasova2 and Lyudmila F. Gulyaeva3

1Molecular carcinogenesis, Institute of MolBiol & Bioph SB RAMS, Novosibirsk, Russia, 2Molecular Carcinogenesis, Institute of MolBiol and Bioph SB RAMS, Novosibirsk, Russia, 3Molecular carcinogenesis, Institute of MolBiol and Bioph SB RAMS, Novosibirsk, Russia

Hormone-dependent cancer such as endometrial and ovarian cancer shows a growing incidence rate all over the world. Xenoestrogens may have an important role in increasing the risk of these hormone-dependent cancers. In this study we investigated time-dependent gene expression of CYP19, ERa and ERb in ovary and uterus of female rats treated with known xenoestrogen dichlorodiphenylthrichloroethane (DDT). CYP19 mRNA was undetectable in uterus of control and DDT-treated rats. A clear activation of CYP19 gene was observed in ovary from DDT-treated rats during 2, 4 and 6 days, whereas CYP19 mRNA level was reduced to control level after 8 days. A detectable ERa and ERb mRNA levels were measured in uterus and ovary of untreated rats. Tissue-specific effects of DDT treatment were observed in these tissues. Our results demonstrated activation of ERa gene in rat uterus after 8 days DDT treatment only, whereas DDT suppressed its expression in ovary after 2 days. On 4, 6 and 8 days DDT did not have any remarkable effect on ERa gene expression in this tissue. In parallel, ERb gene expression was increased in uterus after 4 and 6 days treatment. On the other hand, after 8 days treatment with DDT resulted in reduction of this gene expression in uterus. At the same time DDT treatment evoked to significant decrease of ERb gene expression after 6 and 8 days. Thus, our results give evidence about tissue-specific effects of DDT on expression of genes, which participate in hormonal carcinogenesis.

This work was supported by RFBR grant 10-04-00930-à, Russia.

P33. Modification with Plant′S Product Xenobiotics Induced CHROMOSOMAL Instability IN the PLANTS and Anymals CELLS

Rena Agabeyli1 and Urkhan Alakbarov2

1Institute of Botany, Baku, Azerbaijan, 2National Academy of Sciences, Baku, Azerbaijan

The influence on cells and protective properties of the sum of saponins (SS) obtained from leafs of Yucca gloriosa L. and their ability to decrease the level of chromosomal instability induced by gamma-rays (GR) and N-methil-N-nitro-N-nitrozoguanidine (MNNG) has been studied on meristematic cells of the Allium cepa L. (I), Triticum durum L. (II) and marrow cells of rats (Vistar). The studied SS contents about 2 % of steroid saponins, derivatives of the thygogenine. The influence of the SS in concentrations 0.001 – 100 mkg/ml on spontaneous and induced by GR, and MNNG mutability and spectrum of chromosomes aberrations, mitotic activities has been studied. In experiments with GR seeds of the (I) have been irradiated by doses 10 Gy, (II) – in doze 6.5 Gy. MNNG used in doze 3mM (treatment - 3 hours, washing – 3 hours) with following incubation in the SS solutions. The rats have been treated with MNNG (5 mg/100g of animal′s weight, one time acute intoxication). The high antimutagenic efficiency (AE) of the SS has been shown in experiments with decreas-ing of the mutability induced by chemical xenobiotics and GR. The studied SS decreased also the level spontaneous mutations induced by the ageing process in the cells of plants and animals. Aberrations of the chromosomes, c-mitoses, inhibition of the mitotic activities, poly nucleus cells, induced by GR and chemically xenobiotics modified and stabilized by SS with high efficiency. These effects have been observed in experiments in which SS has been administrated before the xenobiotics. The studied sum of saponins decreased the level of chromosomes aberration induced by xenobiotics in the plants and animals cells with high efficiency (64 - 87 %). The antimutagenic efficiency of the SS is growing with increasing of their concentrations and antioxidant activities. The decreasing of the xenobiotics induced damages may be resulted also from apoptosis of cells having many damages. The results of experiments demonstrated that the SS is one of elements of the natural autoantimutagenic system of the plants. The Yucca gloriosa L. may be used as the source

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of the natural compounds which are effective for decreasing of the xenobiotics toxicity and prevention of the genetic instability induced with irradiation and ageing.

P34. Predicting the Genotoxicity of Allylbenzene Compounds

Alex Cayley and Richard Williams

Lhasa Limited, Leeds, United Kingdom

Allylbenzenes are a class of organic compound prevalent in nature and present in numerous different foodstuffs. A number of these compounds induce positive responses in in vitro genotoxicity assays as well producing positive results in rodent carcinogenicity studies. Activity within the class, however, is far from universal and for those compounds with genotoxic potential, activity is generally not consistent between assays. In this work the available literature relating to the genotoxicity of the allylbenzenes has been reviewed, and the findings from these studies used to extend and refine some existing in silico structural alerts for allylbenzenes in an attempt to more accurately predict the activity of the compounds in different in vitro assays. Initial investigations were made into the activity of the compounds in the Ames test, unscheduled DNA synthesis (UDS) assay and chromosome aberration test. Following this analysis structure activity relationships were developed, taking into account mechanistic and metabolic factors. It was found that different structure activity relationships were required to describe the activity of allylbenzenes in the individual in vitro tests, highlighting the subtle differences between the assays. Most allylbenzenes require metabolic activation in order to produce electrophilic species which are thought to promote their genotoxic activity. When comparing the UDS assay (when carried out in rat hepatocytes) and the Ames test one might hypothesise that the differences in results may be a consequence of variation in metabolic capability between the two test systems. In the case of the chromosome aber-ration test a significantly different structure activity relationship was developed which suggested that activity in this assay may be related to the formation of quinone metabolites - a biotransformation considered to be a detoxification pathway in the Ames and UDS assays. Our work demonstrates that analysis of detailed genotoxicity data for a class of toxic compound, an understanding of the mechanism by which a compound causes toxicity and a good knowledge of the limitations of different test systems can, in some cases at least, lead to more accurate prediction of the toxicity of these compounds in various different in vitro assays. This information can then be used as part of an integrated testing strategy for such compounds, where knowledge of the appropriate test required to evaluate genotoxic activity is important to make meaningful risk assessments.

P35. Predictive Gene-expression Signatures of Non-Genotoxic Carcinogen Responses in Mouse Embryonic Stem Cells

Bob van de Water1, Ilse Huijskens1, Martijs Jonker2, Timo Breit2 and Harry Vrieling3

1Div. of Toxicology, LACDR, Leiden University, Leiden, Netherlands, 2MicroArray Department, University of Amsterdam, Amsterdam, Netherlands, 3Department of Toxicogenetics, Leiden University Medical Center, Leiden, Netherlands

Non-genotoxic (NGTX) carcinogens do not physically cause DNA mutations, but induce tumorigenesis through interactions via various cellular receptors or response elements and subsequent activation of various pathways in a tissue and species specific manner. At present, there is a limitation of predictive models to identify novel chemi-cal entities with potential NGTX carcinogenic hazard for humans. We attempt to assemble biomarker-based cell reporter systems for detecting chemicals with potential NGTX carcinogenic features by toxicogenomic studies. Since stem cells are vital in cancer development, we have used mouse embryonic stems (mES) cells as a model. For our study we selected 20 NGTX carcinogens belonging to 10 different chemical classes based on the following rationale: (a) group 1 and group 2A/2B carcinogens recognized by IACR (international agency for research on cancer), (b) differ in mechanisms, (c) carcinogenic in both in vivo and in vitro studies. NGTX carcinogens used in this study include: cyclosporine A/FK-506, hexachlorocyclohexane/heptachlor epoxide, Wyeth-14643/clofibrate, tetrachlorodibenzo-p-dioxin/aroclor1254, bis-dichloropyridyloxy-benzene/phenobarbital, phorbol-myristate-acetate/phorbol-dibutyrate, carbon tetrachloride/trichloroethane, okadaic acid/calyculin A, sodium arsenite/lead acetate, diethylstilbestrol/estradiol. Genotoxic carcinogens and non-genotoxic noncarcinogens were included as

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control compounds. Single treatment concentrations were systematically determined based on cytotoxic responses in mES cells. Transcriptomics analysis was performed at 8 hr post treatment on Affymetrix gene expression micro-array. NGTX carcinogenic responses were compared with transcriptomics profiles of genotoxic carcinogens and non-genotoxic noncarcinogens. Bioinformatics analysis resulted in ∼70 potential probe sets that showed clear transcriptional changes for those NGTX carcinogens with statistically significant difference in expression. We will evaluate these candidate NGTX carcinogens biomarkers in extensive time and dose-response exposure experiments followed by RT qPCR studies. Candidate genes will be used to generate BAC transgene-omics based mES reporter cell lines.

P36. Who is most susceptible to arsenic-induced internal malignancies?

Ling-I Hsu and Chien-Jen Chen

Genomic Research Center, Academia Sinica, Taipei, Taiwan

Arsenic exposure is an important public health issue worldwide. Skin lesions including hyperpigmentation, hyperkeratoses and Bowen¡¦s diseases are hall mark signs for chronic arsenic exposure. To test the hypothesis that individuals with a personal history of arsenic-induced skin lesions may have an increased risk of subsequent internal malignancies, a cohort of 2448 subjects from arsenicosis-endemic area with at least one skin examination at the end of 1980s has been followed for 17 years. The subjects were divided into 5 skin groups: Group I(1666 persons): no lesions and no Bowen/NMSC; Group II (276 persons): hyperpigmentation with neither palmoplantar hyperkeratosis nor Bowen/NMSC; Group III (123): palmoplantar hyperkeratosis but no Bowen/ non melanoma skin cancer (NMSC); Group IV (262): Bowen/NMSC but not palmoplantar hyperkeratosis and Group V (121): both Bowen/NMSC and palmoplantar hyperkeratosis. Cox regression analysis with time-dependent covariates was used to determine the hazard ratio of internal malignancies associated with previously-diagnosed skin lesions. Compared with the subjects with no history of skin lesions (Group I), Group III, VI and V had significantly increased risk of lung cancer with RR=2.6, 3.2 and 4.1, respectively. The magnitude of association became stronger after adjustment of several putative risk factors including cigarette smoking and arsenic exposure. Smokers in Group III, IV and V had striking high risk of lung cancer when compared to reference ¡§nonsmoker and no lesions¡¨ and the interaction was statistically significant under multiplicative model. Conclusions: Our study is the first perspective follow-up study to estimate the association of arsenic-induced skin lesions with subsequent internal malignancies. The results provide evidence for an association of palmoplantar hyperkeratosis and Bowen/NMSC with increased risk of lung cancer after adjustment of other putative risk factors. The smokers with either hyperkeratosis or Bowen/NMSC had strikingly increased lung cancer risk. We suggested Bowen/NMSC as well as hyperkeratosis were susceptible markers of lung cancer and interaction between cigarette smoking and occurrence of these lesions was statistically significant.

P37. Age-Dependent Disposition of Micafungin in Humans: Effect of Serum Protein Binding

Souzan B. Yanni1, Philip B. Smith2, Daniel K. Benjamin Jr.3, Patrick Augustijns4, Dhiren R. Thakker5 and Pieter Annaert4

1School of Pharmacy-Division of Molecular Pharmaceutics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 2Department of Pediatrics, Duke Clinical Research Institute, Duke University, Durham, NC, USA, 3Department of Pediatrics, Duke University, Durham, NC, USA, 4Laboratory for Pharmacotechnology and Biopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium, 5School of Pharmacy-Division of Pharmacotherapy & Experimental Therapeutics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Micafungin, a new echinocandin antifungal agent, has been widely used for the treatment of various fungal infections in human populations. Micafungin is predominantly cleared by biliary excretion and binds extensively to serum pro-teins (>99.5%). Both clearance and volume of distribution are higher in neonates than in adults, but the mechanisms

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underlying these differences are not understood. Previous work revealed the roles of sinusoidal uptake (Na+-Taurocholate Co-transporting Peptide, NTCP; Organic Anion Transporting Polypeptide, OATP) as well as canalicular efflux (Bile Salt Export Pump, BSEP; Breast Cancer Resistance Protein, BCRP) transporters in micafungin hepatobiliary elimination. In the present study, the relative protein expression of hepatic transporters was compared between liver homogenates from neonates and adults. Also, the extent of micafungin binding to serum from neonates and adults was measured in vitro. The results indicate that relative expression levels of BSEP, BCRP, OATP and MRP3 (Multidrug Resistance-associated Protein 3) were similar in neonates and in adults. However, micafungin fraction unbound (fu) in neonatal serum was about 8-fold higher than in adult serum (0.033 +/- 0.012 vs. 0.004 +/- 0.001, respectively). While there was no evidence for different intrinsic hepatobiliary clearance of micafungin between neonates and adults, our data suggest that age-dependent differences in protein binding of micafungin are responsible for its higher clearance in neonates compared to adults.

P38. Evaluation of the human prediction of clearance from hepatocyte and microsome intrinsic clearance for 52 drug compounds

Anna-Karin Sohlenius-Sternbeck1, Lovisa Afzelius1, Peteris Prusis1, Jan Neelissen1, Janet Hoogstraate1, Jenny Johansson1, Eva Floby1, Annelie Bengtsson1, Olle Gissberg1, John Sternbeck2 and Carl Petersson1

1DxDMPK, Local Discovery CNS Pain, AstraZeneca R&D Södertälje, Sweden, 2WSP Environmental, Stockholm, Sweden

We compare three different approaches to scale clearance from human hepatocyte and microsome CLint

(intrinsic clearance) for 52 drug compounds. By using the well-stirred model with protein binding included only 11% and 30% of the compounds were predicted within 2-fold and the average absolute fold errors (AAFE) for the predictions were 5.9 and 4.1 for hepatocytes and microsomes, respectively. When predictions were performed without protein binding, 59% of the compounds were predicted within 2-fold using either hepatocytes or microsomes and the AAFE was 2.2 and 2.3, respectively. For hepatocytes and microsomes there were significant correlations (p=8.7x10−13, R2=0.72; p=2.8x10−9, R2=0.61) between predicted CL

int in vivo (obtained from in vitro CL

int) and measured CL

int in vivo(obtained using the well-

stirred model). When clearance was calculated from the regression, 76% and 70% of the compounds were predicted within 2-fold and the AAFE 1.6 and 1.8 for hepatocytes and microsomes, respectively. We demonstrate that microsomes and hepatocytes are in many cases comparable when scaling of clearance is performed from regression. By using the hepatocyte regression, clearance for 73% of the compounds in an independent test set (n=11) were predicted within 2-fold (AAFE 1.6). We suggest that a regression line that adjusts for systematic under-predictions should be the first-hand choice for scaling of clearance.

P39. Prediction of clearance from the HepaRG cell system: comparison with human hepatocytes for a range of substrates

Ugo Zanelli1, Nicola Pasquale Caradonna1, David Hallifax2 and James Brian Houston3

1Screening & Technologies, Siena Biotech SpA, Siena, Italy, 2School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom, 3School of Pharmacy & Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom

In this work, we compare the intrinsic clearance (CLint) obtained for a set of 40 well known drugs using a donor pool of human cryopreserved hepatocytes with those obtained with the hepatoma-derived cell line HepaRG. HepaRG have been proposed as an alternative complete model to evaluate hepatotoxicity with potential as a quantitative model for prediction of metablic clearance. Differentiated cryopreserved HepaRG cells and cryopreserved hepatocytes were evaluated using the same procedure for CLint determination involving a relatively low cell density (250,000/ml) and low substrate concentration (1µM). On average, the hepatocytes underpredicted unbound CLint derived from in vivo plasma clearance, with differences in average fold error (AFE) being dependent on liver model: 1.9 and 1.4 for the well

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stirred (WS) and parallel tube (PT) respectively. Average prediction of unbound CLint from HepaRG was marginally lower, with AFE of 2.7 and 1.8 for the WS and PT model, respectively. For hepatocytes, under-prediction in this study was not as great as has been reported in similar assessments. However, in both systems, AFE was dependent on clear-ance, with increasing underprediction observed with increasing clearance. AFE for each of the separate in vivo CLint ranges <10, 10-100, 100-1000 and >1000 ml/min/kg was 0.4, 1.4, 2.7 and 8.5, respectively, for hepatocytes and 1.6, 1.4, 2.8 and 7.2, respectively, for HepaRG (using the WS model). Moreover, both systems gave greater under-prediction with increasing protein binding irrespective of liver model, possibly reflecting correlation of prediction accuracy with clearance. In summary, the HepaRG cell system was quantitatively comparable with human hepatocytes, in terms of CLint, for a wide range of substrates indicating potential for evaluation as a completely functional substitute for the human donor dependent hepatocyte model.

This work was partially supported by the European Union: FP6 PRIORITY LSH-2005-2.02.07: “Development of new in vitro tests to replace animal experimentation”. Li.Int.Op. Project (Optimization of Liver and Intestine in-vitro models) Contract number LSBH-CT-2006-037499 (http://www.liintop.cnr.it)

P40. Prediction of Oral Pharmacokinetics of cMet Kinase Inhibitors in Humans: Physiologically-Based Pharmacokinetic Modeling versus Traditional One-compartment Model

Shinji Yamazaki1, Judith Skaptason1, David Romero1, Sylvia Vekich1, Hannah Jones2, Weiwei Tan3, Keith Wilner3 and Tatiana Koudriakova1

1Pharmacokinetics, Dynamics & Metabolism, Pfizer, San Diego, CA, USA, 2Pharmacokinetics, Dynamics & Metabolism, Pfizer, Sandwich, United Kingdom, 3Clinical Pharmacology, Pfizer, San Diego, CA, USA

In recent years, there has been a growing interest in physiologically-based pharmacokinetic (PBPK) models, which provide the disposition profiles in various species to be predicted from physico-biochemical properties of compounds with the species-specific physiological parameters. The objective of this study was to assess PBPK models for predict-ing plasma concentration-time profiles of orally available cMet kinase inhibitors, PF02341066 and PF04217903, in humans. The prediction accuracy of pharmacokinetics (PK) by the PBPK modeling was compared to traditional one-compartment PK model based on allometric scaling. The predicted clearance values from allometric scaling with the correction for the interspecies differences in protein binding were used as representative comparison, which showed more accurate PK prediction in humans than the other methods. Overall the PBPK modeling provided a better predic-tion of the area under the plasma concentrationtime curves for both PF02341066 and PF04217903 compared to the one-compartment PK model. More importantly, the simulated plasma concentration-time profiles of PF02341066 and PF04217903 by the PBPK modeling appeared to be consistent with the observed profiles showing multi-exponential declines, resulting in more accurate prediction on the apparent half-lives. Therefore, the PBPK modeling has the potential to be more useful and reliable for the PK prediction of PF02341066 and PF04217903 in humans compared the traditional one-compartment model prediction. In summary, the present study has shown examples that the PBPK models can be used to predict PK profiles in humans. *Current address: Drug Metabolism & Pharmacokinetics, Johnson & Johnson, San Diego, CA

NH

HON N

N

N NN

NN

N

NN

NH2

O

Cl

Cl

PF02341066 PF04217903F

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P41. Prediction of the hepatic and renal clearance of anionic drugs in humans based on in vitro uptake experiments

Takao Watanabe1, Hiroyuki Kusuhara2, Tomoko Watanabe2, Yasuyuki Debori2, Kazuya Maeda2, Tsunenori Kondo3, Hideki Nakayama3, Shigeru Horita3, Zhuohan Hu4 and Yuichi Sugiyama2

1Pharmacokineticks Research Laboratories, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan, 2Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 3Department of Urology, Tokyo Women′s Medical University, Tokyo, Japan, 4Research Institute for Liver Diseases (Shanghai) Co., LTD., Shanghai, China

Prediction of the pharmacokinetic properties of drugs in humans is an important issue in drug development to avoid failure in the subsequent clinical stages because of poor pharmacokinetic properties. Drug-metabolizing enzymes and transporters play pivotal roles in drug disposition. The overall elimination of anionic drugs is characterized by the coop-eration of uptake transporters, and efflux transporters or drug metabolizing enzymes. The activities of uptake transporters are critical for the systemic exposure of drugs, while the impact of the efflux transporters or drug metabolizing enzymes on the systemic exposure depends on the rate-determining process. We previously reported that the majority of anionic drugs are efficiently excreted into the bile or urine from inside the cells and, accordingly, uptake becomes the rate-determining process in the overall elimination. Comparison of the uptake clearance with the intrinsic hepatic clearance suggests that hepatic elimination of statins is uptake-limited in humans. So, measuring the uptake clearance would allow the successful prediction of the overall hepatic and renal clearances of anionic drugs. The present study examined the predictability of renal and hepatic clearance using freshly prepared human kidney slices and cryopreserved human hepatocytes. The saturable uptake of seven drugs (rosuvastatin, pravastatin, valsartan, olmesartan, trichlormethiazide, p-aminohippurate and benzylpenicillin) by human kidney slices was extrapolated to in vivo parameters. These correlated well with the cor-responding overall intrinsic clearances of tubular secretion by introducing a scaling factor of 10, except for valsartan the tubular secretion of which was underestimated. As for the hepatic uptake, extrapolation of the in vitro uptake clearance of four statins and three sartans in cryopreservedhuman hepatocytes could predict a clearance comparable with the observed hepatic clearance although fluvastatin and rosuvastatin required a scaling factor of 11 and 6.9, respectively. The present study suggests that uptake is the rate-determining process in the overall elimination of most anionic drugs in human liver and kidney. In vitro uptake assays using human hepatocytes and kidney slices provide a good estimation of the hepatic and renal clearances and the major elimination pathway from the systemic circulation.

P42. Using SimCYP® to Project Human Oral Pharmacokinetic Variability in Lead Development to Mitigate Mechanism-based Adverse Events in the Clinic

Christopher L. Shaffer1, Renato J. Scialis1, Haojing Rong1 and R. Scott Obach2

1PDM-Neuroscience, Pfizer Inc, Groton, CT, USA,2PDM-Biotransformation, Pfizer Inc, Groton, CT, USA

Positive allosteric modulators (“potentiators”) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor have been shown to display a mechanism-mediated exposure-response continuum in preclinical species with electrophysiologic and nootropic effects (“efficacy”) at low exposures and motor coordination disruptions at progressively higher exposures (Shaffer, CL et al. Soc Neurosci Abstr 2009, 883.17). Due to the dose-capping nature of such motor coordination deficits, a threshold-mediated adverse event (AE), the adequacy of exposure separation between the maximal plasma concentration (C

max) at a projected clinically efficacious oral dose and this AE was

explored with three high-interest potentiators in lead development. In vitro metabolism studies with each compound in human liver microsomes and human hepatocytes demonstrated their metabolic clearance was predominately cytochrome P450 (CYP)-mediated. Thus, for each compound′s projected clinically efficacious dose(s), human oral variability in C

max was assessed using SimCYP® software, which combines its virtual human populations database

using extensive demographic, physiological and genomic information with routinely collected compound-specific in vitro biochemical data to simulate and predict drug distribution (Rostami-Hodjegan, A and Tucker, GT Nature Rev Drug Discov 2007, 6, 140–148). Using a combination of experimentally determined recombinant human CYP intrinsic clearances for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, in vitro-derived binding factors,

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expected fraction absorbed and projected steady-state volume of distribution, SimCYP® simulations demonstrated that two of the three evaluated compounds had acceptable projected C

max variability (i.e. the 95th percentile C

max did

not encounter the AE threshold). This evaluation resulted in the selection of these two compounds for preclinical progression, and the termination of the third potentiator due to its unacceptable therapeutic index.

P43. A Novel Method for the Production of N- and O-Glucuronides Using Recombinant Fission Yeast (Schizosaccharomyces pombe)

Daniela Buchheit, Calin-Aurel Dragan and Matthias Bureik

PomBioTech GmbH, Saarbrücken, Germany

UDP- glucuronosyl transferases (UGTs) are the main enzymes of human phase II metabolism. Metabolites generated by those enzymes are essential for the assessment of drug caused side- effects or toxicity, especially in the context of metabolites in safety testing (MIST). Since chemical metabolite synthesis has limits concerning the stereoselectivity of conjugation, a variety of biological approaches for glucuronide production have been developed. Those, in turn, are limited by the membrane- dependency of UGT activity as well as the requirement of the expensive cofactor uridine diphospho- glucuronic acid (UDP- GA). We have developed a whole- cell biotransformation system using recombinant fission yeast, circumventing all above mentioned limitations (Dragan Ca, Buchheit D, Bischoff D, Ebner T, Bureik M: Glucuronide production by whole-cell biotransformation using genetically engineered fission yeast schizosaccharo-myces pombe. Drug Metab Dispos 38(3), 509-515 (2010)).The system is based on the coexpression of a human (or rat) UGT isoform and UDP glucose- 6- dehydrogenase (UGDH), which provides the essential cofactor UDP- GA in vivo and permits a completely self- sufficient glucuronide production. This glucuronide production system has been extended to comprise all 19 human UGTs and was shown to be applicable for the production of different classes of glucuronides. Several standard O- glucuronides such as 4- methylumbelliferone- glucuronide and testosterone- glucuronide were produced as well as isotope- labeled metabolites, the latter being labeled either in the aglycon or the glucuronic acid. N- glucuronides tend to be disproportionate metabolites i.e. they are present much more in humans than in animals (Anderson S, Luffer-Atlas D, Knadler Mp: Predicting circulating human metabolites: How good are we? Chem Res Toxicol 22(2), 243-256 (2009)) and are therefore of special interest for pharmacological toxicity investigations. In a recent research project with a pharmaceutical company, the formation of an N- glucuronide at the imidazole moiety of a farnesyl transferase inhibitor could be achieved using a UGT1A4 and UGDH expressing fission yeast strain in a whole- cell biotransformation. The identity of the metabolite was confirmed by LC- MS. These results underline the suitability of fission yeast based whole- cell biotransformations for the preparative production of glucuronides.

P44. Casodex Up-Regulates The Expression Of The Androgens Inactivating Enzymes UDP-Glucuronosyltransferases 2B15 And 2B17 Expression In Prostate Cancer Cells

Laurent Grosse, Sophie Paquet, Alain Belanger and Olivier Barbier

Endocrinology and genomics, CHUQ Research Center and Faculty of Pharmacy, University Laval, Quebec, QC, Canada

Androgens play a major role in prostate cancer initiation and development. Anti-androgens, such as Casodex (Bicalutamide®), constitute the first line therapy for metastatic prostate cancers. Casodex is a non-steroidal androgen antagonist that binds the androgen receptor (AR), thus blocking its activation by potent androgens (Dihydrotestosterone and Testosterone). We recently observed that AR activation with natural and synthetic androgens abolishes the expres-sion of the two androgen-conjugating enzymes, UDP-glucuronosyltransferases (UGT) 2B15 and 2B17 in prostate cells [1]. By converting androgens into inactive glucuronide metabolites, these 2 enzymes play a major role in the control of the androgen signaling in these cells. Indeed, siRNA-mediated knock-down of these genes in the prostate cancer cell line LNCaP provokes an intracellular accumulation of DHT which causes an improved cell proliferation rates, when compared to cells treated with non-target siRNA [2]. These results were in agreement with correlation stud-ies identifying a low activity UGT2B15 polymorphism as an important determinant in the development of prostate cancer [3]. Based on these observations, we recently tested the hypothesis that pharmacological inactivation of AR

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may result in an increased UGT2B15/B17 expression and in an improved inactivation of the endogenous hormones. To confirm this hypothesis, two androgen-dependent prostate cancer cell lines, LNCaP and LAPC-4, were treated with increasing doses of Casodex (1nM to 20µM). Real-Time PCR analyses showed a dose-dependent induction of mRNA level up to 2.5 fold for UGT2B15 in both cell lines. While being not expressed in LAPC-4, UGT2B17 mRNA levels were also increased in LNCaP upon Casodex exposure. In LNCaP cells, the induction of UGT2B15 expression was also confirmed at the protein level. Time-course experiments in both LNCaP and LAPC-4 cells revealed that the induction in UGT expression occurs as soon as 12 hours of exposition to Casodex and reaches a maximal expres-sion after 48H. In conclusion, these results confirm that an unexpected effect of Casodex is to stimulate androgens inactivation in prostate cells. Considering that most patients become eventually resistant, a deeper characterization of the anti-androgens′ effect in prostate cancer cells will be helpful for future studies aimed at designing novel and more efficient therapeutic strategies.

References

1. Barbier, O. and A. Belanger, Inactivation of androgens by UDP-glucuronosyltransferases in the human prostate. Best Pract Res Clin Endocrinol Metab, 2008. 22(2): p. 259-70.

2. Chouinard, S., O. Barbier, and A. Belanger, UDP-glucuronosyltransferase 2B15 (UGT2B15) and UGT2B17 enzymes are major determinants of the androgen response in prostate cancer LNCaP cells. J Biol Chem, 2007. 282(46): p. 33466-74.

3. Park, J., et al., Asp85tyr polymorphism in the udp-glucuronosyltransferase (UGT) 2B15 gene and the risk of prostate cancer. J Urol, 2004. 171(6 Pt 1): p. 2484-8.

P45. Comparison of biotransformation of hydroxylated warfarins (OHWars) by human sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs)

Anna Radominska-Pandya1, Josie L. Falany2, Ian T. Cook2, Stacie M. Bratton1, Vi-Huyen Le3, Kan Hui Yiew1, Anna Gallus-Zawada1, Charles N. Falany2 and Jeffery H. Moran3

1Department of Biochem & Molec Biol, Univ of Arkansas for Med Sci, Little Rock, AR, USA, 2Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA, 3Department of Health, AR Public Health Laboratory, Little Rock, AR, USA

Targeted metabolomics of Coumadin [R/S-Warfarin (War)] in urine from patients analyzed by LC-MS/MS show significant amounts of Phase II metabolites. Our recent work showing that hydroxywarfarin (OHWars) can potently inhibit CYP2C9 activity in human liver microsomes demonstrates the importance of fully delineating the conju-gative pathways responsible for War inactivation and elimination. The purpose of this work is to determine the significance of Phase II metabolism of War and its hydroxylated derivatives by human recombinant UGTs and SULTs. Here, we hypothesize that effective OHWar metabolism by Phase II enzymes results in elimination of the OHWar generated from the active drug and decreases anticoagulant activity; however, altered drug conjugation would lead to accumulation of OHWar. The metabolic capacities and affinities of human recombinant UGTs and SULTs were evaluated with racemic 6-, 7-, and 8-OHWar as well as their separated enantiomers. Glucuronidation activity was detected for UGT1A family members 1A1, 1A8, 1A9, and 1A10, and sulfation activity was detected with the SULT1 family members 1A1, 1A3, 1B1, and 1E1, with racemic 6-, 7- and 8-OHWar. No sulfation activ-ity was detected with the SULT2 or hydroxysteroid SULTs. Enzymatic products were confirmed with LC-MS/MS analysis. Kinetic analyses confirmed the high affinity/low capacity role of SULTs as compared to the low affinity/high capacity role of UGTs by comparing K

m values (SULT: 7-21 µM; UGT: 44-400 µM) and V

max values (SULT:

20-800 pmol/mg protein/min; UGT: 600-25,000 pmol/mg protein/min). Data for the glucuronidation of R- and S-8-OHWar with UGT1A10 showed that this isoform had a similar affinity for each enantiomer, but a much higher capacity to glucuronidate the S-enantiomer (29,000 vs. 5,900 pmol/mg protein/min for S and R, respectively). SULTs 1B1 and 1E1 also showed a higher capacity for the S-enantiomer, but with much lower V

max values (15.5

and 7.1 pmol/mg protein/min, respectively with no measurable activity toward the R form). Similar differences were observed using purified enantiomers of 6- and 7-OHWar. Further studies with more enantiomer and isoform combinations focusing on determining inhibition constants are in progress. Data are consistent with the hypothesis that conjugation of OHWars plays an important role in modulation of the metabolic pathways involved in War inactivation and elimination. (NIH-DK60109 to AR-P; Bioterrorism Cooperative Agreement U90/CCU616974-07 and APHL-Fellowship to JHM).

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P46. Development of glucuronide preparation system for xenobiotic metabolites using genetically enginerred budding yeast

Shinichi Ikushiro, Yuuka Masuyama, Kaori Yasuda, Masaki Kamakura and Toshiyuki Sakaki

Toyama Prefectural University, Toyama, Japan

Glucuronidation, which is catalyzed by UDP-glucuronosyltransferases (UGTs), is the most common pathway for detoxi-fication and elimination of hydrophobic xenobiotics occurring in tissues of most mammals. Because of their ubiquitous nature and high physiological significance, development of an efficient in vitro synthesis of glucuronides often becomes critical during studies of drug metabolism undertaken in the development of a new pharmaceutical product. In order to synthesize the glucuronides as drug metabolites, we have developed several mammalian CYP, UGT and UDP-glucose dehydrogenase (UDH) coexpression systems in budding yeast. Mammalian CYP, UGT and UDH were expressed in budding yeast cells, Saccharomyces cerevisiae AH22, using a multicopy plasmid vector and a genome integrated vector. Each enzyme expression in yeast was confirmed by Western blot analysis. Glucuronide formation in yeast cells was performed in reaction buffer containing 0.1M KPi (pH7.4) and 8% glucose. Using genetically engineered yeast strain containing human UGT1A6 and rat UDH, glucuronide formation of 7-hydroxycoumarin (7HC) as a model substrate was examined. Most glucuronide of 7HC was found in reaction medium with time-dependent production, suggesting the functional expression of both enzymes and the presence of endogenous transport system for glucuronide in yeast. Optimization of the reaction conditions resulted in 95% conversion of 7HC into its gulucuronide. Compared with fis-sion yeast system as host cell, budding yeast appears to be more competent for glucuronide formation. Mycophenolic acid with multiple glucuronidating sites was conjugated as UGT isoform-dependent formation, suggesting that the regiospecific glucuronides of several drugs could be obtained using UGT1A and 2B isoforms. In order to synthesize glucuronide from CYP-dependent metabolite during Phase I and II processes, rat CYP1A1 and yeast NADPH-P450 reductase were coexpressed with UGT in yeast. The resultant recombinant yeast cells with xenobiotic metabolizing enzymes are able to produce directly the glucuronide from 7-ethoxycoumarin via 7HC. This coexpression system of mammalian CYP, UGT and UDH in budding yeast would be a powerful tool for enzyme-assisted synthesis of various xenobiotic metabolites including glucuronides.

P47. Dichloroacetic acid protects against acetaminophen toxicity in mice

Angelo Theodoratos, Anneke Blackburn, Jean Cappello and Philip Board

Structural Biology, John Curtin School of Medical Research, Canberra, Australia

Mice that are genetically deficient in glutathione transferase Zeta (GSTZ1-1) have low hepatic glutathione levels and are very sensitive to hepatic acetaminophen toxicity. Dichloroacetic acid (DCA) is a substrate for GSTZ1-1 but can also act as a mechanism -based inactivator and the treatment of mice with DCA can create a severe deficiency of GSTZ1-1. We considered that chemically-induced deficiency of GSTZ1-1 may sensitise mice to the acetaminophen hepatotoxicity observed in GstZ1 knock out mice. This potential toxicity is of particular interest as it has been pro-posed that DCA may be a useful agent for the treatment of cancer and it has been used as an experimental drug for the treatment of lactic acidosis. As there is a growing incidence of cancer patients self medicating with DCA (www.thedcasite.com) and it is highly likely that these patients may also take acetaminophen we considered that there may be a risk of hepatotoxicity if the doses of DCA are high enough to inactivate most of the GSTZ1-1. To evaluate this possibility, we treated mice with DCA to inactivate GSTZ1-1 then exposed the mice to a dose of acetaminophen that causes severe hepatotoxicity in GstZ1-deficient mice. The release of ALT is a measure of hepatic toxicity and was significantly elevated in control mice 3 hours after receiving acetaminophen (300 mg/kg IP). Unexpectedly, treatment of mice with DCA for 5 days (250mg/kg/day) prior to treatment with acetaminophen prevented the release of ALT into plasma that occurred in wild type control mice not treated with DCA. This protective response is closely correlated with the levels of hepatic glutathione that were completely depleted in control mice 3 hours after the acetaminophen dose but remained at near normal levels in mice that had been treated with DCA. Treatment with DCA along with or after the administration of acetaminophen did not provide protection against hepatotoxicity suggesting that a change in gene expression is required. The protection may be mediated by an increased capacity to synthesize GSH since DCA increased the levels of the catalytic subunit of glutamate cysteine ligase, the rate limiting enzyme in GSH synthesis. The capacity of DCA to maintain GSH levels in the presence of acetaminophen

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metabolites suggests that it may have a therapeutic role in protecting the liver against other hepatotoxins or drugs that oxidize or are conjugated to GSH.

P48. Glucuronidation of arbidol: identification of human UDP-glucuronosyltransferases and interaction potential

Zhong-Ze Fang1 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Arbidol is an antiviral drug indicated for the prevention and treatment of all types of influenza infection and some other kinds of acute respiratory infections. It was marketed in Russia in 1993 and in China in 2006. Previous reports (Wang et al., 2008) have demonstrated that glucuronidation conjugation reaction was a major elimination pathway of arbidol. However, the UDP-glucuronosyltransferases (UGTs) involved in this process remains to be investigated. The present study aimed at identifying unambiguously the UGT isoforms involved in the production of arbidol O-glucuronide. Arbidol O-glucuronide was firstly isolated from a reaction mixture consisting of arbidol and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and elucidated by HPLC-MS/MS. The kinetic param-eters were determined for pooled human liver microsomes (HLMs) and V

max and K

m values were calculated to be

2.03 ± 0.05 nmol/min/mg protein and 8.0 ± 0.7μM respectively. Assessment of a panel of recombinant UGT isoforms revealed the arbidol glucuronosyltransferase activity of UGT1A1, UGT1A3, UGT1A9. The results obtained from kinetic studies and chemical inhibition all demonstrated that UGT1A9 was a predominant UGT isoform involved in the glucuronidation of arbidol in HLM. Considering that UGT1A9 and UGT2B7 could metabolize many clinical drugs, inhibitory effects of arbidol on these two UGT isoforms were investigated. The results demonstrated that arbidol competitively inhibited UGT1A9 and UGT2B7. K

i values were calculated to be 3.5 μM (for UGT1A9) and

0.5 μM (for UGT2B7) when 4-MU was used as substrates. When propofol and 3′-azido-3′-deoxythymidine (AZT) were utilized as substrates for UGT1A9 and UGT2B7 respectively, K

i values were 29.7 μM (for UGT1A9) and 2.8 μM

(UGT2B7). All these results were helpful for better understanding of arbidol′s pharmaceutical behaviour and its DDI potential.

P49. Identification of UDP-Glucuronosyltransferases involved in human hepatic glucuronidation of diethylstilbestrol

Liangliang Zhu, Guangbo Ge, Huixin Liu and Ling Yang

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Diethylstilbestrol (DES), a synthetic estrogen, was once prescribed to pregnant women to reduce the risk of preg-nancy complications and losses, and now in treatment for breast and prostate cancers and locally for estrogen deficiencies. It has been proved that DES can cause a series of cancers and other health problems. Previous studies revealed that monoglucuronidation in human was a major metabolic pathway [1], which is generally believed to be a detoxification process [2]. However, this pathway of DES in human remained poorly understood. This study was focused on hepatic glucuronidation of DES by human liver microsomes (HLMs) and a panel of 12 recombinant UDP- glucuronosyltransferases (UGT) including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17. DES was extensively glucuronidated in pooled HLMs, and one monoglucuronide was observed by HPLC and LC/MS/MS. HLMs exhibited substrate inhibition kinetics for DES, with K

m, K

si, and V

max value of 2.44 μM, 19.4 μM

and 1.12 nmol/min/mg protein, respectively. Assays with 12 recombinant UGT isoforms revealed that UGT1A1, 1A3, 1A8 and 2B7 can catalyze the DES glucuronide formation. Each isoform displayed substrate inhibition kinetics, and UGT2B7 showed the highest UDP-Glucuronosyltransferases activity towards DES with K

m, K

si, and V

max value of 2.16

μM, 12.9 μM and 0.31 nmol/min/mg protein, respectively (similar to that of pooled HLMs). DES glucuronidation (2 μM) in HLMs from 11 donors correlated strongly (r=0.90, p<0.001) with AZT glucuronidation (a probe reaction for

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UGT2B7 activity), while no significant correlation (r=0.52, p=0.10) was observed with estradiol-3-glucuronidation (a probe reaction for UGT1A1 activity). In chemical inhibition study, a series of inhibitors including estradiol, mefenamic acid, and androsterone showed similar inhibitory effects towards HLMs and UGT2B7 mediated glu-curonidation. In summary, this study characterized the hepatic glucuronidation pathway of DES, during which UGT2B7 played an important role.

References

1. Dodgson KS, Williams RT. Isolation of stilboestrol monoglucuronide from human urine. Nature 1948;161:604-5.2. Metzler M. The metabolism of diethylstilbestrol. Crc Critical Reviews in Biochemistry 1981;10:171-212.

P50. Improvement of In Vitro Approach to Study UDP-Glucuronosyltransferases 1A1 and 1A9 by Comparison of Absolute Protein Quantification and Metabolic Activity Measured Using Estradiol and Propofol as Selective Probe Substrates

Staffan Thorén1, Wojciech Krol2, Marie Wallin1, Philip C. Smith3, Maria Ribadeneira4, Diansong Zhou5 and Pawel Baranczewski6

1Clinical Pharmacology & DMPK, AstraZeneca R&D Södertälje, Södertälje, Sweden, 2Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA, 3Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA, 4Discovery DMPK, AstraZeneca R&D Wilmington, Wilmington, DE, USA, 5Clinical Pharmacology & DMPK, AstraZeneca, Wilmington, DE, USA, 6Clinical Pharmacology & DMPK, AstraZeneca R&D Södertälje, Södertälje, Sweden

Current in vitro methods underestimate the in vivo clearance and magnitude of drug-drug interactions (DDIs) related to metabolic reactions catalyzed by the UDP-glucuronosyltransferases (UGTs). One of the reasons for this failure is lack of appropriate factors to translate data obtained under in vitro conditions to the in vivo environ-ment. In order to improve in vitro predictions of the contribution of UGT1A1 and UGT1A9 to metabolic clearance and DDIs, metabolic activity was compared to absolute protein quantification in a set of individual human liver microsomes (HLMs) from 20 subjects. The catalytic rates were measured under pre-determined optimal incuba-tion and analytical (UPLC-MS/MS) conditions for each selective probe substrate, estradiol (EST) for UGT1A1 and propofol (PRO) for UGT1A9. An absolute protein quantification method was developed to measure UGT1A proteins. After trypsin digestion of 20 µg HLM, signature peptides for UGT1As were measured by isotope dilution mass spectrometry relative to heavy labeled peptide standards. The limits of quantification (<0.5 pmol/mg HLM protein with intra- and inter-day variability <20%) were determined by NanoUPLC-ESI-MS/MS performed with an ABI 5500 QTrap operated in the MRM mode. A S50 value of 10.5 µM was obtained for EST in incubations with HLMs using a sigmoidal model. The following concentrations of EST were chosen for estimation of UGT1A1 activity: 2.5, 10 and 100 µM. The EST 3-glucuronidation rate obtained at the substrate concentration of 100 µM varied between 65 and 781 pmol/min/mg (average 302). The absolute UGT1A1 protein quantification assay revealed values between 3.8 and 59.2 pmol/mg of HLM proteins. The correlation, r = 0.84, between UGT1A1 activity and protein abundance was statistically significant at p<0.0001. Additionally, the relative ratio between the maximal and minimal values was similar: 12-fold for UGT1A1 activity and 15-fold for protein abundance, respectively. A Km value 55 µM was determined for PRO in HLMs using Michaelis-Menten plot. The UGT1A9 activity measurement was performed at final concentrations of PRO: 25, 250 and 1000 µM. The minimal and maximal activity at the substrate concentra-tions of 1000 µM were 278 and 1264 pmol/min/mg (average 660), respectively. The UGT1A9 protein abundance was estimated between 5.1 and 19.1 pmol/mg of HLM protein. The correlation between UGT1A9 activity and pro-tein abundance was statistically significant (p<0.0001) at r of 0.80. Furthermore, the ratio between maximal and minimal values for the UGT1A9 activity was 4.5 and for UGT1A9 abundance 3.7, respectively. The combination of analysis of specific probe substrates of hepatic UGTs in HLMs and novel absolute quantification of UGT proteins will enable translation of data obtained with HLMs to the whole liver and thus may significantly improve IVIVE for glucuronidation of drugs in development.

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P51. Influence of Assays Conditions to Glucuronidation Activity in Vitro: Psilocin Study

Nenad Manevski1, Mika Kurkela2, Camilla Höglund2, Timo Mauriala2, Michael H. Court3, Jari Yli-Kauhaluoma1 and Moshe Finel2

1Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Helsinki University, Helsinki, Finland, 2Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Finland, 3Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA, USA

Human UDP-glucuronosyltransferases (UGTs) are phase II metabolic enzymes responsible for the elimination of numerous endo- and xenobiotics from the body. The ability to accurately estimate the glucuronidation activity in vivo by performing cost-effective in vitro experiments is crucial for modern drug development. The chemical insta-bility of substrate or the presence of inhibitors within the incubation mixture can result in erroneous estimation of glucuronidation activity by both recombinant enzymes and liver microsomal fractions. Lately it was reported that long-chain unsaturated fatty acids that, may be released during in vitro incubation, are potent inhibitors of UGTs 2B7 and 1A9 (Rowland et al., 2007, and 2008). We have recently found that psilocin, a hallucinogenic indole alkaloid that is highly prone to non-enzymatic oxidation in solution (Anastos et al., 2006), is mainly glucuronidated by human UGTs 1A9 and 1A10 (Manevski et al., 2010). We have found that inclusion of up to 2.5 mM DL-dithiothreitol (DTT) in the reaction mixture prevented both the degradation of psilocin and psilocin glucuronide, leading to higher meas-ured glucuronidation rates. Direct effects of DTT on the activity of the psilocin-glucuronidating UGTs was excluded by control assays in which chemically stable substrates, either beta-estradiol or 4-hydroxyindole, were employed. Therefore, it appears that DTT may be used as an efficient antioxidant in glucuronidation studies using substrates that are prone to oxidative degradation. In order to remove potential UGT inhibitors from the incubation mixture, we have included up to 2% fatty-acids-free bovine serum albumin (BSA) in the psilocin glucuronidation reactions. The presence of BSA increased psilocin glucuronidation activity in both UGT1A9 and UGT1A10. In addition, enzyme kinetic analysis revealed that “albumin effect” is more complex then described previously (Rowland et al., 2008) and consist of both Km decrease and Vmax increase. Observed changes in kinetic parameters are consistent with removal of one or more inhibitors from the test UGTs. One or more of these inhibitors may act in a noncompetitive manner. Further studies are needed for complete characterization of tentative inhibitors and inhibition modality. This study demonstrates that careful optimization of assay conditions in vitro is essential for correct determination of UGT activity.

P52. Kinetic and Enzymatic Characterisation of Human Hepatic R- and S- Lorazepam Glucuronidation

Verawan Uchaipichat1, Chuthamanee Suthisisang2, Peter I. Mackenzie3 and John O. Miners4

1Department of Pharmacy Practice, Khon Kaen University, Khon Kaen, Thailand, 2Department of Pharmacology, Mahidol University, Bangkok, Thailand, 3Department of Clinical Pharmacology, Flinders University, SA, Australia, 4Department of Clin Pharmacol, Flinders Univ Sch of Med, Adelaide, Australia

The hypnosedative-anxiolytic agent lorazepam (LZP) is eliminated in humans by glucuronidation. This study aimed to characterize the glucuronidation kinetics of R- and S-LZP by human liver microsomes (HLM) and identify the human UDP-glucuronosyltransferase (UGT) enzyme(s) responsible for hepatic glucuronidation. LZP glucuronides (R- and S-LZPG) were quantified by a stereo-selective HPLC method. Recombinant human UGT1A (viz. UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10) were expressed in HEK293 cells whereas UGT 2B4, 2B7, 2B15 and 2B17 were from a commercial source (Supersomes). R- and S-LZP glucuronidation by HLM (n=4) exhibited substrate inhibition kinetics. Mean (±SD) K

m and V

max values were 29 ± 8.5 µM and 7 ± 1.8 pmol/min.mg for R-LZPG, and 42 ± 20 µM and

11 ± 5.0 pmol/min.mg for S-LZPG. Substrate inhibition with both metabolites was weak; Ksi values were approxi-

mately 8- to 14-fold higher than Km

s. Of the twelve recombinant human UGTs screened for activity, UGT 2B4, 2B7 and 2B15 were the only hepatically expressed enzymes that catalyzed R-LZP glucuronidation. S-LZP glucuronidation was also catalyzed by UGT 2B4, 2B7 and 2B15. However, highest activity was associated with UGT2B15. At an S-LZP

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concentration of 250 µM, UGT2B15 activity was about 4-times higher than those observed with UGT 2B4 and 2B7. R-LZP glucuronidation by UGT 2B4, 2B7 and 2B15 was well described by the Hill, Michaelis-Menten, and substrate inhibition equations, respectively. In contrast, the kinetics of S-LZPG formation by UGT 2B4, 2B7 and 2B15 were well modelled using the Hill equation. Derived K

m or S

50 values for UGT 2B4, 2B7 and 2B15 catalyzed R-LZP were 43, 13

and 44 µM, while the respective S50

values for S-LZP glucuronidation were 46, 17 and 23 µM. Derived Km

or S50

values observed with recombinant UGTs and HLM were comparable. It is concluded that UGT 2B4, 2B7 and 2B15 are likely to be the major enzymes responsible for human liver microsomal R,S-lorazepam glucuronidation. S-Lorazepam appears to be a relatively selective substrate for UGT2B15.

P53. Metabolic Capacity of UDP-glucuronosyltransferase in Rat Brain

Kuniyuki Imai, Miki Katoh, Yuya Kondo and Masayuki Nadai

Faculty of Pharmacy, Meijo University, Nagoya, Japan

UDP-glucuronosyltransferase (Ugt) catalyzes the conjugation of several drugs that have pharmacological effects in the brain. We investigated the mRNA expression pattern of the Ugt1a subfamily isoforms in 9 regions of the rat brain. The results of the study indicated that the Ugt1a subfamily isoforms were expressed in the rat brain; this finding may represent an important role in drug metabolism. The present study aimed to elucidate the enzyme activity of Ugt in the rat brain. Further, we conducted kinetic analyses of Ugt activities and compared the kinetic parameters of the cerebellum and the liver. The brains from male and female Sprague-Dawley rats were divided into 9 regions: the cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, olfactory bulb, striatum, and thalamus. Each region was pooled (n = 10). Kinetic analyses of Ugt activities were performed using 2 substrates (mycophenolic acid and p-nitrophenol) in the cerebellar and liver microsomes. Mycophenolic acid O-glucuronidation and p-nitrophenol glucuronidation were detected in all the regions; however, the metabolic capacities were different for each region. These findings are indicative of the metabolism catalyzed by Ugt in the rat brain. With regard to kinetic analyses, mycophenolic acid O-glucuronidation fitted Hill′s equation. The kinetic parameters of the cerebellar and liver microsomes from the male rats were as follows: S

50, 153 µM and 536 µM, respectively; Vmax, 0.02 nmol/min/

mg and 2.65 nmol/min/mg protein, respectively; CLmax, 0.05 µl/min/mg and 2.49 µl/min/mg protein, respectively; Hill coefficient, 1.7 and 1.9, respectively. The CLmax value of the cerebellum was 0.02-fold relative to that of the liver. p-Nitrophenol glucuronidation showed biphasic kinetics on an Eadie-Hofstee plot. The kinetic parameters of high- and low-affinity components in the cerebella of the male rats were as follows: Km, 9.2 µM and 129.4 µM, respectively; Vmax, 0.5 nmol/min/mg and 4.0 nmol/min/mg protein, respectively; CLint, 58.7 µl/min/mg and 30.7 µl/min/mg protein, respectively. In contrast, the kinetic parameters of high- and low-affinity components in the livers of the male rats were as follows: Km, 83.9 µM and 582.2 µM, respectively; Vmax, 10.9 nmol/min/mg and 35.6 nmol/min/mg protein, respectively; CLint, 129.8 µl/min/mg and 61.1 µl/min/mg protein, respectively. The kinetic parameters in the cerebellum appeared to be different from those in the liver. Furthermore, sex differences in the kinetic study were also observed. We elucidated the kinetic analyses of glucuronidation in the rat cerebellum, indicating that the rat brain has significant Ugt metabolic activity.

P54. Phylogeny of arylamine N-acetyltransferases in fungi

Marta Martins, Julien Dairou, Fernando Rodrigues-Lima, Jean-Marie Dupret and Philippe Silar

Sciences du Vivant, Universite Paris Diderot, Paris, France

Previous studies have shown that Eumycetes fungi can acetylate arylamine thanks to arylamine N- acetyl transferases, xenobiotic-metabolizing enzymes also found in animals and bacteria. Here, we present the results of mining 96 avail-able fungal genome sequences for arylamine N-acetyl transferase genes and propose their phylogeny. The filamentous Pezizomycotina are shown to possess many putative N-acetyl transferases, while these often lack in other fungal groups. The evolution of the N-acetyl transferases is best explained by presence of at least one gene in the opisthokont ancestor of fungi and animal, followed by recurrent gene losses and gene duplications. A possible horizontal gene transfer event may have occurred from bacteria to the basidiomycetous yeast Malassezia globosa.

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P55. Prediction of in vivo drug-drug interactions involving UDP-glucuronosyltranferases using DDI Predict 2009® application

Patrice Dehanne, Estelle Rapine, Olivier Barberan and Cristina Lopez

Aureus Pharma, Paris, France

Drug-drug interactions (DDIs) can lead to severe side effects, drug toxicities and have resulted in refusal of approval, severe prescribing restrictions, and withdrawal of drugs from the market. Many DDIs are due to the co-administration of drugs that can alter drug metabolism. Glucuronidation is a major metabolism pathway, representing fifteen percent of the cleared drugs [1]. Significant advances have been made in the last years in the determination of UDP-glucuronosyltransferases (UGT) isoforms involved in the metabolism of compounds (recent availability of commercial recombinant enzymes, identification of probe substrates). Nevertheless, determination of UGTs in vitro parameters such as fraction metabolized (fm

UGT), metabolic intrinsic clearance (Clint or Clmax),

inhibitory constant (Ki), fraction unbound in microsomes (fu(m)

) remains a critical issue due to specificities of UGTs (latency, long-chain fatty acid inhibition, atypical kinetics). These UGTs features were taken into account in DDI Predict 2009® Edition to increase prediction accuracy. DDIs predictions involving UGT metabolized compounds were performed. Impact of UGTs contribution was discussed based on prediction of buprenorphine DDIs, an opioid partial agonist, used to treat opioid dependence. Finally, these predictions were compared to published in vivo interactions.

References

1. Williams et al, Drug Metab Dispos (2004) 32:1201

P56. Regional and Sex Differences in Expression of Ugt1a Subfamily in Rat Brain

Miki Katoh, Kuniyuki Imai, Yuya Kondo and Masayuki Nadai

Faculty of Pharmacy, Meijo University, Nagoya, Japan

UDP-glucuronosyltransferase (Ugt) is a phase II biotransformation enzyme, which catalyzes the conjuga-tion of several endogenous substrates and xenobiotic drugs. Some isoforms of the Ugt1a subfamily catalyze glucuronidation of several anti-epileptic, anti-depressant, central anesthetic, and narcotic analgesic drugs, which have pharmacological effects in the brain. To date, there are only a few reports regarding the expres-sion of the Ugt1a subfamily isoforms in the brain. It is surmised that the expression of the Ugt1a subfamily in the brain plays an important role in drug metabolism. However, it is still unclear as to which regions they are highly expressed in. In rats, the Ugt1a subfamily consists of 10 isoforms: Ugt1a1, Ugt1a2, Ugt1a3, Ugt1a4, Ugt1a5, Ugt1a6, Ugt1a7, Ugt1a8, Ugt1a9, and Ugt1a10. Ugt1a4 and Ugt1a9 do not code for functional protein and are called pseudogenes. In the present study, we aimed to elucidate the regional and sex differences in the expression levels of the Ugt1a subfamily isoforms. The brains from male and female Sprague-Dawley rats were divided into 9 regions: the cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, olfactory bulb, striatum, and thalamus. Each region was pooled (n = 5). In this study, we measured the expression levels of Ugt1a1, Ugt1a2, Ugt1a3, Ugt1a5, Ugt1a6, Ugt1a7, and Ugt1a8 mRNAs by real-time polymerase chain reaction with specific primers for each isoform. The expression level of each mRNA was corrected with that of beta-actin mRNA. All the above-mentioned isoforms were detected in all the regions of the rat brain. However, the expression levels of these isoforms were different in each region. The majority of the isoforms, particularly Ugt1a3 and Ugt1a7, showed high expression levels in the hippocampus and the medulla oblongata than in the other regions. With regard to sex differences, the expression levels of Ugt1a7 in the frontal cortex and the hippocampus in the female rats were higher than those in male rats. In conclusion, the present study provide evidence for the expression of Ugt1a1, Ugt1a2, Ugt1a3, Ugt1a5, Ugt1a6, Ugt1a7, and Ugt1a8 in the rat brain. In addition, these data indicate that sex differences in the expression levels of the isoforms of the Ugt1a subfamily likely exist.

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P57. Role of Human Glutathione S-Transferases on the Inactivation of Reactive Drug METABOLITES of Clozapine

Sanja Dragovic, Jan Simon Boerma, Laura van Bergen, Nico P. E. Vermeulen and Jan N. M. Com-mandeur

Division of Molecular Toxicology, Vrije Universiteit, Amsterdam, Netherlands

Conjugation of reactive intermediates (RIs) to glutathione (GSH) is considered an important detoxification mecha-nism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). Given this role and the high abundance of GSTs in the liver and other tissues, genetically determined deficiencies (polymorphisms) in GSTs might be risk factor for adverse drug reactions (ADRs) related to the formation of reactive drug metabolites. Although formation of multiple GSH conjugates has been demonstrated in many in vitro studies [1,2], the role of GSTs in the detoxification of RIs of clozapine, a drug causing idiosyncratic drug reactions (IDRs) in humans, has remained unexplored. Here, the role of (polymorphic) GST-isoenzymes in catalyzing the trapping of c.q. protection against RIs of clozapine by GSH was studied. The ability of four recombinant human GSTs (GST A1-1, GST M1-1, GST P1-1, and GST T1-1) on formation of clozapine GSH adducts in vitro by human and rat liver microsomes, three human recombinant P450s and a drug metabolising P450 BM3 mutant M11his was investigated. Purified M11his was selected because it is able to bioactivate CLZ to all relevant metabolites at much higher activity than human and rat liver microsomes [1]. In presence of three of the hGSTs, hGST P1-1, hGST M1-1 and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. Highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of clozapine RIs. Interestingly, addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates, previously only found in in vivo studies [2] were completely dependent on the presence of hGSTs, which explains their absence in vitro. These results demonstrated that human GSTs might play a significant role in the inactivation of RIs of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced ADRs.

References

1. Damsten, M.C., Van Vugt-Lussenburg, B.M., Zeldenthuis, T., De Vlieger, J.S., Commandeur, J.N.M., and Vermeulen, N.P.E. (2008) Application of drug metabolizing mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites. Chem.-Biol. Interact. 171, 96-107.

2. Maggs, J.L., Williams, D., Piromohamed, M., and Park, B. K. (1995) The Metabolic Formation of Reactive Intermediates from Clozapine, a Drug Associated with Agranulocytosis in Man, J. Pharmacol. Exp. Ther. 275,1463-1475.

P58. Abstract Withdrawn.

P59. UGT2A2: a human polymorphic UDP-glucuronosyltransferase with unusual properties

Nina Sneitz1, Xiuling Zhang2, Xinxin Ding2, Michael H. Court3 and Moshe Finel1

1Centre for Drug Research, University of Helsinki, Helsinki 79, Finland, 2Wadsworth Center, NYSDOH, Albany, NY, USA, 3Department of Pharmacology/M & V 308, Tufts Univ, Boston, MA

UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that catalyze the conjugation of glucuronic acid to various aglycone substrates. The 19 functional members of human UGT enzymes are divided into 3 subfamilies, 1A, 2A and 2B. We have recently characterized UGT2A2 enzyme, possibly the last orphan human UGT. UGT2A2 has never been expressed as recombinant enzyme nor have its activities been character-ized. Only its first exon was found in human genomic DNA and due to this, UGT2A2 was suggested to have exon sharing with UGT2A1 (Mackenzie et al., 2005). Exon sharing with UGT2A1 also means that UGT2A2 possesses the same polymorphisms within exons 3 and 5 that were previously found in UGT2A1 (Iida et al., 2002). Tissue

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specific RT-PCR studies were performed, and they revealed that UGT2A2 is mainly expressed in nasal mucosa, but trace amounts of UGT2A2 mRNA were also found in small intestine, lung and liver. Subsequent sequencing of RT-PCR products from human nasal mucosa mRNA confirmed the accuracy of the constructed sequence and exon sharing between UGT2A1 and UGT2A2. The UGT2A2 enzyme was expressed as recombinant protein in Sf9 insect cells and its enzymatic activities were analyzed. Based on the exon sharing proposal, full length cDNA was constructed by amplifying UGT2A2 exon 1 from human genomic DNA and ligating it with exons 2-6 of UGT2A1 that were amplified from nasal cDNA library. UGT2A2 was found to have broad substrate specificity, but the glucuronidation rates were, with few exceptions, generally low, 0.1-10 pmol/min/mg. (Sneitz et al., 2009) Interestingly, UGT2A2 was found to be largely resistant to inhibition by Triton X-100 at concentrations that abolish the activity of other human UGTs. This feature might have important applications, since the difficulties in extraction of monodispersed and fully active UGTs from membranes with detergents has so far been a major obstacle in UGT protein crystallization.

References

1. Iida A, Saito S, Sekine A, Mishima C, Kitamura Y, Kondo K, Harigae S, Osawa S, Nakamura Y (2002) Catalog of 86 single-nucleotide polymorphisms (SNPs) in three uridine diphosphate glycosyltransferase genes: UGT2A1, UGT2B15, and UGT8. J Hum Genet 47; 505-10

2. Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, Owens IS, Nebert DW (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet Genomics 15: 677-85

3. Sneitz N, Court MH, Zhang X, Laajanen K, Yee KK, Dalton P, Ding X, Finel M (2009) Human UDP-glucuronosyltransferase UGT2A2: cDNA con-struction, expression, and functional characterization in comparison with UGT2A1 and UGT2A3. Pharmacogenet Genomics 19: 923-934

P60. Evaluation of Chimeric Mice with Humanized Liver to Predict Human Intrinsic Clearance of Drug Molecules at Preclinical Phase

Guangqing Xiao, Tonika Bohnert, Cheryl Black, Lewis Klunk and Liang-Shang Gan

Department of PKDM, Biogen Inc, Cambridge, MA, USA

Chimeric mice with humanized liver are obtained via injecting urokinase-type plasminogen activator (upa+/+/) / severe combined immunodeficient (SCID) mice (20-30 days after birth) with human hepatocytes. The severe liver failure in these upa+/+ / SCID mice makes it ideal to obtain mice, which have approximately 70-80% human liver. Extent of replacement, represented by a number called RI (replacement index), of mouse liver by human liver is assessed by measuring the concentration of human albumin (hAlb) in the resulting mice blood and mice which have > 70% replacement by human liver are deemed suitable for metabolism studies. Due to similar expression of major human CYPs (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 3A4) at their comparable metabolizing capacities in the chimeric mice, this in vivo model has gained significant popularity recently to investigate human specific metabolism, CYP-inhibition, CYP-induction, effect of CYP-polymorphism, and toxicity. Correct human clearance (Cl) prediction is pivotal before initiation of any clinical trial but accurate human Cl prediction is very challenging due to significant differences in drug disposition and clearance, frequently observed between preclinical species and humans. A pilot study was initiated to evaluate this model for its ability to predict human intrinsic clearance (Clint) of four well-known drugs with a varying range of Cl reported in humans. Three of these drugs are reported to be substrates of the major human drug-metabolizing enzyme CYP3A4: antipyrine (low Cl in humans), midazolam (moderate Cl in humans), and verapamil (high Cl in humans). The fourth investigative drug, zidovudine, is a known-human UGT2B7 substrate, which demonstrates high Cl in humans but low Cl in mice, due to absence of any UGT2B7 mouse ortholog. It was ensured that the drugs under current evaluation are all hepatically cleared in humans since the goal of this study was to investigate whether human systemic Cl can be predicted from systemic Cl in chimeric mice with human liver. The preliminary results indicated that the Clint values, calculated from the in vivo Cl of the CYP3A4 substrates in the chimeric mice were significantly higher (5-20 fold) than expected human Clint values, calculated from literature reported human Cl values. In contrast, the observed Clint value of the UGT2B7 substrate in the chimeric mice was approximately 2-fold lower than the literature reported human Cl of zidovudine. In addition, the rank order of Clint values obtained from reported human Cl data (antipyrine < midazolam < verapmil < zidovudine) was also not maintained in chimeric mice (antipyrine < zidovudine < verapamil < midazolam). Studies are planned to further explore the use of chimeric mice to predict human clearance of investigative drugs.

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P61. Assessment of Effects of IR and IPC on Activities of Cytochrome P450 Isoforms in Rats by a Five-Drug Cocktail Approach

Jianshi Lou, Ying Liu, Qin LI, Jianjie Jiao and Caili Zhang

Department of pharmacology, Tianjin Medical University, Tianjin, China

Hypothesis: To assess whether changes of cytochrome P450 (CYP) activity occur in differently injured rat liver in vivo and to evaluate the changes in activities of CYP1A2, CYP2D6, CYP2E1, CYP2C9 and CYP3A4 with five probe drugs as a “cocktail”, caffeine (2.5mg/kg), metoprolol (10mg/kg), chlorzoxazone (5mg/kg), tolbutamide (2.5mg/kg) and midazolam (5mg/kg), intravenously injected in combination. Methods: “Cocktail” approach was used to evaluate the influence of ischemia reperfusion (IR) and ischemic preconditioning (IPC) on the activities of five CYP isoforms, which were indirectly reflected by the changes of pharmacokinetic parameters of 5 probe drugs respectively. Rats were randomly divided into IR, IPC and Sham groups, and then injected the mixture of 5 probe drugs through vena caudalis. Before and after injection, the blood samples were collected through inferior caval vein at a series of time-points, the concentrations of five probe drugs in plasma were measured by a HPLC method with UV detection in a single run. The pharmacokinetic parameters were calculated by the software of 3P97 (Practice Pharmacokinetic Program 1997 edition, provided by Chinese Pharmacological Society). Results: The parameters including t

1/2β, CLs, AUC and K10

exhibited similar tendency for both IR and IPC groups. Compared with Sham group, (1) CLs of five probe drugs were significantly lower in IR group (for chlorzoxazone, tolbutamide, metoprolol and midazolam, P<0.01; for caffeine, P<0.05). (2) AUC of five probe drugs were significantly increased in IR group (P<0.01). (3) T

1/2β of chlorzoxazone, metoprolol and midazolam were prolonged in IR group (P<0.01 or P<0.05). (4) K

10 of chlorzoxazone, tolbutamide, metoprolol and midazolam were significantly decreased in IR group (P<0.01 or

P<0.05). Compared with IPC group, (1) CLs of five probe drugs were decreased in IR group (for chlorzoxazone and tolbutamide, P<0.01; for caffeine, metoprolol and midazolam, P<0.05). (2) AUC of five probe drugs were significantly increased in IR group (P<0.01 or P<0.05). (3) T

1/2β of five probe drugs were prolonged but there were no statistic significance between IR and IPC group (P>0.05). (4) K

10 decreased and statistic differences were found only in

chlorzoxazone and tolbutamide between IR and IPC group (P<0.01 or P<0.05). Conclusion: (1) The pharmacokinetic parameters derived from five probe drugs should be valid indicators for evaluating drug-metabolizing functions of hepatic CYP isoforms after IR and IPC. (2) IR could decrease the activities of CYP isoforms in rats, and this decrease could be attenuated by IPC.

P62. Functional Characterization of Recombinant NADPH-P450 Reductase Protein from Candida albicans

Hyoung-Goo Park1, Young-Jin Chun2 and Donghak Kim1

1Biological Sciences, Konkuk University, Seoul, South Korea, 2College of Pharmacy, Chung-Ang University, Seoul, South Korea

Candida albicans is a major pathogenic fungus that causes opportunistic oral and vaginal infections in humans. Analysis of its genome found NCP1 gene coding a putative NADPH-P450 reductase (NPR) enzyme. It appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. Here, we report the characterization of functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein containing a 6X(His)-tag was heterologously expressed in Escherichia coli and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm indicating the feature of oxidized flavin cofactor, which was decreased by adding NADPH. It also exhibited NADPH-dependent cyto-chrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results suggest that purified C. albicans NPR is an orthologous reductase protein to support cytochrome P450 or heme oxygenase enzymes in C. albicans. (This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea, A084005).

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P63. Involvement of Rabbit Liver Cytochrome P4502B4 in the Bioreduction of Idarubicin in Reconstituted Systems Containing Highly Purified P450 Reductase and P4502B4

Haydar Çelik1 and Emel Arinç2

1Department of Food Engineering, Faculty of Engineering, Erciyes University, Kayseri, Turkey, 2Graduate Programme in Biochemistry and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

Idarubicin is a second-generation anthracycline anticancer drug which is clinically effective against breast can-cer and some hematological malignancies including acute myelogenous leukemia, multiple myeloma and non-Hodgkin′s lymphoma. Among the mechanisms proposed for the antitumor effects of anthracyclines, free radical generation via bioreductive activation and subsequent redox cycling under aerobic conditions is considered as having an important contributing role on the effectiveness of these chemotherapy agents. Free radical generation by anthracyclines involves their bioreductive activation by cellular oxidoreductases. The main objective of the present study was to investigate the possible role of cytochrome P450 in the bioreduction of idarubicin using reconstituted systems of purified rabbit liver cytochrome P4502B4 and either purified rabbit liver or beef liver cytochrome P450 reductase. In this study, all the experiments were also repeated under the same reaction condi-tions using mitomycin C, and the results were compared. Mitomycin C was chosen as a model compound because it is an extensively studied quinone-containing anticancer drug with respect to its molecular pharmacology and chemistry. The reconstitution experiments performed with varying amounts of purified rabbit liver cytochrome P4502B4 and purified rabbit liver P450 reductase under aerobic conditions revealed that reconstituted rabbit liver CYP2B4 produced about 1.5 and 1.4 fold rate enhancements in idarubicin and mitomycin C reductions catalyzed by P450 reductase alone, respectively, as shown by drug-induced NADPH oxidations. Thus, cytochrome P450 reductase, besides its direct role in catalyzing the one-electron reduction of idarubicin and mitomycin C, can transfer electrons from cofactor NADPH to cytochrome P450 which in turn may catalyze the reduction of these anticancer drugs as well. However, to what extent the electrons from NADPH are utilized by cytochrome P450 for the reduction of idarubicin and mitomycin C or for the reduction of oxygen through uncoupling of the monooxy-genase cycle is a matter of speculation. In order to clarify this point, the initial rates of idarubicin and mitomycin C reductions were determined under anaerobic conditions in reconstituted systems of purified rabbit liver CYP2B4 and beef liver P450 reductase. The data obtained from experiments under anaerobic conditions demonstrated that the relative contribution of reconstituted rabbit liver CYP2B4 to the reduction of idarubicin (as measured by drug-induced NADPH oxidation at 340 nm) and mitomycin C (as measured by the decrease at 375 nm based on the disappearance of quinone moiety of the drug) was almost the same with that observed under aerobic condi-tions. In conclusion, our findings demonstrate that rabbit liver CYP2B4, although not efficient as P450 reductase, is capable of reductively activating both idarubicin and mitomycin C which may play an important role in the cytotoxicity of these anticancer drugs.Acknowledgements: Supported by a grant from TUBITAK (Project ID: 106T139). This study was carried out in Middle East Technical University (METU).

P64. Expression and Inducibility by Phenobarbital of CYP2C33, CYP2C42 CYP2C49, CYP2B22 and CYP3As in Liver, Kidney, Small Intestine and Nasal Tissues of Pig

Emanuela Puccinelli1, Pier Giovanni Gervasi1, Margherita la Marca2, Pascale Beffy1 and Vincenzo Longo2

1Institute of Clinical Physiology, CNR, Pisa, Italy, 2Institute of Agricultural Biotechnology, CNR, Pisa, Italy

Cytochrome P450s (CYPs) play an important role in the oxidative metabolism of numerous xenobiotics as well as endogenous compounds. In humans, the CYP2C subfamily accounts for about 20% of the total hepatic CYPs and has a prominent role in the metabolism of 20-30% of all drugs [1]. Despite the fact that pig has become of interest because it has been proposed as a new model for pharmacological and toxicological studies, little information is available on its metabolic system, and in particular on its CYPs expression. In the present study, the expression and inducibility of

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CYP2C33, 2C42 and 2C49 along with CYP2B22, 3A22, 3A29 and 3A46 were investigated at activity and transcriptional level in liver, kidney, small intestine, respiratory and olfactory nasal mucosa of control and phenobarbital (PB)-treated pigs. Four male castrated Large White x Landrace hybrid pigs (25-30 kg body weight) were utilized as controls and four were treated i.p. with phenobarbital (20 mg/kg/day in water for 3 days). Total RNA was extracted, retrotranscripted and amplified by PCR using specific primers. The CYP content, along with the oxidation of testosterone and selective monooxygenase activities markers of CYP2Bs, CYP2Cs and CYP3As, were evaluated in microsomes of both liver and extra-hepatic tissues. An immunoblot experiment was carried out with polyclonal antibodies anti-human CYP2C9. The PB treatment resulted in an up-regulation of mRNA levels of all analysed CYPs in liver, of CYP2C42 and 2C49 in kidney, of CYP2C42, 2C49, CYP2B22 and CYP3As in small intestine. In liver microsomes, these transcriptional activations were accompanied by an increase of the analysed marker activities whereas, among the extrahepatic tissues, a significant induction by PB was observed only in kidney for the marker activities of CYP2C9. In liver microsomes, the PB treatment caused a strong increase of two immunoreactive protein bands with anti-human CYP2C9, indicating that at least two CYP2C enzymes are present in this tissue. Taken together, our results demonstrated that the PB administration in pig induced at least in liver, in addition to CYP2B22 and 3As, the expression of CYP2C33, 2C42 and 2C49 at transcriptional and activity levels, as reported in human liver through a regulation mechanism involving CAR, PXR and HNF4alpha [2]. Furthermore, our findings showed that the catalytic activities and the inhibition profiles of porcine CYP2Cs are different amongst themselves and with respect to the human counterparts. Thus, the use of pig as a model for humans in studies with drugs either substrates and/or inducers of CYP2Cs should be considered carefully.

References

1. Guengerich F.P., 2005. Human cytochrome P450 enzymes. In Cytochrome P450, Structure, Mechanism and Biochemistry. Third Ed. (P.R. Ortiz de Montellano Ed.) pp. 377-463. Kluwer Academic/Plenum Publishers, New York.

2. Ferguson S.S., Chen Y., LeCluyse E.L., Negishi M., Goldstein J.A. (2005) Human CYP2C8 is Transcriptionally Regulated by the Nuclear Receptors Constitutive Androstane Receptor, Pregnane X Receptor, Glucocorticoid Receptor, and Hepatic Nuclear Factor 4. Molecular Pharmacology 68: 747-757.

P65. Absolute Quantification of CYP27A1 and CYP46A1 in the Human Brain and Retina

Illarion V. Turko1, Wei-Li Liao1, Gun-Young Heo2, Rachel E. Reem2, Nathan G. Dodder3, Pier Luigi DiPatre4 and Irina A. Pikuleva2

1Center for Advanced Research in Biotechnology, Rockville, MD, USA, 2Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, USA, 3Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD, USA, 4Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA

Accumulating evidence indicates a link between cholesterol and neurodegenerative diseases, such as Alzheimer′s disease and age-related macular degeneration. It has been established that cholesterol elimination from the brain is achieved primarily via enzymatic conversion to 24S-hydroxycholesterol by CYP46A1. Cholesterol 27-hydroxylation, cata-lyzed by CYP27A1, also takes place in the brain, yet plays only a minor role in cholesterol removal. The systematic eluci-dation of cholesterol homeostasis in the retina began only recently. We have developed and optimized a nanoLC-MS/MS workflow for the absolute quantification of CYP27A1 and CYP46A1 and used this methodology to measure the target protein expression in the temporal lobe, cerebellum, and retina. The measurements were performed in multiple reaction monitoring (MRM) mode on a 4000 QTRAP (ABI/MDS-Sciex). The total membrane pellet was analyzed using 15N-labeled CYPs as internal standards. Two peptides per protein and three transitions per peptide were monitored. Quantitative results from each MRM transition were obtained and statistically compared with each other for validation. In the temporal lobe and cerebellum, the concentrations of CYP27A1 were similar at approximately 100-130 fmol of CYP/mg of total tissue protein. However, in the retina the concentration of CYP27A1 was higher and ranged from 390-470 fmol of CYP/mg of total protein. The concentrations of CYP46A1 in the temporal lobe and cerebellum were in the range of 310-390 and 70-100 fmol of CYP/mg of total tissue protein, respectively. In the retina, the concentration of CYP46A1 was in a range of 60-70 fmol of CYP/mg of total protein. These data are in good agreement with measured levels of cholesterol metabolites in the brain and retina, and demonstrate that cholesterol metabolism in the retina is significantly different from that in the brain. The described method is applicable towards the quantification of other CYPs.

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P66. Probabilistic GALAS Models For The Prediction Of The Human Cytochrome P450 Inhibition

Remigijus Didziapetris, Justas Dapkunas, Andrius Sazonovas and Pranas Japertas

ACD/Labs, Inc., Vilnius, Lithuania

This study presents a series of in-silico models for the prediction of probable inhibitors of CYP450 isoforms 3A4, 2D6, 2C9, 2C19, and 1A2 developed using a novel GALAS (Global, Adjusted Locally According to Similarity) modeling methodology. A very important feature of this modeling methodology is the possibility to quantitatively evaluate prediction quality using calculated Reliability Index (RI) values. Inhibition constant thresholds of 10 and 50 uM were used to classify compounds in the initial data sets ranging from ca. 5000 to 8000 compounds for five considered enzyme isoforms. For the predictions with acceptable RI values, the accuracy approaches 90% in all five internal test sets (20% of the corresponding initial database). All models have been externally validated using the latest data from PubChem screening program. As an example, in case of CYP3A4 inhibition, it yielded the results similar to the model testing on internal test set (88% accuracy when RI > 0.3). Model trainability feature was assessed in an attempt to train the CYP3A4 inhibition model based on literature dataset with PubChem library, while reserving half of the PubChem data as a test set. After adding 5% of the training library the number of test set predictions with acceptable reliability (RI > 0.3) was below 50% while the number of high reliability predictions (RI > 0.5) barely exceeded 10%. Subsequent additions of library portions gave a steady increase in these numbers reach-ing ca. 85% and 60% correspondingly with whole library added. Obtained models represent the valuable computational filters in early drug discovery to identify compounds that may have unwanted cytochrome P450 inhibition liability. GALAS modeling methodology utilized in this work enables fast and efficient training of the obtained models, i.e. extending their applicability domain, adjusting them to screen proprietary databases for potential CYP inhibitors.

P67. Dexamethasone Enhances CYP3A-mediated Inactivation of Active Vitamin D3 Metabolite in Mouse Liver

Subrata Deb, Mitali Pandey, Hans Adomat and Emma Guns

Vancouver Prostate Centre, Vancouver, BC, Canada

Epidemiological studies have shown that lower serum levels of vitamin D3 are associated with increased risk of prostate

cancer, colon cancer and breast cancer. 1α,25-(OH)2D

3, the dihydroxy metabolite of vitamin D

3, is the biologically active

form of vitamin D3. Inactivation of 1α,25-(OH)

2D

3 by cytochrome P450 (CYP) enzymes can be an important determinant

of it′s circulating serum and tissue levels. Patients receiving dexamethasone, a prototypical CYP inducer, as premedica-tion before administration of docetaxel experience vitamin D-deficiency like symptoms. The purpose of the present study was to compare the effects of two glucocorticoid steroids- namely dexamethasone and prednisone- on inactivation of 1α,25-(OH)

2D

3 in mice. Adult CD-1 mice were treated with either vehicle (50% ethanol), dexamethasone (80 mg/

kg/day) or prednisone (80 mg/kg/day) for three consecutive days by intraperitoneal (i.p.) injection. A different group of mice were pre-treated with dexamethasone (50 mg/kg twice a day for three consecutive days; i.p.) and then with docetaxel (10 mg/kg; single tail-vein injection) to mimic the clinical dosing regimen of docetaxel. Livers were quickly excised and were used for preparation of microsomes by differential ultracentrifugation. A liquid chromatography-mass spectrometry (LC/MS) method was developed to analyze metabolite formation and substrate depletion. In vitro reac-tion mixtures contained 100 mM potassium phosphate buffer (pH 7.4), 0.045 mg of mouse hepatic microsomal protein, NADPH-regenerating system, and varying concentrations of 1α,25-(OH)

2D

3 (2-80 µM) in a final volume of 0.3 ml incu-

bated for 30 min. A fixed concentration of 20 µM 1α,25-(OH)2D

3 was used to compare the treated samples. Incubations

were also carried out using human hepatic microsomes and human recombinant CYP3A4 (co-expressed with b5) for

comparative purposes. Hydroxy metabolites of 1α,25-(OH)2D

3 were resolved using a LC/MS on a 100 mm BEH MS C

18

column. Authentic metabolite standards of 1α,24R,25-(OH)3D

3 and 1α,23S,25-(OH)

3D

3 (obtained from Dr. Toshiyuki

Sakaki, Toyama, Japan) were used for method development and identification of metabolites. Metabolite analysis indi-cated that the Km value of hydroxy metabolite formation in pooled vehicle-treated hepatic microsomes is ∼31 µM. Hydroxy metabolite formation pattern was significantly altered with mouse hepatic microsomes from dexamethasone-treated mice. The formation of hydroxy metabolites is stimulated by approximately 50- and 2- fold with the treatment of dexamethasone in comparison to vehicle-treated and prednisone-treated groups, respectively. Co-incubation with ketoconazole, a potent mouse CYP3A inhibitor, led to ∼98% suppression of hydroxy metabolite formation in pooled vehicle-treated microsomes. Substrate depletion analysis at a lower concentration of 1α,25-(OH)

2D

3 (1.6 µM) with

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hepatic microsomes from mice receiving dexamethasone and docetaxel suggest that dexamethasone is more efficient than untreated mice in inactivating 1α,25-(OH)

2D

3. Dexamethasone-mediated enhanced depletion of 1α,25-(OH)

2D

3

was completely blocked by co-incubation with ketoconazole. Similarly, triazolam hydroxylation (1`-OH and 4-OH), a mouse CYP3A marker assay, and CYP3A protein expression were significantly increased in dexamethasone-treated microsomal samples. In summary, our results suggest that dexamethasone augments CYP3A-mediated inactivation of 1α,25-(OH)

2D

3 in mice and alters the metabolite formation profile.

P68. Influence of CYP2C9 polymorphism on metabolism of Valproate and its hepatotoxin metabolite in Iranian patients

Noushin Amini-Shirazi1, 5, Mohammad Hossein Ghahremani1, Reza Ahmadkhaniha2, Ali Mande-gary3, Armin Dadgar2, Mohammad Abdollahi1,Shahin Shadnia4, 5, Hossein Pakdaman5 and Abbas Kebriaeezadeh1

1Department of Toxicology and Pharmacology, Tehran University of Medical Sciences, Tehran, Iran, 2Department of Medicinal Chemistry, Tehran University of Medical Sciences, Tehran, Iran, 3Department of Toxicology and Pharmacology, Kerman University of Medical Sciences, Kerman, Iran, 4School of Medicine and Toxicological Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran, 5Legal Medicine Organization, Tehran, Iran

Sodium Valproate (VPA) has 16 known metabolites in human. The 2-ene-VPA has anticonvulsant efficacy and 4-ene-VPA reported to contribute in VPA hepatotoxicity. The formation of 4-ene-VPA is catalyzed by cytochrome P450 2C9 (CYP2C9). CYP2C9 Allele mutation is closely related to the attenuation of the enzymatic activity and 4-ene-VPA pro-duction. In the present work VPA, 2-ene-VPA, and 4-ene-VPA in serum of patients receiving VPA were determined and the correlation between CYP2C9 polymorphism and 4-ene-VPA concentration was examined. Blood samplings in 68 patients were performed at two time- points(peak and trough) and one sample blood obtained from 50 healthy volun-teers for genotype evaluation. Patients were divided into three groups (22 cases of monotherapy, 19 cases of enzyme inducer therapy and 27 cases of polytherapy). There was a significant reduction in concentration of VPA and 4-ene-VPA between peak and trough time. In peak concentration, there was a significant correlation between 2-ene-VPA and VPA in all groups. The concentration of 4-ene-VPA in enzyme inducer and polytherapy group was significantly higher than that of monotherapy group. The allele frequency of CYP2C9*1, CYP2C9*2 and CYP2C9*3 were 88.97%, 8.09 %, and 2.94 % in patient group and 91%, 6%, and 3% in normal group, respectively. There was no significant difference in allele frequency in two groups. Mutated alleles didn′t have any significant effect on 4-ene-VPA production. No patient showed toxic level of 4-ene-VPA or saturation of ß-oxidation pathway. In conclusion, the role of CYP2C9*2 and CYP2C9*3 in attenuation of 4-ene-VPA formation cannot be confirmed.

P69. The inhibitory effect of quercetin on rat hepatal CYP2D2

Ondrej Zendulka, Jan Jurica and Alexandra Sulcova

Department of Pharmacology, Masaryk University, Brno, Czech Republic

Quercetin is one of the most abundant polyphenolic compounds in human diet and is also included in dietary supple-ments with many proven beneficial effects. Some of them are explained by changes in CYP activity as quercetin is known as a potent cytochrome P450 (CYP) 1A2 inhibitor. Thus, there is a high probability of interaction with metabolism of drugs when quercetin intake is high and co-administered drugs are substrates for particular CYP isoenzymes. Although such interactions can be of high clinical relevance the influence of quercetion on specific CYP isoenzymes is not described in details yet. The aim of present work was to evaluate the influence of quercetin on the activity of cytochrome P450 (CYP) 2D2 isoenzyme in rats, which is an orthologue enzyme of human CYP2D6. We determined the effect of intraperitoneal 10 day quercetin administration on the marker for CYP 2D2 activity - dextrometorphan biotransformation in the model of isolated perfused rat liver. Quercetin was dissolved in 30% dimethylsulfoxide and was administered at the dose of 5 mg/kg/day to adult Wistar albino male rats. Control group was administered 30% dimethylsulfoxide solution alone. Levels of dextromethorphan (DM) and CYP2D2 specific metabolite dextrorphan (DX) were measured in the 30th, 60th

74

and 120th minute of perfusion. Metabolic ratios DM/DX were also calculated and compared. Quercetin administration significantly (P < 0.01) elevated the levels of DM in the perfusion medium. The increases were represented by: 78.5 % in the 30thmin, 175.3 % in the 60thmin and 626.1 % in the 120thmin of the perfusion. This fact was not reflected by the concentrations of metabolite DX as it was decreased only in the 30th minute. The metabolic ratios, the more accurate parameter for the CYP2D2 metabolic activity, were also significantly different (P< 0.05) through all monitored intervals. Values of metabolic ratios were decreased in the control animals indicating inhibition of metabolic activity caused by quercetin. We conclude that quercetin is a potent inhibitor of CYP 2D2 in rats and its pharmacokinetic interactions with other drug from CYP 2D family substrates are highly probable and may have clinical impact on individualization of optimal dosing regimen.

The work was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic (MSM 0021622404).

P70. Humanized CYP3A Mice: (1) Construction of Humanized MODEL Mice CONTAINING the CYP3A Cluster for Drug Screening

Yasuhiro Kazuki1, Satoshi Abe1, Masato Takiguchi1, Hidetoshi Hoshiya1, Naoyo Kajitani1, Toko Yoshino1, Shoko Takehara1, Chie Ishihara1, Kinya Kubo1, Takeshi Oshima2, Kazuma Tomizuka2, Sasitorn Aueviriyavit3, Kaoru Kobayashi3, Kan Chiba3, Yasuko Tsukazaki4, Naoto Senda4 and Mit-suo Oshimura1

1Graduate School of Medical Science, Tottori University, Yonago, Japan, 2Inovative Drug Research Laboratories, Kyowa Hakko Kirin CO. Ltd, 3Graduate School of Pharmaceutical Sciences, Chiba Univestiry, Chiba, Japan, 4Analytical Technology Research Department, Mitsubishi Chemical Medience Corporation, Tsukuba, Japan

Human CYP3A is the most abundant P450 isozyme present in human liver and small intestine, and metabolites around 50% of medical drugs on the market. In order to generate ‘humanized′ trans-chromosomic (TC) mice with the human CYP3A cluster for drug screening, a human artificial chromosome (HAC) containing the CYP3A cluster (CYP3A-HAC) was constructed using the Cre/loxP-mediated chromosome-cloning system. The CYP3A-HAC was then introduced into mouse embryonic stem (ES) cells using the microcell-mediated chromosome transfer (MMCT) technique, and healthy chimeric mice containing the CYP3A-HAC were generated from the ES cells, and the CYP3A-HAC was transmitted through germline. CYP3A4, CYP3A5, and CYP3A7 on the CYP3A-HAC were expressed mainly in the liver and intestine of the CYP3A-HAC mice, consistent with the expression profile in humans. Furthermore, adult specific CYP3A4 and fetal specific CYP3A7 were expressed in adult stages and fetal stages, respectively. Dehydroepiandrosterone (DHEA) was metabolized to 16alpha-hydroxy dehydroepiandrosterone (16alpha-OH DHEA), one of CYP3A7-specific metabolites by CYP3A-HAC fetal mouse liver microsome but not observed 7beta-OH DHEA, one of major metabolites by CYP3A4. Next, to investigate the CYP3A function, the CYP3A-HAC was introduced in the endogeneous Cyp3a-knock out (KO) background. In this meeting, our colleagues will present the functional analysis using the CYP3A-HAC/Cyp3a KO mice. In conclusion, the TC mice containing the full CYP3A cluster are the first model mice showing the tissue-specific and developmental stage-specific expression of the CYP3A genes, and the functional expression of the CYP3A genes. These mice may provide a useful model to test novel drugs metabolized by the CYP3A enzymes.

P71. Humanized CYP3A mice: (3)Metabolism of CYP3A4 substrates detected by LC/MS system

Yasuko Tsukazaki1, Manabu Nishii1, Naoto Senda1, Kazuki Yasuhiro2, Kinya Kubo3, Kaoru Kobayashi4, Kan Chiba4, Shigeru Yamada5, Tetsuo Kokaji5, Masakazu Takahashi6, Hiroshi Igarashi6 and Mitsuo Oshimura2

1Analytical Technology Research Department, Mitsubishi Chemical Medience Corporation, Tsukuba-shi, Ibaraki, Japan, 2Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan, 3Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan,

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4Graduate School of Pharmaceutical Sciences, Chiba University, Chuo-ku, Chiba, Japan, 5K. K. AB Sciex., Chuo-ku, Tokyo, Japan, 6GL Sciences Inc., Iruma, Saitama, Japan

We evaluated the metabolic profiles of several CYP3A4 substrates which were metabolized by the humanized CYP3A transchromosomic mouse (CYP3A-HAC/Cyp3a KO mouse) liver microsome. It is confirmed that this mouse has human CYP3A gene cluster (CYP3A4, CYP3A5, CYP3A7, CYP3A43) (Kazuki, Y. et al., a poster presenta-tion in this meeting). Metabolites of typical substrates for CYP3A4 (triazolam, midazolam, and testosterone) were detected by an LC-MS/MS system (AB SCIEX QTRAP® 5500 System equipped with the turbulence flow system, produced by GL Sciences Inc. made to order). Compound-specific predicted MRM methods using Lightsight® Software were applied in order to detect the related metabolites from substrate described the above. Samples were digested by the CYP450 Protein Assay Kit from AB SCIEX, and the protein amounts of human CYPs 1A2, 2B6, 3A4 and 3A5, were quantified using MRM Pilot™ and MultiQuant™ Software. Using the predicted MRM methods, the triazolam metabolites, 1′-hydroxytriazolam and 4-hydroxytriazolam, were detected from every analyte metabolized by human liver microsome, wild type mouse liver microsome, Cyp3a KO mouse liver micro-some, or CYP3A-HAC/Cyp3a KO mouse liver microsome. Exact masses of those metabolites, metabolized by CYP3A-HAC/Cyp3a KO mouse liver microsome were measured using enhanced resolution mass scanning. We assumed that those were triazolam metabolites. –Although, the “assumed” exact masses were slightly differ-ent from the “estimated” exact masses (-6.108 ppm and -4.715 ppm, respectively), those were acceptable range of error. The metabolites of midazolam and testosterone, namely 1′-hydroxymidazolam, 4-hydroxymidazolam, and 6b-hydroxytestosterone, were detected from the analytes which were prepared from samples including CYP3A-HAC/Cyp3a KO mouse liver microsome. The “assumed” exact masses to these metabolites were also slightly different from the “estimated” exact masses (4.982 ppm, -0.5689 ppm, and -3.3268 ppm, respectively). Those “assumed” metabolites had the same Retention times and MS/MS spectra to referential standards of each metabolite. Furthermore, we measured the amount of several kinds of human CYP isoforms in each kind of liver microsome. Human CYP3A4 and CYP3A5 were detected from CYP3A-HAC/Cyp3a KO mouse liver microsome. No human CYPs 3A4 and 3A5 in wild type mouse liver microsomes and in Cyp3a KO mouse liver microsomes were also detected. In conclusion, our examination supported that a part of human CYP3A4 metabolic pathway was expressed in CYP3A-HAC/Cyp3a KO mice.

P72. CYP2E1 INDUCTION and Oxidative STRESS IN CEREBELLAR Granule Neurons

Ana Carolina Valencia-Olvera1, Julio Morán2, Rafael Camacho-Carranza1 and Jesús Javier Espino-sa-Aguirre3

1Departamento de Toxicología Ambiental y Medicina Genómica, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico, 2Neurodesarrollo y Fisiología, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico, 3Departamento de Toxicología Ambienta y Medicina Genómica, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico

Although CYP content in brain is low as compared to hepatic levels, the bioactivation of xenobiotics into oxidants or radicals within the brain is relevant, considering the limited regenerative capacity of the neurons and its vulnerability to oxidative damage. CYP2E1 is considered the most important isoform that is able to form reactive oxygen species leading to lipid peroxidation and cell death. The aim of this study was to determine if CYP2E1 induction by xenobiotics is capable to induce oxidative damage. Exposition of primary neuron cultures to different inducers of CYP2E1 (ethanol, isoniazid and acetone) was traduced in a CYP2E1 increase as determined by immunocitochemistry. This increase was accompanied by a slight production of reactive oxygen species (ROS) and cell death. On the other hand, treatment of cells with buthionine sulfoximine, an agent that reduces glutathione levels, resulted in a significant increase in ROS and cell death after isoniazid treatment. This effect was reverted by simultaneous exposure to diallyl sulfide (CYP2E1 inhibitor) or to the antioxidant MnPTyP. These results suggest that CYP2E1 is a potential promoter of neuronal oxidative damage. Further experiments are planned to elucidate the mechanisms by which CYP2E1 induction could progress in oxidative brain toxicity.

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P73. Cryopreserved HUMAN Hepatocytes as A MODEL for the EVALUATION of TOXICITY and Drug-Drug Interactions

Lysiane Richert1, Audrey Baze1, Coraline Desbans1, Celine Parmentier1, Bruno Heyd2, Georges Mantion2, Philippe Bachellier3, Jean-Christophe Weber4, Thomas Zacharias5, Sandrine Simon6, Anna-Lena Ungell7, Pieter Annaert8 and Eliane Alexandre1

1KaLy-Cell, Illkirch, France, 2Centre de Transplantation Hépatique, Service de Chirurgie Viscérale et Digestive, Besançon, France, 3Hôpital de Hautepierre, Centre de Chirurgie Viscérale et de Transplantation, Strasbourg, France, 4Clinique de l′Orangerie, Strasbourg, France, 5CHU Mulhouse, Mulhouse, France, 6DMPK Department, F. Hoffmann-La Roche Ltd., Basel, Switzerland, 7DMPK and Bioanalytical Chemistry, AstraZeneca R&D, Mölndal, Sweden, 8Laboratory for Pharmacotechnology and Biopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium

Human hepatocytes are recognized as an excellent model for predicting drug safety in man. The limited access of fresh human hepatocytes has driven the development of use of Cryopreserved Human Hepatocytes (CryoHeps). We would like to state that, when used in suspension in 96-well plates under optimal shaking conditions as recently described (Simon et al., 2009), CryoHeps display equivalent CYP activities to their fresh human hepatocytes coun-terparts. Similarly, intrinsic clearance of a series of reference compounds obtained using suspended CryoHeps were equivalent to intrinsic clearance in the suspended fresh hepatocytes of origin. In addition, pools of CryoHeps display mean uptake transporter activities, CYP activities and intrinsic clearances corresponding to calculated averages. We can also show that CryoHeps appear to be suitable for studying drug-drug interactions, i.e. inhibi-tion studies with suspended CryoHeps and induction studies with plateable CryoHeps. Finally, we show that CryoHeps are useful for the screening of metabolism-dependent cytotoxicity and the determination of oxidative stress-related toxicity.

P74. Bupropion Hydroxylation As A Selective Marker Of Rat Cytochrome P450 2B1 Catalytic Activity

Dumrongsak Pekthong1, Alexandre Bonet2, Lysiane Richert1 and Hélène Martin2

1KaLy-Cell, Strasbourg, France, 2UFR des Sciences Médicales et Pharmaceutiques Besançon, Laboratoire de Toxicologie Cellulaire, Besançon, France

Benzyloxyresorufin-O-dealkylation (BROD) is usually used as a specific marker of CYP2B1 in rat. However, some pub-lished results show that CYP1A2 is also highly implicated. In a previous study1 (Richert et al., 2009), we found a poor correlation between BROD activity and CYP2B1 mRNA expression in rat hepatocytes after 24 and 72h of culture. By contrast, bupropion (BUP) hydroxylation correlated well with CYP2B1 mRNA expression at both time points. The pur-pose of the present study was to establish BUP hydroxylation, but not BROD, as a selective in vitro marker of CYP2B1 catalytic activity. IC50 for BROD and BUP hydroxylation were equivalence (40.8 and 41.8 µM, respectively) when using liver microsomes from phenobarbital (PB)-pretreated rats in the presence of metyrapone, a CYP2B1-selective inhibitor. However, when using liver microsomes from rats pretreated with β-naphthoflavone (BNF), in the presence of CYP1A1/2-selective inhibitor α-naphthoflavone, we found an IC50 of 2.5 10−3 µM for BROD while >100 µM for BUP hydroxylation. These results suggest that CYP2B1 is similarly involved in both activities, whereas CYP1A1/2 is involved in BROD activ-ity, but not in BUP hydroxylation. BUP hydroxylation was assessed in microsomes from baculovirus-infected insect cells (SUPERSOMES) co-expressing NADPH-P450 oxidoreductase and CYP1A1, CYP1A2, CYP2A1, CYP2A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2C13, CYP2D1, CYP2D2, CYP2E1, CYP3A1 or CYP3A2 using BUP at 500 µM. We found that BUP hydroxylation was predominantly catalyzed by CYP2B1 (3.84 pmol/min/pmol CYP) whereas low activity was detected with CYP2C6 and CYP2C11 (0.29 and 1.39 pmol/min/pmol CYP, respectively) and activity was almost undetectable with the other CYP isoforms. Finally, the mean apparent Km for BUP hydroxylation in liver microsomes from PB-treated rats (163 µM) was close to the Km for cDNA-expressed CYP2B1 (148 µM). Taken all together, these results demonstrate the selectivity of BUP hydroxylation by CYP2B1, thereby validating its use as a diagnostic in vitro marker of CYP2B1 catalytic activity in rat.

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References

1. L. Richert, G. Tuschl, C. Abadie, N. Blanchard, D. Pekthong, G. Mantion, J.-C. Weber, S.O. Mueller (2009). Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes. Toxicology and Applied Pharmacology. 235, 86–96.

P75. Metabolic Ratio of Dextromethorphan/dextrorphan in CYP2D6 Phenotyping - Identification of Cut-off Values in Serum and Correlation With Urine Levels

Jan Jurica1, Ondrej Zendulka1, Richard Bartecek2, Alexandra Zourkova2, Eva Flodrova3, Jana Zrustova3 and Alexandra Sulcova4

1Department of Pharmacology, Masaryk University, Faculty of Medicine, Brno, Czech Republic, 2Department of Psychiatry, Faculty Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic, 3Department of Medical genetics, Faculty Hospital Brno, Brno, Czech Republic, 4Department of Pharmacology, Masaryk University, Brno, Czech Republic

CYP2D6 plays a pivot role in metabolism of xenobiotics, especially psychotropic drugs. Predicting its metabolic activ-ity in patients may help to personalize pharmacotherapy.Eight-hour urinary ratio of dextromethorphan/dextrorphan molar concentrations (MR

DEM/DOR) serves as a measure of CYP2D6 activity with 0.3 as a cut-off value separating poor

(PM) and extensive metabolizers (EM) (1). Unfortunately, collecting of urine for 8 hours may be difficult in psychiatric patients. The assessment of MR

DEM/DOR in serum is less used since correlation with urine MR

DEM/DOR and cut-off value in

serum is not well established. The aim of this study was to find out correlation between MRDEM/DOR

in serum and urine and to identify cut-off value separating EM from PM and possibly also EM from intermediate metabolizers (IM) and IM from PM. Correlation was done in 28 healthy volunteers, blood samples were withdrawn 3 hours after ingestion of 30 mg of dextromethorphan hydrobromide and urine was collected for 8 hours. Concentrations of dextromethor-phan and dextrorphan in serum and urine samples were measured and MR

DEM/DOR was calculated. Cut-off value was

assessed from CYP2D6 genotype and serum MRDEM/DOR

levels in 41 subjects. Capability of serum MRDEM/DOR

levels to discriminate EM from IM, EM from PM and IM from PM was evaluated using ROC (Receiver-Operator-Characteristic) curve statistical analysis. Cut-off values were assessed on the basis of greatest AUC of ROC curve. Both MR

DEM/DOR in

serum and in urine correlated strongly (r2= 0.929). Cut-off value was found to be 0.0638 for discrimination PM from EM, 0.0208 for discrimination EM from IM, and 0.741 for discrimination IM from PM. Strong correlation between serum and urine MR

DEM/DOR support the suggestion that it is possible to assess CYP2D6 metabolic phenotype from MR

DEM/DOR

in serum. Based on the AUC values of ROC curve analysis, the cut-off value separating EM from PM seems to be reli-able, however, the cut-off values separating IM from PM and EM from IM should be further specified on the basis of a greater number of subjects analyzed. This simpler method of CYP2D6 phenotyping (single collection of blood) is very convenient, especially in psychiatric patients, and could help clinicians to adjust the pharmacotherapeutical dosing schedule according to the patient phenotype.

References

1. Lotsch J, Rohrbacher M, Schmidt H, Doehring A, Brockmoller J, Geisslinger G. Can extremely low or high morphine formation from codeine be predicted prior to therapy initiation? Pain 2009;144: pp.119-24.

The study was supported by the grants of Czech Ministry of Health No. NS 9676-4/2008 and by Czech Ministry of Education Project MSM 0021622404.

P76. Abstract Withdrawn.

P77. The “Albumin Effect” on In vitro Kinetics of Various Cytochrome P450-Catalyzed Reactions: In vitro to In vivo Extrapolation

Nitsupa Wattanachai1, Wichittra Tassaneeyakul1, Verawan Uchaipichat2 and John O. Miners3

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1Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 2Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand, 3Department of Clinical Pharmacology, Flinders University School of Medicine, Adelaide, Australia

The addition of bovine serum albumin (BSA) to in vitro incubations improves estimation of kinetic parameters, intrinsic clearance (CL

int) to in vivo clearance, mainly via a reduction of the K

m for drugs metabolized by cytochrome

P450 2C9 (CYP2C9). The universal effect of BSA on other CYP isozymes, however, is unclear. The aims of this study were to characterize the effect of BSA on kinetics of specific pathways for CYP2C8, 2C19, 2E1, and 3A4 whether the inclusion of BSA could improve the prediction of in vivo clearance from in vitro kinetics data. Using human liver microsomes as an enzyme source; 6α-hydroxypaclitaxel, 5-hydroxyomeprazole, 6-hydroxychlorzoxazone and omeprazole sulfone formations were used as markers for CYP2C8, 2C19, 2E1, and 3A4 activity. In the presence of 2% BSA, the mean CL

int for paclitaxel 6α-hydroxylation was significantly increased by decreasing K

m with a minor

effect on mean Vmax

. In the presence of 2% BSA, the mean Km1

and Vmax1

for omeprazole 5-hydroxylation were decreased which resulted in increases in the mean CL

int1. BSA decreased CL

int for chlorzoxazone 6-hydroxylation

mainly via an increase of the Km

. The mean Km

and Vmax

for CYP3A4-mediated omeprazole sulfoxidation were sig-nificantly increased in the presence of 2% BSA and therefore the mean CL

int was not significantly changed. Taken

together, extrapolation of CLint

values for CYP2C8-mediated paclitaxel 6α-hydroxylation in the presence of 2% BSA were more predictable as compared to in vivo clearance. In conclusion, the effect of albumin on individual CYP isoforms was variable; the use of BSA to improve prediction of in vivo intrinsic clearance seems to be possible with only CYP2C8. BSA showed a minor effect on the CYP2C19-mediated omeprazole 5-hydroxylation whereas it had no effect on CYP2E1-mediated chlorzoxazone 6-hydroxylation and 3A4-mediated omeprazole sulfoxidation for improving clearance prediction.

P78. A simple sampling strategy to assess activity of CYP3A in vivo using deoxyschizandrin as a probe

Jing-jing Wu1, Yan-Yan Zhang2, Ying Hu2, Si-Cheng Liang2 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

The common method used for the determination of phenotyping of drug-metabolizing enzymes in vivo is using the parent compound depletion curve method to obtain the total clearance (CLt) under first-order (linear) conditions. CLt indirectly reflected the intrinsic clearance (CLint), a parameter representing the metabolic enzymes (or/and transporters) activity. However, such total clearance is the surrogate of the catalytic activity of a certain enzyme (CLint,met) only under these limitations: ¢Ù hepatic metabolism is the primary clearance pathway; ¢Ú hepatic clearance (CLh) is enzyme-capacity limited rather than blood-flow limited; ¢Û hepatic uptake is not a rate-limiting step in the elimination. An alternative approach was suggested where the CLint is substituted for the approximate maximal initial formation rate (Vmax, app) of a specific reaction metabolite in vivo, which can accurately reflect the specific enzyme activity and exclude other enzyme and non-metabolism factors. In contrast to the traditional method, this novel method is in need of saturated substrate concentration (¡Ý 5 Km generally) to acheive zero-order (non-linear) conditions. Our previous data have demonstrated the deoxyschizandrin (DS) was selectively metabolized to schizandrin (SZ) by CYP3A in human and rat. Applying the Vmax, app determination method, zero-order SZ formation kinetic period of 10∼60 min in perfusate can be found at saturated substrate concentrations (40∼85 ¦ÌM) with recirculating perfused rat livers. In addition, albumin added to the perfusate did not affect the Vmax, app, in situ. In vivo results indicated that linear formation of SZ in plasma can be measured up to 15∼25 min after intravenous administration of DS (5, 10, 25 mg/kg). Excellent correlation was observed among Vmax, in vitro (54.89¡À4.24 nM/min) with rat liver microsomes, Vmax, app, in situ (63.4¡À6.78 nM/min) with perfused rat livers and Vmax, app, in vivo (63.48¡À6.58 nM/min/kg) with rat. We speculated that at least two blood samples would be needed to determine the Vmax, app in vivo. In conclusion, a simple sampling strategy using DS as a probe was developed to assess activity of CYP3A in vivo.

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P79. Characterization of in vitro bioactivation of rutaecarpine in human liver microsomes (HLMs)

Zhong-Ze Fang1 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Reactive drug metabolites have been always considered mediators of drug-induced toxicities and risk would increase with the increasing reactive metabolites. Rutaecarpine is a main alkaloid isolated from Evodia rutaecarpa (Wu-chu-yu), which has been used as a herbal medicine for the treatment of gastrointestinal disorder and headache. Previous reports have demonstrated that rutaecarpine mainly underwent cytochrome P450 (CYP)-mediated oxidative metabolism. Of significant interest in many biotransformation is the detection and characterization of 3-hydroxyrutaecarpine which might undergo a two-electron oxidation leading to formation of an electrophilic quinine imine intermediate. To test these hypotheses, we examined the bioactivation potential of rutaecarpine in human liver microsomes (HLMs) and recombinant CYP isoforms. LC-MS/MS analysis of extracts of human liver microsomal incubations containing rutae-carpine (200 μM), NADPH and GSH revealed the addition of a glutathionyl moiety to the 3-hydroxyl metabolite of ruteacarpine. Among tested recombinant CYP isoforms, CYP3A4 had the highest activity to catalyze the bioactivation of rutaecarpine. CYP1A2 and CYP2D6 also catalyzed the formation of GSH adduct of ruteacarpine. Previous studies showed that ruteacarpine exhibit mechanism-based inhibition towards CYP3A4 (Iwata et al., 2005). Ruteacarpine-induced immunosuppression was proposed to be associated with its bioactivation (Jeon et al., 2006). The present results might provide a potential explanation for these two phenomena.

P80. Characterization of in vitro bioactivation of Noscapine in Human Liver Microsomes

Zhong-Ze Fang1 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Aims: To investigate the formation of catechol metabolite and corresponding GSH conjugates of noscapine and to identify the CYP isoforms involved in the formation of the catechol intermediate.

Methods: In vitro HLM incubation system in the presence or absence of NADPH and reduced glutathione was utilized to investigate the formation of catechol metabolite and corresponding GSH adduct. Chemical inhibition study and recombinant CYP isoforms assay were employed to identify the enzymes involved in the formation of noscapine′s catechol metabolite.

Results: LC-MS/MS analysis of samples of human liver microsomal incubations containing noscapine (200 μM), NADPH, and GSH revealed the presence of catechol metabolite and corresponding GSH conjugate of noscapine. The generation of catechol metabolite was NADPH-dependent and the formation of GSH adduct was NADPH- and GSH-dependent. The generation of catechol metabolite catalyzed by HLMs obeyed the typical Michaelis-Menten kinetics and the kinetic parameters (K

m and V

max) were 8.2 ± 0.9 μM and 335.5 ± 13.1 pmol/min/mg pro, respectively. A combination

of chemical inhibition study and recombinant CYP isoforms assay demonstrated that CYP3A4 and CYP2C9 were the enzymes involved in the formation of noscapine′s catechol metabolite.

Conclusions: Biotransformation of noscapine by CYP3A4 and CYP2C9 leads to a catechol intermediate which could undergo further oxidation to ortho-quinone which spontaneously reacts with reduced glutathione.

P81. Simultaneous Evaluation of Influence of Scutellaria Baicalensis Georgi on CYP450 by Cocktail Probe Drugs

Qin Li, Jianjie Jiao, Xiaowen Han and Jianshi Lou

Department of pharmacology, Tianjin Medical University, Tianjin, China

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Simultaneous Evaluation of Influence of Scutellaria Baicalensis Georgi on CYP450 by Cocktail Probe Drugs Hypothesis: To evaluate the variations in activities of cytochrome P450 (CYP450) isoforms CYP1A2, CYP2D6, CYP2E1 and CYP 2C9 with four probe drugs as a ¡°cocktail¡±, caffeine (2.5mg/kg), metoprolol (10mg/kg), chlorzoxazone (5mg/kg) and tolbutamide (2.5mg/kg), intravenously injected in combination after administration of Scutellaria baicalensis Georgi in rats.

Methods: The rats were randomly divided into two groups, the control group and test group. The rats in the test group were given Scutellaria baicalensis Georgi plant extracts once a day orally for seven days, while the rats in the control group were given normal saline orally. On the eighth day, the cocktail of 4 probe drugs was intravenously injected to all the rats. Before and after injection, the blood samples were collected through inferior caval vein at a series of time-points and the concentrations of four probe drugs in plasma were determined by a HPLC method with UV detection in a single run. The main pharmacokinetic parameters were calculated by the DAS 2.0 software (Drug And Statistics 2.0 edition, provided by Chinese Pharmacological Society).

Results: The AUC and t1/2 of coffein were 55.83 ¡À 6.91 ¦Ìg/ml/h and 9.02 ¡À 1.80 h in test group, which were significantly higher than those in control group ( 40.31 ¡À 6.52 ¦Ìg/ml/h and 7.47 ¡À 2.40 h, P<0.05 ). The CL of coffein was significantly lower in test group than that in control group ( 0.044 ¡À 0.01 L/kg/h vs 0.067 ¡À 0.01 L/kg/h, P<0.05 ). Meanwhile, the AUC and t1/2 of tolbutamide were significantly lower and CL of tolbutamide was significantly higher in test group than those in control group ( 151.61 ¡À 14.52 ¦Ìg/ml/h vs 189.87 ¡À 24.32 ¦Ìg/ml/h; 10.97 ¡À 1.71 h vs 12.22 ¡À 2.54h; 0.017 ¡À 0.002 L/kg/h vs 0.014 ¡À 0.002L/kg/h; P<0.05). There were no significant differences for the main pharmacokinetic parameters of chlorzoxazone and metoprolol between these two groups (P>0.05).

Conclusion: Scutellaria baicalensis Georgi can inhibit the activity of CYP1A2 and induce the activity of CYP2C9, but has no influence on the activities of CYP2E1 and CYP2D6. This work was supported by a grant from Tianjin Natural Science Foundation key project of China, No. 09JCZDJC21500.

P82. Engineering Bacillus megaterium CYP102A1 Mutants into a Prototype with Human P450 Enzyme Activity

Sun-Ha Park, Dong-Hyun Kim and Chul-Ho Yun

School of Biological Sciences and Technology, Chonnam National University, Gwangju, South Korea

It is well known that human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. P450 BM3 (CYP102A1) enzyme from Bacillus megaterium is a native fusion protein with a mammalian NADPH-P450 reductase-like domain. It is also found that some P450 BM3 mutants could oxidize several human P450 substrates. In this study, we examine the possibility that bacterial P450 BM3 mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction (PCR) was carried out on the heme domain-coding regions of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into E.coli for expression of the CYP102A1 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level with fluorescent substrates, some of the blue colonies, but not the white colonies, showed apparent oxidation activity toward typical human P450 substrates, including coumarin, 7-ethoxycoumarin, phenacetin, chlorzoxazone and testosterone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational find-ings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

References

[1] S. H. Park, D. H. Kim, D. Kim, H. C. Jung, J. G. Pan, T. Ahn, C. H. Yun, Drug Metab Dispos 2010, 38, 732.[2] D. H. Kim, T. Ahn, H. C. Jung, J. G. Pan, C. H. Yun, Drug Metab Dispos 2009, 37, 932.[3] D. H. Kim, K. H. Kim, D. Kim, H. C. Jung, J. G. Pan, Y. T. Chi, T. Ahn, C. H. Yun, Journal of Molecular Catalysis B-Enzymatic 2010, 63, 179.[4] C. H. Yun, K. H. Kim, D. H. Kim, H. C. Jung, J. G. Pan, Trends Biotechnol 2007, 25, 289.

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P83. High level expression of cytochrome P450 2C in white blood cells of mouse and rats induced by type I diabetes

Ki Tae Park1, Eun Yi Cho1, Taeho Ahn2 and Chul-Ho Yun1

1School of Biological Sciences and Technology, Chonnam National University, Gwangju, South Korea, 2School of Biological Sciences and Technology, Chonnam National University, Gwang-Ju, South Korea

We investigated the phenotype changes of cytochrome P450 (CYP) enzymes in white blood cells of mouse and rats with type I diabetes induced by streptozotocin(STZ). STZ specifically induced the high level expression of CYP2C type in the microsomal fractions of the rodent WBCs compared to that of control revealed by immunoblotting and quantitative RT-PCR. The enzyme activity was increased by about 5-fold as well. The specific induction of CTP2C was also confirmed by using 2-D electrophoresis and mass spectroscopy. However, other CYP types were not changed when the RT-PCR was perfomed for CYP1A1/2, CYP2A6, CYP2B1/2, CYP2D6, CYP2E1, and CYP3A2. These result suggest that CYP2C can be used as specific enzyme marker of type I diabetes although the experiment was not undertaken with human samples and for other CYP types.

P84. Interactions between Di-n-Butyl- (4-Chlorobenzohydroxamato) Tin(IV)Chloride (DBDCT), a New Antitumor Compound with Rats Liver Cytochrome P-450

Qing-Shan Li, Li-Feng Zhang and Yao-Dong Ping

School of Pharmaceutical Sciences, Shanxi Medical University, Taiyuan, China

Background DBDCT is a new antitumor diorganotin(IV) compound. Our previous studies showed that DBDCT has strong antitumor activity against S

180, H

22 and EAC via intravenous injection. Tumor cells apoptosis induced by DBDCT

may be the p53 apoptosis signal conductive pathway. The apoptosis of SGC-7901 cells induced by DBDCT was related to the mitochondria and death receptor pathway. The biotransformation pathways of DBDCT in vivo perhaps comprise of hydrogen reduction, ethylization reaction, glucuronides and elimination process of ligand and ethyl. Through hepatic microsomal enzyme, DBDCT was gradually metabolized and eventually eliminated from kidney and bile. Objective The present studies were to characterize subtype enzymes of cytochrome P450 (CYP450) in the metabolism of DBDCT and evaluate the effects of DBDCT on CYP450. It is significant to understand the metabolic mechanism of DBDCT, and predict pharmacokinetic drug-drug interactions. Method Liver microsomes of rats induced by dexamethasone (Dex), Phenobarbital (PB), b-Naphthoflavone (BNF) and DBDCT were prepared by using ultracentrifuge method. Metabolic rate of DBDCT was determined by incubation with different rats liver microsomes. Specific CYP450 inhibitors were added in the incubation system to characterize subtype enzymes of CYP450 in the metabolism of DBDCT. The effects of DBDCT on the activiation of CYP450 were evaluated by substrate experiments. Results Metabolic rate of DBDCT in vitro in different enzyme sources showed that, PB, DEX group had significant deviation compared with the control group, and BNF group had no significant deviation compared with the control group. In the inhibitory experiment, ketocona-zole showed strong inhibitory effect on metabolism of DBDCT, sulfamethoxazole also had some inhibitory effect, and inhibition of fluvoxamine, trimethoprim, and omeprazole on the metabolism of DBDCT was weak. In the substrate tests, the metabolic rate of nifedipine in DBDCT treated rat liver microsomes had significantly deviation compared with the control group. The metabolic rate of phenacetin, Diclofanac Sodium and omeprazole had no significant difference compared with the control group. Conclusions CYP3A is the chief subtype enzymes of cytochrome P450 that involved in the metabolism of DBDCT. CYP2C may partly participate in metabolic reactions. CYP1A has no significant catalytic effect. DBDCT has strong inhibitory effect on CYP3A, and has no significant effect on CYP1A, CYP2C9 and CYP2C19.

P85. Hepatic Ischemia-Reperfusion Injury Impairs Drug Metabolism and Treprostinil, A Prostacyclin Analog, Improves the Functional Capacity of the Liver Graft Post-Transplantation

Nisanne Ghonem1, Wenchen Zhao1, Shimin Zhang1, Noriko Murase2, Junichi Yoshida2, Donna B. Stolz3, Abhinav Humar2, Thomas E. Starzl2 and Raman Venkataramanan4

1Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA, USA, 2Surgery, University of Pittsburgh, Pittsburgh, PA, USA,

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3Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA4Pharmaceutical Sciences, Pathology, University of Pittsburgh, Pittsburgh, PA, USA

Background: The expression and activities of cytochrome P450 (CYP) enzymes are down-regulated in the liver during the host response to inflammation, leading to impaired drug metabolism. Inflammatory-related mediators, includ-ing pro-inflammatory cytokines, have been associated with decreased activity and/or expression of CYP enzymes. Ischemia-reperfusion (I/R) injury, a result of the inflammatory response during liver transplantation, contributes significantly to the impaired function of the transplanted liver graft, thus it is important to understand the effect of I/R injury on the activity of hepatic CYP enzymes in an orthotopic liver transplant (OLT) model. The objectives of this study are: 1) examine the effect of I/R injury on the activity of phase I CYP enzymes in liver grafts post-OLT, and 2) evaluate the protective effect of treprostinil against I/R injury. HYPOTHESIS: The activation of pro-inflammatory cytokines in the liver, as a result of I/R-induced hepatic injury during OLT, will alter the activity of CYP enzymes and that treprostinil will ameliorate I/R-induced impairment in the activity of CYP enzymes.

Methods: OLT was performed in syngeneic Lewis rats with 18 hours preservation in UW (4 °C) solution. Donor and recip-ients received treprostinil (100 ng/kg/min) or placebo for 24 hours before and up to 48 hours post-OLT. Recipients were sacrificed 1 - 48 hours post-OLT. Rat liver microsomes were used to measure the formation of 6′-hydroxychlorzoxazone, 2α-hydroxytestosterone, and 1′-hydroxymidazolam, which served as markers of CYP2E1, CYP2C11, and CYP3A activity, respectively.

Results: At 24 hours post-OLT, serum ALT and AST levels (mean ± SEM) in placebo group peaked at 2810 ± 201.9 and 4445 ± 950.7 IU/L, respectively. Treprostinil administration significantly reduced ALT and AST levels to 807 ± 140.1 and 1231 ± 111.6 IU/L, respectively. The areas under the curve from 6 to 48 hours post-reperfusion of ALT and AST in treprostinil group were lower vs. placebo group. Early post-transplantation, hepatic mRNA levels of ICAM-1, TNFα, IL-6, and IFNγ, were significantly increased in placebo group vs. normal. Treprostinil significantly reduced mRNA expression levels of these pro-inflammatory cytokines and increased mRNA levels of the anti-inflammatory cytokine IL-10. At 48 hours post-transplantation, CYP2E1 activity was reduced to 35% of normal in placebo group vs. 73% in treprostinil group. CYP2C11 and CYP3A activity was significantly reduced to 18% of normal in placebo group vs. 55% in treprostinil group.

Conclusion: Treprostinil effectively ameliorated I/R-induced hepatic injury and improved the metabolic activity of the liver graft post-transplantation. Treprostinil has the potential to serve as a therapeutic option to protect the liver graft against I/R injury and improve its functional capacity in patients undergoing OLT.

P86. Confluent Growth of Huh7 Human Hepatoma Cells Induces Transcriptional Activation of the CYP3A4 Gene

Louise Sivertsson1, Monica Ek1, Malin Darnell2, Irene Edebert3, Magnus Ingelman-Sundberg4 and Etienne P.A. Neve1

1Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden, 2AstraZeneca R&D Mölndal, Clinical Pharmacology and DMPK, Mölndal, Sweden, 3AstraZeneca R&D Södertälje, Safety Assessment, Department of Molecular Toxicology, Södertälje, Sweden, 4Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden

Drug-induced hepatotoxicity is one of the most important single cause for disapproval or withdrawal of drugs, and the need for reproducible in vitro systems for the prediction of such toxicity and metabolism is urgent. Previously, we have shown that the expression of P450s is increased in human B16A2 cells during differentiation and we hypothesized that confluent growth of the human hepatoma cell line Huh7 would result in a similar differentiation into a more hepatocyte-like phenotype. Indeed, we show that confluent growth of the human hepatoma cell line Huh7 up to 5 weeks generates increased gene and protein expression of several CYPs, UDP-glucuronosyltransferases, transcription factors, transporters, as well as several liver specific genes. The most striking effect is observed for CYP3A4 with about 1000-fold increase in gene expression after 4 weeks of confluence, as measured by quantitative real-time PCR. Western blot analysis revealed increased CYP3A4, NADPH cytochrome P450 reductase and albumin protein levels with increasing time of confluence. By using the CYP3A4 specific substrates luciferin 6′ benzyl ether, testosterone, and midazolam, we

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can confirm that the increased CYP3A4 gene expression also is accompanied by a similar increase in catalytic activ-ity. The CYP3A4 enzyme activity is effectively inhibited by ketoconazole and inducible by rifampicin, both selective CYP3A4 modulators. In confluent Huh-7 cells, we can also induce CYP3A4 dependent cytotoxic activation of aflatoxin B

1, which is inhibited by ketoconazole. Three regions in the 5′ upstream regulatory region of the human CYP3A4 gene

are shown to be important for the increased CYP3A4 promoter activity in the confluent cells; the proximal promoter region (PROX), the xenobiotic response element (XREM) and the constitutive liver enhancer module (CLEM). Using a cell based reporter assays we observe that CYP3A4 promoter activity is up-regulated in the confluent cells, whereas no activity is seen in non-confluent cells. In conclusion, we propose that this hepatocyte cell model could be a useful addi-tion to the existing in vitro model systems, to study CYP3A4-dependent metabolism and toxicity of drugs. In addition, it provides a useful tool for analyzing the transcriptional regulation of the CYP3A4 gene.

P87. Inter-Laboratory Evaluation Of HepaRG® Cells For Determining CYP Induction

Rebecca L. Denton1, Gill Elliott1, Matthew Soars1, Dermot McGinnity1, Arnaud M. Bruyère2 and Françoise Brée2

1Discovery DMPK, AstraZeneca R&D Charnwood, Loughborough, United Kingdom, 2Xénoblis, Saint Grégoire, France

Induction of human cytochrome P450 (CYP) enzymes can result in significant clinical consequences via reduced expo-sure leading to suboptimal efficacy of co-administered drugs and/or enhanced bioactivation. Therefore, assessment of CYP induction in drug discovery is essential to enable potential induction-mediated drug-drug interactions to be minimized. CYP induction data can be generated using primary human hepatocytes, but limited availability of quality cells and inter-individual variability somewhat limits routine application within a drug discovery setting. The recently developed HepaRG® human hepatoma cell line has been evaluated as an alternative in vitro system (1, 2, 3). The aim of the work presented here was to extend the validation of HepaRG® cells and assess inter-laboratory variability and reproducibility. HepaRG® cells were cultured and incubated with 3 prototypical inducers and 9 proprietary AstraZeneca (AZ) compounds at two laboratories (AZ R&D Charnwood and Xenoblis). CYP induction was assessed following 24h incubation, using a mRNA endpoint, determined using single and multiplex quantitative reverse transcriptase-polymer-ase chain reaction (qRT-PCR). The data obtained at the two sites was in good agreement with <3-fold variation for both the prototypical inducers and the AZ compounds. Rifampicin maximally induced CYP3A4 mRNA 47 and 66-fold and omeprazole maximally induced CYP1A2 mRNA 92 and 129-fold at Xenoblis and AZ, respectively. The data presented further demonstrates that HepaRG® cells are an excellent surrogate for evaluation of CYP1A, CYP2B6 and CYP3A4 induction in primary human hepatocytes and that robust, reproducible data can be obtained using separate batches of cells at different laboratories.

References

(1) Kanebratt KP and Andersson TB (2008). HepaRG cells as an in vitro model for evaluation of cytochrome P450 induction in humans. Drug metabolism and disposition 36: 137-145.

(2) McGinnity DF, Zhang G, Kenny JR, Hamilton GA, Otmani S, Stams KR, Haney S, Brassil P, Stresser DM and Riley RJ (2009). Evaluation of multiple in vitro systems for assessment of CYP3A4 induction in drug discovery: human hepatocytes, pregnane X receptor reporter gene, and Fa2N-4 and HepaRG cells. Drug metabolism and disposition 37: 1259-1268.

(3) Antherieu S, Chesne C, Li R, Camus S, Lahoz A, Picazo L, Turpeinen M, Tolonen A, Uusitalo J, Guguen-Guillouzo C and Guillouzo A (2009). Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug metabolism and disposition 38: 516-525

P88. Effect of Organic Solvents on BM3-P450 Biotransformation

Lifang Sun1, Caroline Decker1, Peter Grootenhuis1, John Saunders1, Jan N. M. Commandeur2, Jelle Reinen2 and Nico Vermeulen2

1Drug Innovation, Vertex Pharmaceuticals, San Diego, CA, USA, 2Division of Molecular Toxicology, Vrije Universiteit Amsterdam, Amsterdam, Netherlands

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BM3-M11, a CYP-102 from BM3-M11 mutant1 was used to generate milligrams of two mono-hydroxylated azabicy-cloheptan compounds. To optimize the yield during incubation, various organic solvent additives were investigated. For acetonitrile, methanol, acetone, dimethylsufoxide, and dimethylformamide, a maximum yield of mono-hydroxylated compounds was observed at 10%-20% (v/v) organic solvent composition. Methanol gave the most significant effect, with ten-fold increase of one compound at 20% (v/v). A further investigation revealed that the addition of organic solvents greatly suppressed the formation of secondary dihydroxylated compounds. Therefore, the increase of monohydroxylated compounds with the increase of organic solvent is likely due to reduced conversion of monohydroxylated compounds to dihydroxylated compounds. This hypothesis was supported by the results on solvent stability of P450 from BM3-M11, v

max/K

mmeasurements, and solvent effects on other substrates.

References

1. van Vugt-Lussenburg BMA, Stjernschantz E, Lastdrager J, Oostenbrink C, Vermeulen NPE and Commandeur JNM (2007) Identification of Critical Residues in Novel Drug Metabolizing Mutants of Cytochrome P450 BM3 Using Random Mutagenesis. J. Med. Chem. 50:455-461.

P89. a-Naphthoflavone as an inducer or the CYP1A subfamily

Mariana Flores Torres Medicina Genómica y Toxicologia Ambiental,

Instituto de Investigaciones Biomedicas, UNAM, Mexico City, Mexico

The cytochrome P450 (CYP) is a large family of well-conserved integral membrane enzymes localized in the membrane of the endoplasmic reticulum, where they metabolize a variety of endogenous and exogenous compounds. The CYP1A subfamily plays an important role in the bioactivation and transformation of toxic, mutagenic and/or carcinogenic compounds, like polycyclic aromatic hydrocarbons. Well-known CYP1A subfamily inducers and aryl hydrocarbon receptor (AhR) agonist are known including: TCDD, B(a)P and 3-MC, called classic inducers, all utilize the AhR path-way for the CYP1A induction. Other type of inducers has been also described as non classic inducers, for example benzimidazol molecules like: omeprazole, thiabendazole and albendazole (ABZ). Many in vitro studies have shown that a-naphthoflavone (a-NF) prevents the induction of the CYP1A by the classical inducer TCDD, but there are not enough in vivo observations that corroborate this effect. The aim of this study is to explore the in vivo modulation effect of ?-NF on CYP induction by a classical (3-MC, B(a)P) and a non classical inducer (ABZ). Wistar rats were treated with a single dose of a-NF (5, 10, 40mg/Kg), ABZ (50mg/Kg), B(a)P (50mg/Kg), 3-MC (40mg/Kg) or in combination with a-NF: a-NF-ABZ, a-NF -B(a)P, a-NF-3-MC. In the combined protocol, a-NF was administrated 1h prior the inducer. Animals were sacrificed 24h after administration of the inducer. Our results shown that a-NF and ABZ are in vivo inducers of CYP1A, and that the latter possess a similar induction potential as the classic inducers 3-MC and B(a)P. Compared with the other compounds tested, a-NF is a CYP1A1 weak inducer but when it is administrated in a combined treatment, the result depend on the used dose. High a-NF doses (40mg/Kg) exert an additive effect with ABZ, 3-MC and B(a)P, but at low doses (5mg/Kg) the additive effect is only apparent with ABZ but not with 3-MC.

P90. Differential gene expression of CYP3A-isoforms in equine intestines and liver

Eva Tydén, Maria Löfgren, Hans Tjälve and Pia Larsson

Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Uppsala, Sweden

Introduction

CYP3A in the intestinal mucosa is considered to play an important role for oral bioavailability of substrate drugs. Thus, following oral intake drugs may encounter sequential CYP3A mediated first pass metabolism in the small intestines and liver. We have previously shown high levels of CYP3A, as a blanket term for all members of the subfamily, in the small intestines in horse (Tydén E et al. CYP3A in horse intestines, Toxicol Appl Pharmacol, 201:112-119, 2004). There

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exist large species differences of the CYP3A-isoenzymes in the expression, the tissue distribution as well as the substrate specificity. The equine CYP3A family consists of three known members, CYP3A89, CYP3A96 and CYP3A97, whereas in human there are four members and in dog two members. In the present study we have examined the gene expression of these CYP3A-isoforms in the small intestine and the liver in horse. Materials and Methods Tissue samples of the small intestines and the liver were collected from slaughtered horses and total RNA was isolated. qRT-PCR was used to examine the gene expression of CYP3A89, CYP3A96 and CYP3A97. Results In the liver CYP3A97 represented about 40 %, CYP3A89 about 36 % and CYP3A96 about 24 % of the total CYP3A gene expression. In contrast CYP3A96 was the dominating isoform in the intestine with about 99 % of the total CYP3A gene expression. There was almost no gene expression of CYP3A89 or CYP3A97 in the intestines, the levels representing about 0.1 % and 0.01 % of the total CYP3A gene expression. The level of CYP3A96 gene expression in the intestines was at a level similar to that observed in the liver. Conclusion The results from the present study show that CYP3A96 represents almost the entire CYP3A mRNA pool in the intestines. In contrast, all three CYP3A-isoenzymes are markedly expressed in the liver. Studies in dog have also shown a differential CYP3A gene expression in the intestines and the liver, with domination of CYP3A12 in the intestines and CYP3A26 in the liver (Mealey K.L et al. Differential expression of CYP3A12 and CYP3A26 mRNAs in canine liver and intestine, Xenobiotica, 38(10):1305-1312, 2008). In man the expression of CYP 3A4 dominates over CYP3A5 both in the intestines and in the liver. (Paine M.F. et al. Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism, J Pharmacol Exp Ther, 283:1552-1562, 1997). The differential expression of CYP3A-isoforms between the intestines and the liver, as observed in horse and dog, may have implication as regards the disposition of orally vs. parenterally administered drugs. In addition, it appears that marked species differences may exist regarding CYP3A expression in the intestine and liver. Acknowledgement Supported by the Swedish-Norwegian Foundation for Equine Research (Stiftelsen Hästforskning).

P91. Improved Predictions of Selective Estrogen Receptor Modulator and Glucocorticoid Metabolism by CYP3A4/5/7 Enzymes using Integrated Computational Models

Kiumars Shahrokh1, Chad D. Moore2, Thomas E. Cheatham III3 and Garold S. Yost4

1Pharmacology & Toxicology, University of Utah, Salt Lake City, UT, USA, 2Pharmacology & Toxicology, University Of Utah, Salt Lake City, UT, USA, 3Departments of Medicinal Chemistry and of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, USA, 4Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, USA

Objectives: We have used P450-specific metabolism of the Selective Estrogen Receptor Modulators (SERM): tamoxifen & raloxifene and of several inhaled glucocorticoids (GC) as molecular probes of competing metabolic pathways by the three CYP3A enzymes to refine an integrated computational approach for predicting drug metabolism and bioactivation. The unstable products of these bioactivation reactions are electrophilic intermediates that are linked to biomolecular adduct formation which results in toxicities that range from altered drug metabolism (mechanism-based inactivation) to carcinogenesis.

Methods: To remove structural artifacts potentially introduced by the crystallography conditions, molecular dynam-ics (MD) simulations were performed with the x-ray structure of CYP3A4 (PDB code: 1TQN) and homology models of CYP3A5 & CYP3A7along multiple trajectories. Each MD trajectory was then subjected to cluster analysis, and one representative structure was generated from the largest cluster of each trajectory. These structures were then modified with quantum mechanics (QM)-based parameters for the heme during different stages of the P450 catalytic cycle and were used as docking templates for multiple SERMs and GCs.

Results: An integrated approach of docking using QM optimized substrate structures and MD-refined protein structures modified with QM-based heme parameters produced as the most highly populated conformations those that correctly predicted observed metabolism of SERMs and GCs by the three CYP3A enzymes (CYP3A4/5/7). The accuracy of these models was validated by site-directed mutagenesis and in vitro incubations. Specifically, for CYP3A4,-arginine 212 and for CYP3A5-lysine212 within the active site play important roles in determination of competing reaction mechanisms catalyzed by the CYP3As.

Conclusions: An integrated computational approach for the structure-based prediction of metabolism and bioactiva-tion chemically distinct drugs by the three CYP3A enzymes has been developed. This computational approach of greatly

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improved predictions over x-ray structure-based modelling, and confirmed the importance of accurately modelling the electronic and thermodynamic contributions to both substrate and enzyme structures to predict P450-mediated metabolism of xenobiotics.

Supported by NIH grants # GM074249 from the National Institute of General Medical Sciences, NICHD Grant # HD060559 and # NCRR 1 S10 RR17214-01 from the National Center for Research Resources, NSF grant #MCA017S027 and the University of Utah Center for High Performance Computing.

P92. A continuous spectrophotometric assay for the reductase domain of cytochrome P450 BM3 (BMR) activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5-cyano-2,3-ditolyl tetrazolium chloride

Seon Ha Park and Chul-Ho Yun

School of Biological Sciences and Technology, Chonnam National University, Gwangju, South Korea

P450 BM3 is a self-sufficient monooxygenase where the heme domain and the reductase domain are fused in a single polypeptide, a beneficial characteristic for efficient electron transfer. Because of the solubility and high expres-sion of P450 BM3, it is an excellent model for studies on microsomal P450s. In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by the reductase domain of P450 BM3 (BMR) was measured as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to the substrates of BMR, MTT and CTC. Tetrazolium salts are used extensively to measure cell viability, metabolic activity, and oxidative reactions. Reduction of tetrazolium salts produces highly colored products called formazans and therefore the formazans are easily observed with naked eyes. MTT and CTC are monotetrazolium salts, which form formazans upon reduction. The MTT and CTC reduction is then assessed spectrophotometrically in the reaction medium. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (k

cat = 57 sec−1, K

m = 94 uM for MTT and k

cat = 3425 sec−1, K

m = 40 uM, for CTC). A continuous

assay using MTT and CTC allows a simple measurement of BMR activity in short analysis time. Moreover, this method is also applicable with the reductase domain of bacterial P450s from Bacillus Subtilis, CYP102A2 and CYP102A3.

Reference

1. Kim DH, Yim SK, Kim KH, Ahn T, Yun CH. 2009. Biotechnol Lett 31: 271-5 2. Yim SK, Yun CH, Ahn T, Jung HC, Pan JG. 2005. J Biochem Mol Biol 38: 366-9

P93. Characterization of CYP106A1 genetic variants from various Bacillus megaterium strains

Ga-Young Lee and Chul-Ho Yun

School of Biological Sciences and Biotechnology, Chonnam National University, Gwangju, South Korea

Cytochrome P450 (P450 or CYP for a specific isoform) enzymes are remarkably diverse oxygenation catalysts found throughout all classes of life. An extreme diversity of their substrates and catalytic reactions is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of several natural variants of CYP106A1 within a species of bacteria, Bacillus megaterium. Extensive amino acid substitutions were identified from the variants. CYP106A1 genes were cloned from Bacillus megaterium variants, expressed in Escherichia coli, and purified. We studied the potential electron partner and possible substrates of bacterial CYP106A1 by examining oxidation activity toward several kinds of chemicals. Its activity in vitro could not be achieved by using rat NADPH-cytochrome P450 reductase, and putidaredoxin (Pdx)-reductase (Pdr) pair. However, the spinach redox proteins, ferredoxin (Fdx)-reductase (Fdr) pair from spinach, were found to be a redox partner for CYP106A1. We also examined ‘peroxide shunt′ pathway toward steroid hormone testosterone, progesterone and other substrates using hydrogen peroxide. These results suggest the presence of a number of natural variants within a bacterial species. Possible applications of the natural variants have been discussed.

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Reference

1. Berg A, Gustafsson JA, Ingelman-Sundberg M. 1976. J Biol Chem 251: 2831-8 2. Hannemann F, Virus C, Bernhardt R. 2006. J Biotechnol 124: 172-81 3. Furuya T, Shibata D, Kino K. 2009. Steroids 74: 906-12

P94. Oriented Attachment of Cytochrome P450 Enzymes on Electrodes: Implications for Biosensor Development

Elizabeth Schneider1 and Douglas S. Clark2

1UC San Francisco/UC Berkeley Joint Graduate Group in Bioengineering, University of California Berkeley, Berkeley, CA, USA, 2Chemical Engineering, UC Berkeley, Berkeley, CA, USA

By immobilizing cytochrome P450 enzymes on an electrode NADPH is no longer needed as the electron source and the P450 can accept electrons directly from the electrode. An increase in the current upon application of a potential can be correlated to catalytic activity of the enzyme against a candidate substrate, and the immobilized P450 can be used as a biosensor. Using this methodology, we aim to develop a novel biosensor microarray of mammalian P450 enzymes on gold electrodes as a high-throughput screen of pharmaceuticals in development for P450 reactivity. Arrays of very small size can be made using microfabrication technology such that thousands of electrodes can fit onto the area of a microscope slide, allowing for the screening of thousands of molecules at once against multiple P450 enzymes. Such a device would be useful in the pharmaceutical industry as a tool for initial screens of drug candidates for P450 reactivity and potential drug-drug interactions. However, electrically wiring the P450 enzyme to an electrode in a manner that allows for fast electron transfer through the enzyme to the heme co-factor buried within remains a challenge that must be overcome in order to build an accurate biosensor. Modification of the electrode via formation of self assembled monolayers (SAMs), polyelectrolytes, or lipid bilayers can prevent enzyme denaturation on the surface and enhance electron transfer by providing an environment that mimics the in vivo P450 microenvironment. In addition, controlling the orientation of the enzyme on the electrode could improve the electron transfer rate by ensuring that all enzyme molecules on the surface accept electrons from the electrode in the same manner. We hypothesize that orienting the P450 on the electrode surface such that the proximal side of the heme faces the electrode will improve electroactivity by orienting the P450 in the same way it orients for electron transfer in vivo, where it receives electrons from P450 Reductase, which binds on the proximal side. In this study, we investigate oriented immobilization of a model P450 (CYP2C9) on a single gold electrode and its effects on electron transfer by comparing three different immobilization techniques using electrochemical and surface characterization techniques: non-oriented and random attachment of 2C9 to a carboxy terminated SAM, C-terminal oriented attach-ment of 6x His-tagged 2C9 to a Ni-NTA surface, and proximal side oriented attachment of 2C9 via cysteine mutations on the reductase binding site. The ability of each immobilization method to promote electron transfer and produce catalytically active 2C9 will elucidate the role orientation plays in enzyme electrochemistry and how it can be used to build a more effective P450 biosensor.

P95. Characteristic of Bacillus megaterium cytochrome P450 variants

Seon-Ju Choi and Chul-Ho Yun

School of Biological Sciencs and Biotechnology, Chonnam National University, Gwangju, South Korea

The soluble CYP102A1 (P450 BM3) enzyme from Bacillus megaterium appear to be a good biocatalyst candidate for use in the pharmaceutical industry. In this enzyme, the soluble reductase domain is fused to the C-terminus of the catalytic heme domain in a single polypeptide. The fusion of these two enzymatic activities makes soluble CYP102A1 an ideal model for mammalian, and in particular human, P450 enzymes. In this work, the possibility of the presence of natural variants of P450 BM3 has been examined. At first twelve(12) different strains of B.megaterium obtained from rice straw in Korea were screened if they have BM3 variants with typical PCR and cloning techniques. Six variants of BM3 enzyme were obtained and all the six strains showed some variation from BM3WT. The amino acid changes of

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variants were from ten to thirty three among total 1,050 amino acids of BM3WT. These analyses indicate that the vari-ation of natural BM3 enzymes can go up to 3% difference of amino acid sequence. The mutation rate of heme domain was much higher than that of reductase domain. The natural variants of BM3 were cloned into Escherichia coli and expressed. The purified BM3 variants were used for the catalytic activity towards fatty acids and other chemicals. This suggests the presence of a large set of natural variants of BM3. A possible application of the natural variants of BM3 has been discussed.

Reference

1. Patricia Fajardo-Cavazos and Wayne Nicholson.2006. ASM. Rev. 2856-2858 2. Li QS, Ogawa J, Schmid RD, Shimizu S. 2001. Appl Environ Microbiol 67: 5735-5739

P96. Comarative metabolism of albendazole in finfish species

Badar Shaikh1, Nathan G. Rummel1, Charles Gieseker1, Renate Reimschuessel1 and Joseph Kawalek2

1Office of Research, US Food & Drug Administration, Laurel, MD, USA, 2Office of Research, Food and Drug Administration, Laurel, MD

Metabolic and residue depletion profiles of albendazole (ABZ) and its three major metabolites: albendazole sulfox-ide (ABZSO), albendazole sulfone (ABZSO

2) and albendazole aminosulfone (ABZ-2-NH

2SO

2) were studied in finfish

species of importance to US aquaculture. The species were: rainbow trout, Atlantic salmon, tilapia, channel catfish, largemouth bass and hybrid striped bass. The fish were grown and housed in large re-circulating fiberglass tanks con-taining fresh water at appropriate temperature for each species and provided ad libitum a commercial fish diet. The fish were cultured until they reached a market weight range suitable for dosing experiments. A single oral dose, 10mg/kg body weight, of ABZ was given to fish species for each withdrawal period. Six fish from each species were sacrificed at designated withdrawal periods, necropsy performed and muscle fillets collected. The muscle samples were homog-enized and powdered in dry ice and stored at - 80 °C, until assayed. One gram of powdered tissue sample was subjected to liquid-liquid extraction and clean-up procedures. The final sample extracts were analyzed by high performance liquid chromatography (HPLC) for the determination of parent drug ABZ and its major metabolites. The results on the metabolic profile indicate that four fish species: rainbow trout, Atlantic salmon, tilapia and largemouth bass bio-transform albendazole into three of its major metabolites: ABZSO, ABZSO

2 and ABZ-2-NH

2SO

2, whereas, catfish and

hybrid striped bass into two metabolites: ABZSO and ABZSO2. The depletion data show that parent drug albendazole

was metabolized quickly: by 8 h post dose in both catfish and hybrid striped bass, by 12 h in both tilapia and rainbow trout, by 24 h in Atlantic salmon, and by 48 h in largemouth bass. The pharmacologically active metabolite ABZSO was detectable until 48 h in three species: rainbow trout, tilapia and hybrid striped bass; however, it was retained longer, until 96 h, in both Atlantic salmon and largemouth bass. Among the inactive metabolites, ABZSO

2 was present in all

species at most withdrawal periods, however, ABZ-2-NH2SO

2 was present only in four species: rainbow trout, Atlantic

salmon, tilapia and largemouth bass, suggesting that it either does not form or only at very low concentrations in other two species: catfish and hybrid striped bass.

P97. Comparison of the Formation Kinetics of Hydroxymethyltolbutamide in Chimpanzee, Cynomolgus Monkey, Marmoset, Rhesus Monkey and Human Liver Microsomes

James Grace1, Michael Sinz2 and Kimberley Lentz3

1Metabolism and Pharmacokinetics, Bristol-Myers Squibb Co, Wallingford, CT, USA, 2Department of PCO-MAP Disc, Bristol-Myers Squibb Co, Wallingford, CT, USA, 3Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, NJ, USA

Non-human primates are widely used in the pharmaceutical industry for the prediction of human pharmacoki-netics and as a preclinical safety model. The metabolism of tolbutamide to hydroxymethyltolbutamide (HMT)

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is regarded as a measure of CYP2C activity. In order to understand species differences in CYP2C-like activity, the formation kinetics of HMT was investigated in vitro using liver microsomes from human and nonhuman primates. The linearity of HMT formation was determined with respect to protein concentration and time to determine optimal incubation conditions for enzyme kinetic analysis. Tolbutamide was incubated in the pres-ence of 0.25 mg/mL of pooled microsomes at 37°C over a range of substrate concentrations (1-5000 µM). After incubation, 100 µL of reaction mixture was quenched with 200 µL of acetonitrile containing internal standard and samples were analyzed by UPLC-MS-MS. Samples were generated in duplicate and the kinetics of HMT formation was estimated by nonlinear regression. The models used were selected based on visual inspection of the fit of the data and the goodness of fit criterion (AIC). The intrinsic clearance was calculated by V

max/K

m. The K

m for the

formation of HMT for human and nonhuman primates was similar across all species. Vmax

, calculated using total CYP450 content, was greatest for human liver microsomes and similar for all nonhuman primates. In general, the higher V

max observed in humans is not consistent with the CYP450 content as human liver microsomes had

the lowest CYP450 content in this study. These results may assist in the understanding of species differences in metabolism and pharmacokinetics observed in non-human primates when compared to humans for compounds that are CYP2C substrates.

P98. Evaluation of the IL-6-Mediated Suppression of Cytochrome P450 and Transporter Activities in Human Cryopreserved Hepatocytes

Brigitte Gerin1, Sylvie Dell′Aiera1, Annick Cauvin1, Helen Neale2, Hilmar Boekens1, Steven Smith1 and Hugues Chanteux1

1UCB Pharma SA, Braine-L′Alleud, Belgium, 2UCB Pharma SA, Slough, United Kingdom

IL6 is well known to down-regulate in vitro the mRNA and protein levels of some P450 enzymes and the activities of hepatic transporters. In addition, there is also evidence of CYP450 suppression in patients with high level of IL-6 (inflammation, sepsis, heart failure, cancer, …). The purpose of the study was to evaluate directly the effect of IL-6 on the enzymatic activities of cytochrome P450 (1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) and on the uptake transporters (sodium taurocholate cotransporting polypeptide or NTCP, organic anion transporting polypeptide or OATPs and organic cation transporter or OCTs). The assay was conducted using cryopreserved human hepatocytes from one representative donor where preliminary assay showed the presence of functional IL-6 receptor (using STAT-3 phosphorylation as the readout). Monolayers were cultured in a sandwich configuration (collagen - Matrigel™) for 24 h, 48 h and 72 h with IL-6 (0.1 ng/mL, 1 ng/mL and 10 ng/mL) renewed every 24 h. At the end of the treatment period, the CYP450 activities were evaluated following in situ incubation with specific probe substrates of P450 enzymes using a cocktail strategy (first cocktail with 10 µM phenacetin (CYP1A2), 100 µM bupropion (CYP2B6), 10 µM diclofenac (CYP2C9) and 3 µM mida-zolam (CYP3A4); second cocktail with 15 µM mephenytoin (CYP2C19) and 5 µM dextromethorphan (CYP2D6)). The function of NTCP, OATPs and OCTs was evaluate using specific substrates of each transporter family i.e. taurocholate (10 µM) for NTCP, estrone-3-sulfate (10 µM) for OATPs and methylphenylpyridinium (10 µM) for OCTs. Incubations were performed in situ at 37°C in Hank′s Balanced Saline Solution (HBSS). IL-6 treatment down-regulated CYP2B6, CYP2C19 and CYP3A4 activities, as well as the NTCP activities, in a time and concentrationdependent manner. The maximal effect was observed at 10 ng/mL IL-6 after a 72 h-treatment (loss of ca. 60%, ca. 50%, ca. 70% and ca. 40% of their control activity, respectively). No effect of the IL-6 treatment was observed on CYP1A2, CYP2C9 and CYP2D6 activities nor on the activities of OATPs and OCTs. Our data confirmed previous published results (obtained at the mRNA and protein levels) and showed that human cryopreserved hepatocytes could be used as an in vitro model to investigate the effect of IL-6 on P450 enzymes and hepatic transporter activities.

P99. Influence of Gender and Ethnicity on Drug Metabolizing Enzyme Activity in Human Hepatocytes

Matthew Sherman, Cornelia Smith, Heather Rollins, Jeanine Fogarty and Jasminder Sahi

ADME/Tox, CellzDirect/Life Technologies, Durham, NC, USA

90

Purpose: Interindividual variability in the pharmacokinetic profile of most drugs is largely the result of differences in liver function caused by factors such as disease state, genetic differences in metabolizing enzymes and drug interac-tions, including enzyme inhibition and induction. Most of the currently used therapeutics are effective in less than 60% of patients, and metabolism of 90% of these drugs is predominantly by CYP1A2, CYP3A4 CYP2C9, CYP2C19, CYP2D6 and CYP2E1. To better understand variable liver function and in particular, drug metabolism, we have developed a data base with 258 human hepatic tissues and characterized these for hepatocyte viability, activity and genotype of the major drug metabolizing enzymes.

Methods: Human hepatic tissues used in this study were from transplant reject livers and liver resections that were harvested for healthy tissue, mainly from patients with hepatic cancers. Within 18 hours of retrieval hepatocytes were isolated and subsequently cryopreserved. Cryopreserved hepatocytes were thawed and characterized for Phase I cytochrome P450 enzyme activities of CYP 1A2, 2C9, 2D6, 3A and Phase II (sulfation and conjugation) activity. Some preparations of hepatocytes were placed in primary culture and enzyme regulation of CYP1A1/2 and CYP3A studied after 3 days of treatment in primary sandwich culture. Genotyping assessment was conducted for the following SNPs: CYP2C19*2, CYP2C9*2 and *3, CYP2D6*4, *6 and *9 and CYP3A5*3.

Results: Our data indicates differences in expression of drug metabolizing enzymes between resections and whole livers, as reflected by higher CYP1A2 and CYP3A4 activity in hepatocytes harvested from resections with CYP1A2: 96.3 vs. 52.7 pmol/min/mg protein and CYP3A4: 631 vs. 444 pmol/min/mg protein. BMI of the donor does not affect drug metabolizing enzyme activity. Another interesting observation is that CYP3A4 and 7-HCG activities (sulfation and glucuronidation) are higher in females vs. males. CYP3A4: 581 vs. 430; 7-HCG 698 vs. 557 pmol/min/mg protein. We also observe up to 3- fold, significant ethnic differences e.g. 7-HCG activity (pmol/min/mg protein) in hepatocytes from Hispanic (1011), Caucasian (640), Asian (604), and African American tis-sues (356). Highest 2C9 and 1A2 activities are found in hepatocytes from Asian donors. CYP2C9 (pmol/min/mg protein) in particular demonstrates race-dependant activity: Asian (125) >Caucasian (112) >African American (76.8) >Hispanic (68.9). This correlates with CYP2C9 polymorphisms, as reflected by our genotyping data with the *2 and *3 SNPs.

Conclusions: Our data highlights that the large interindividual variability in drug clearance and responses in the clinic are likely due to gender and ethnic differences in activities of the major human hepatic drug-metabolizing enzymes. Personalized medicine where these enzymes are evaluated prior to medication will likely result in much higher success and safety of therapeutics.

P100. Interspecies differences acetylation of (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione hydrochloride

Ana I. Loureiro, Carlos Fernandes-Lopes, Lyndon Wright and Patricio Soares-da-Silva

Department of Research & Development, BIAL, S Mamede do Coronado, Portugal

BIA 5-453 (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione hydrochloride is a reversible dopamine-ß-hydroxylase (DBH) inhibitor that decreases norepinephrine (NE) levels in sympathetically innervated tissues and slows down sympathetic nervous system drive. The purpose of the current study was to characterise the potential species differences in BIA 5-453 N-acetylation and the N-acetylation of BIA 5-453 by N-acetyltransferases (NAT1 and NAT2) using a sensitive and specific LC/MS assay. Significant differences of BIA 5-453 N-acetylation levels were observed in mouse, hamster and rat plasma. In addition in vitro studies showed that rat, monkey and human liver enzymes, converted BIA 5-453 to the N-acetylated metabolite with different rates and no acetylation of BIA 5-453 was observed in dog liver enzymes. These results show that BIA 5-453 N-acetylation exhibit marked interspecies differ-ences. Studies performed with human NAT1*4 and NAT2*4 alleles showed that both were able to conjugate BIA 5-453 albeit at different rates. NAT1 had much lower acetylation affinity compared with human liver cytosol and NAT2 (Km 3399 ± 312.0 µM, 124.8 ± 9.0 µM and 17.1 ± 3.6 µM, respectively). Comparing BIA 5-453 N-acetylation by human single donors and sulfamethazine, a selective substrate to NAT2, a significant correlation (r2 = 0.65) was observed. No cor-relation was observed with NAT1 selective substrate, p-aminosalicyclic acid. These data demonstrate that interspecies differences in BIA 5-453 N-acetylation should be considered in BIA 5-453 bioavailability and suggest that BIA 5-453 is a preferential substrate for NAT2.

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P101. Ontogeny of Glutathione Transferase Z1 (GSTZ1) Expression and Activity with Dichloroacetate in Human Liver Cytosol

Margaret O. James1, Ronald N. Hines2, Yuan Gu1, Wenjun Li1, Taimour Langaee3 and Peter W. Stacpoole4

1Medicinal Chemistry, University of Florida, Gainesville, FL, USA, 2Departments. of Pediatrics and Pharmacology/Toxicology, Children′s Research Institute, Medical College of Wisconsin & Children′s Hospital & Health System, Milwaukee, WI, USA, 3Pharmacotherapy and Translational Research, University of Florida, Gainesville, FL, USA, 4Medicine & Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, USA

The orphan drug dichloroacetate (DCA), used to treat lactic acidosis and, recently, solid tumors by overcoming the Warburg effect (1), is metabolized to glyoxylate in a reaction catalyzed exclusively by GSTZ1, also known as maleylac-etoacetate isomerase. A reversible side-effect of DCA, peripheral neuropathy, is observed more commonly in adults than children (2), and children appear to eliminate DCA more effectively than adults following multiple doses (3). To determine if there are age-related changes in GSTZ1, we investigated its pre- and post-natal expression and activity in a bank of human livers previously studied for the ontogeny of other drug-metabolizing enzymes (4). Cytosolic expres-sion was measured by Western blot using a rabbit anti-human GSTZ1C-1C antibody and activity with 0.2 mM 14C-DCA in the presence of 1 mM glutathione (2). DNA of selected samples was subjected to genotyping for the known GSTZ1 human variants (5). Prenatally, both expression and activity were very low. Activity and expression increased in the first two weeks of age then rose further over the first year and attained variable but similar levels between ages 1 and 18. Overall, there was good correlation between activity and expression (r2 = 0.65), however if data for six outliers from this 216 sample bank were removed, the correlation improved to r2 = 0.81. The six outliers were shown to be GSTZ1A-1A homozygous or GSTZ1A-1C heterozygous, and had higher activity than average based on expression. Within this small cohort, there was excellent correlation between activity and expression, r2 = 0.98. This study demonstrated age-related changes in GSTZ1, with a rapid increase immediately after birth and adult levels attained by age 1. The mature level of GSTZ1, combined with the greater liver mass to body surface area in children versus adults could contribute to the age-related differences in DCA pharmacokinetics. Furthermore, we showed that some of the inter-individual variability in expression and activity were related to differences in the GSTZ1 haplotype. Supported in part by the US Public Health Service ES07355 and GM081344.

References

1. Michelakis, ED, Sutendra, G, Dromparis, P, Webseter, L, Haromy, A, Niven, E, Maguire, C, Gammer, T-L, Mackey, JR, Fulton, D, Abdulkarim, B, McMurtry, MS, Petruk, KC Metabolic Modulation of Glioblastoma with Dichloroacetate. Science Translational Medicine 2: 1-8, 2010

2. Shroads, AL, Guo, X, Dixit, V, Liu, HP, James, MO and Stacpoole, PW. Age-Dependent Kinetics and Metabolism of Dichloroacetate: Possible Relevance to Toxicity. J. Pharmacol. Exp. Therap. 324: 1163-1171, 2008

3. Kaufmann, P, Engelstad, K, Wei, Y, Jhung, S. Sano, M.C., Shungu, DC, Millar, WS, Hong, X, Gooch, CL, Mao, X., Pascual, JM, Hirano, M, Stacpoole, PW, DiMauro, S and De Vivo, DC. Dichloroacetate as a treatment for MELAS: a randomized controlled clinical trial. Neurol. 66: 324-330, 2006

4. Hines R. The ontogeny of drug metabolism enzymes and implications for adverse drug events. Pharmacol Therap 118:250-267, 20085. Blackburn AC, Coggan, M, Tzeng, HF, Lantum, H, Poleknina, G, Parker, MW, Anders, MW and Board, PG. GSTZ1d: a new allele of glutathione

transferase zeta and maleylacetoacetate isomerase. Pharmacogenetics 11: 671-678, 2001

P102. Abstract Withdrawn.

P103. A16kDa Plasma Membrane Protein of Mice Liver Parenchymal Cell Recognizes the Peptide Moiety of Human Serum Alpha1-Acid Glycoprotein

Ayaka Suenaga1, Mari Kikuchi1, Koji Nishi1, Kazuaki Matsumoto1, Hisakazu Komori1, Hiroshi Watanabe1, Toru Maruyama1 and Masaki Otagiri2

1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan, 2Laboratory of Pharmacokinetics, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan

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Human alpha1-acid glycoprotein (AGP) is a serum glycoprotein composed of 183 amino acid residues and also contains five glycan chains that account for about 40% of its total mass of 40 kDa. We previously found that liver was the main site of AGP accumulation in mice, and AGP was incorporated into liver via a receptor-mediated pathway. Asialoglycoprotein receptor (ASGPR), which is well known to be one of the C-type lectins, has been known to involve in the uptake of asialo-AGP into liver. It was thought to be a receptor for AGP too. However, AGP did not compete with asialoglycoprotein for the uptake into mice liver, suggesting that receptors other than ASGPR may involve. Thus, in this work, we investigated the effect of glycan chain on the liver uptake of AGP in mice. Recombinant glycan-deficient AGP (r-gdAGP) was prepared from Pichia pastoris by mutation of the five Asn residues that have the five glycan chains of AGP attached. Pharmacokinetic study showed that 111In-AGP and 111In-r-gdAGP disap-peared drastically from blood within 5 min after injection. Meanwhile, the saturated concentrations of 111In-AGP and 111In-r-gdAGP were observed until 30min. This result indicates that 111In-AGP and 111In-r-gdAGP are being distributed to tissues via a receptor-mediated pathway. Majority of r-gdAGP was also found to have incorporated to parenchymal cells when the hepatocellular distribution of r-gdAGP was compared to that of AGP. This result suggests that the receptor of r-gdAGP is present in parenchymal cells same as AGP. Ligand blotting analysis of 125I–r-gdAGP was then performed on mice hepatocyte membrane to identify the receptor responsible for the uptake of r-gdAGP. Interestingly, r-gdAGP was found to have interacted with a 16 kDa protein, suggesting that AGP might also interact with the same protein. In a same experiment using rat liver primary parenchymal cells, we observed that native AGP interacted with this 16 kDa protein. Thus, this 16 kDa protein may function as a receptor for the liver uptake of AGP in mice. The results obtained in this study suggest that the 16 kDa protein recognizes a peptide sequence of AGP via peptide-peptide interaction. Elucidation of the structure of this 16 kDa protein may shed light on the biological functions of AGP.

P104. Brain Exposure Prediction (BEP) from in vitro data

Nicolas Perriere1, Loic Laplanche2, Viviane Bourgougnon1, Bernard Julian2 and Bruno Bournique1

1Brain Research, VigiCell, Villejuif, France, 2Dsar, Sanofi-Aventis, Montpellier, France

The level, time-course and localization of free drug concentration into the brain relatively to targeted receptors′ EC

50 or IC

50 depend on several inter-related parameters: CLin, CLout and flux ratio across the blood brain barrier

(BBB), fu brain, fu plasma, and plasma half-life (Liu X. et al., JPET 313:1254-1262, 2005; Summerfield S. et al., JPET 322:205-213, 2007, Hammarlund-Udenaes M. et al., Pharmaceutical Research 25: 1737-1750 2008; Jeffrey P. and Summerfield S, Neurobiology of Disease 37: 33-37, 2010). Drug discovery would greatly benefit having early access to this comprehensive approach. In the present study, we simulated rat brain pharmacokinetics of 5 molecules substrate of membrane transporters, including P-gp: daunorubicine, digoxine, loperamide, vinblastine, and SAR, a sanofi-aventis compound, and one non-substrate molecule: venlafaxine. We then compared the simulations with published and in-house in vivo rat brain data. Simulations were performed with a brain PBPK model derived from Liu X. et al., JPET 313:1254-1262, 2005. The input parameters and data for the PBPK model and their determination are described below:

CLin, Clout and flux ratio across the BBB: determined • in vitro with our primary syngenic Rat Brain Endothelial Cell (RBEC) BBB model as previously described (Perrière et al., J Neurochem. 93:279-89, 2005; Perrière et al., Brain Res. 1150:1-13, 2007).Fu brain and fu plasma: determined • in vitro by equilibrium dialysis in a 96-wells format, using a cortex homogenate method (Summerfield S. et al., JPET 322:205-213, 2007).Plasma PK data and parameters were determined by • in vivo rat studies and/or literature.

The in vitro results and the in vitro/in vivo correlations will be presented and discussed. Particular attention will be devoted to the functional polarization of the RBEC model, the need to use lower test molecule concentrations than usually implemented in ADME screenings, the adaptability of the PBPK modeling, and the limitations of the approach.

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P105. Bridging traditional LC/MS analysis with radioactivity detection and AMS to support metabo-lite profiling in a human ADME study with [14C]ibipinabant

Ramaswamy A. Iyer1, Anthony Barros1, Wenying Li1, Lisa Christopher1, Erna Hissink2, Karel Stegman2, Marianne Debruijn2 and W. Griff Humphreys1

1Department of Biotransformation-PCO, Bristol-Myers Squibb, Princeton, NJ, USA, 2Department of Clinical Candidate Selection, Abbott Healthcare Products BV, Weesp, Netherlands

This poster summarizes the metabolism profile of [14C]ibipinabant after oral administration to humans. In metabolite profiling studies with radiolabeled compounds, after HPLC separation, radioactivity detection has traditionally been achieved via an online radioactive detector or by offline counting HPLC eluate fractions with a liquid scintillation counter (LSC). In the past decade, Topcount and PE Wallac 96-well plate counters have improved detection limits, however, these sensitivity gains are often insufficient to permit metabolite profiling of samples collected at later time points. Given the pharmacokinetic characteristics of ibipinabant, it was clear that plasma samples from a human ADME study collected at later time points would not contain enough radioactivity to be measured by LSC or Topcount technol-ogy. Accelerator mass spectrometry (AMS) was therefore explored as a method for generating the metabolism profiles of samples collected at later time points (post-48 h). Since AMS analysis is not specific, i.e. it measures a C-12/C-14 ratio in a sample after graphitization and cannot identify metabolites based on mass, like LC-MS, it was important to develop a robust HPLC method that was capable of separating all ibipinabant metabolites. A rat bile sample, which contained the greatest selection of metabolites, was used for generation of an appropriate HPLC method. This method was then utilized for metabolite profiling of plasma samples from the human ADME study collected at early time points (4 and 12 h). The method was then successfully transferred to an AMS lab and was used for generation of metabolite profiles in human plasma collected at 2, 14 and 55 days post dose. This presentation shows the data generated by both traditional radioactivity counting and AMS analysis used in the profiling of samples from a human ADME study with [14C]ibipinabant.

P106. Effect of Exposure of Albumin Solutions to Activated Charcoal and Ion Exchange Resin on Ligand Binding Properties and Esterase-like Activity of Albumin

Tom De Bruyn1, Björn Meijers2, Pieter Evenepoel2, Ruth Laub3, Ludo Willems4, Patrick Augustijns1 and Pieter Annaert1

1Laboratory for Pharmacotechnology and Biopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium, 2Division of Nephrology, Faculty of Medicine, University Hospital Leuven, Leuven, Belgium, 3Central Department of Fractionation, Red Cross, Brussels, Belgium, 4Hospital Pharmacy, University Hospital Leuven, Leuven, Belgium

Patients suffering from acute-on-chronic liver failure can benefit substantially from the use of dialysis-based liver support. The non-biological liver support device, MARS (Molecular Adsorbents Recirculating System), is based on the exchange of albumin-bound toxins between the patient′s blood and a 20% human serum albumin solution circulating in a secondary circuit up to 7 hr. Toxins are continuously removed from the circulating albumin solution by exposure to charcoal and ion exchange resin. However, there is currently limited information on the stability of albumin in the sec-ondary circuit. The aim of the present in vitro study was to determine the impact of exposure to activated charcoal and ion exchange resin on the ligand binding properties of albumin, which contains various levels of stabilizers depending on the supplier (i.e., Baxter, CAF-DCF, Sigma-Aldrich). The albumin binding kinetics for warfarin, diazepam, and sali-cylate were assessed before and after in vitro incubation with adsorbing materials. Esterase-like activity was measured as additional marker of albumin integrity. Baseline (i.e. before incubation) K

d values describing affinity of warfarin for

albumin were about 5 µM and equal for all albumins tested, while the baseline Bmax

value (reflecting binding capacity) amounted to 1.8 for CAF-DCF albumin as compared to 1.4 for the other albumins. These parameters were substantially altered in a supplier-specific way upon exposure of the albumins to adsorbing materials: K

d values for albumins obtained

from Baxter or Sigma increased about 5-fold, while no change in Kd was observed for CAF-DCF albumin. Also the B

max

value for CAF-DCF albumin remained unaltered. In contrast, the Bmax

value for Baxter albumin reached about 3.5 after 7 hr incubation with ion exchange resin. Interestingly, some of these effects were also observed following incubation

94

in the absence of adsorbing materials. This indicated that altered albumin binding properties are due to the incuba-tion time per se, rather than the presence of adsorbing materials. Furthermore, binding properties of salicylate and diazepam appeared to remain largely unaltered, irrespective of the albumin used. Esterase activity associated with the Baxter and CAF-DCF albumins increased about 2-fold following 7 hr exposure to ion exchange resin. Our in vitro data support changes in both ligand binding affinity and binding capacity of albumins, which appear to depend on the supplier, the contact time, the type of adsorbing material and the substrate used. It remains to be investigated to what extent binding properties of albumin in the secondary circuit during a MARS treatment may undergo time-dependent and supplier-specific alterations.

P107. Hepatic Uptake of Alpha1-acid Glycoprotein Through Hemoglobin Beta-Chain Mediated Endocytic Pathway

Hisakazu Komori, Nao Uehara, Koji Nishi, Hiroshi Watanabe, Toru Maruyama and Masaki Otagiri

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

Human alpha1-acid glycoprotein (AGP) is the major binding protein for basic and neutral drugs in blood, and is also known as an acute phase protein. In general, since the tissue distribution of drug largely depends on its unbound concentration in blood, the drug tightly bound to AGP is not highly affected by the blood concentration of AGP, but also kinetics of AGP in the body. Therefore, kinetic analysis of AGP would be necessary for appropriate drug administration plan and drug design. We previously reported that AGP was mainly incorporated into liver in mice. In this study, to clarify the hepatic cellular uptake mechanism of AGP, we explored the specific receptors for AGP on plasma membrane in liver. Ligand blotting was performed using 125I-labeled AGP and crude membrane fraction from mice liver, followed by MALDI-TOF/MS analyses. The expression of the high affinity protein for AGP in human hepatic cell line (HepG2) was detected by Western blotting, immunofluorescent staining and RT-PCR. Protein-protein interaction was assessed by immunoprecipitation. Uptake of AGP into HepG2 cells were evaluated by flow cytometry using FITC labeled AGP (FITC-AGP). Expression of protein was knockdowned by small interfering RNA (siRNA), evaluated by immunofluores-cent staining and quantitative RT-PCR.As a high affinity protein in the mouse hepatic membrane fraction, hemoglobin beta-chain (HBB) was identified by Ligand blotting and MALDI-TOF/MS analyses. Moreover, HBB was detected by protein and mRNA levels in HepG2 cells, respectively. In addition, the direct interaction between AGP and human hemoglobin (Hb) was confirmed by immunoprecipitation. FITC-AGP was incorporated into HepG2 cells in time- and dose-dependent manner. Uptake of AGP was inhibited at low temperature and treatment with endocytosis inhibitors. Similar inhibition was observed in the presence of exogenous Hb. Suppression of HBB expression with siRNA also showed the decreased uptake of AGP into HepG2 cells. These results indicate that interaction of AGP with HBB involves in endocytic pathway of AGP. AGP could be taken up by hepatocytes through HBB-mediated endocytic pathway. Hence, drugs highly bound for AGP might be also internalized by the same route. Present findings could be contributed to the detail understanding of the pharmacokinetics and physiological function of AGP.

P108. Mechanistic RBC Partitioning Studies of MLN4924, a Nedd8-Activating Enzyme inhibitor

Cindy Q. Xia, Susan Chen, Ji Zhang, Jing-Tao Wu, Mark G. Qian, Frank W. Lee and Suresh K. Balani

Dmpk, Millennium Pharmaceuticals, Inc., Cambridge, MA, USA

MLN4924 is a potent and selective inhibitor of Nedd8-Activating Enzyme (NAE), a novel target for anticancer therapy. NAE is considered to play a key role in protein homeostasis disruption of which leads to apoptosis. MLN4924 is currently in clinical trials. Preclinical in vivo data have indicated that MLN4924 has non-linear PK in dog and rat whole blood but not in plasma, and is extensively partitioned into red blood cells (erythrocytes, RBC). The objectives of this study were to investigate the RBC partitioning mechanism of MLN4924 in whole blood from NCR nude mice, Sprague-Dawley rats, Beagle dog, Cynomolgus monkey, and healthy human volunteers and characterize the binding site of MLN4924 in RBC. After incubation of MLN4924 at several concentrations in blood at 37°C for 60 minutes the hematocrit, and blood

95

and plasma concentrations by LC/MS/MS were determined. The dissociation binding constant (Kd) and the maximum

binding rate (Bmax

) of MLN4924 to RBC were determined from the measured total and free concentrations of MLN4924 in whole blood and plasma. The RBC binding site of MLN4924 was characterized by co-incubation with carbonic anhydrase (CA) inhibitors, acetazolamide or chlorthalidone. The RBC binding of MLN4924 was found to be saturable in human, mouse, rat, dog and monkey blood and the concentration ratio of whole blood/plasma was species dependent. The K

d of MLN4924 (free concentration in blood) in the blood from human, mice, rats, dog and monkey was 0.0686,

0.0328, 0.0507, 0.0104, and 0.0126 μM, respectively, and the corresponding blood and plasma concentrations (Cb/C

p)

were 55.2/2.26, 21.6/2.19, 37.2/3.87, 16.9/0.82, and 29.3/0.41 μM. The Bmax

of MLN4924 in the blood from human, mice, rats, dog and monkey was 110, 43.2, 74.4, 33.8, and 58.6 μM, respectively. There was no marked difference observed in MLN4924 binding to the RBC among four individual healthy volunteers tested. The binding site of MLN4924 in the RBC was determined to be carbonic anhydrase (CA), as coincubations with CA inhibitors markedly decreased the accumula-tion of MLN4924 in RBCs. The binding of MLN4924 to CA in RBCs was determined to be saturable and reversible.

P109. Pharmacokinetics and Brain Disposition of ST1936, A Potent 5-HT6 RECEPTOR Agonist, In Rat

Angelo Mancinelli1, Giandomenico Brogin2, Silvia Pace2 and Franco Borsini1

1C&PNS and General Pharmacology, Sigma-Tau Spa, Pomezia, Italy, 2Pharmacokinetics and Metabolism, Sigma-Tau Spa, Pomezia, Italy

ST1936 (2-[5-chloro-2-methyl-1H-indol-3-yl]-N,N-dimethylethanamine) is a potent 5-HT6 receptor agonist with activity in

animal pharmacological models for central nervous system disorders. In this study, plasma and brain pharmacokinetics of ST1936 and its N-oxide (ST4166) and N-demethyl (ST5523) metabolites were evaluated in rat after a pharmacological active dose of ST1936. Plasma and brain concentrations of the compound and metabolites were measured by a HPLC-MS/MS validated method. The retention times were approximately 2.5, 4.5, and 6.5 min for ST4166, ST5523, and ST1936, respectively, with no interference from drug-free plasma and brain homogenate. The calibration curves were linear over the concentration range considered with a correlation coefficient > 0.99 for plasma and brain. The inter-assay precision and accuracy were lower than 5.1 % (precision) and within 87 – 109 %, respectively for all analytes. The limit of quanti-fication was 1.98 ng/ml for all the analytes in plasma and brain with the exception of ST4166 19.8 ng /ml in plasma. The pharmacokinetic analysis was carried out according to a non- compartmental approach for sparse data sampling by using WinNonlin version 5.0.1 software program. After an intraperitoneal dose of 20 mg/kg, the parent compound attained mean maximum plasma and brain concentrations (C

max) within 0.25 hr (first sampling time)of dosing (T

max), decreasing thereafter

to below the limit of quantification from 8 hr after dosing according a mono-exponential decay. The mean elimination half-life of ST1936 and its metabolites was approximately 1 hr in both plasma and brain. ST4166 reached the C

max of plasma

and brain values within 0.25 - 0.5 h (Tmax

) declining with a rate similar to that of the parent compound. A similar behaviour was observed for ST5523 in plasma (T

max = 0.25 h), whereas in brain its C

max appeared slightly later with T

max value of 1 hr. The

examination of the area under the curve from zero to infinity (AUC) between brain and plasma resulted in brain/plasma ratio values of 53, 0.10, and 11 for ST1936, ST4166, and ST5523, respectively. These findings suggest a very high distribu-tion of ST1936 in the target organ. In a similar manner, the N-dimethyl metabolite also showed a good brain penetration, whereas, as expected, a very low distribution was found for the N-oxide metabolite in the brain.

P110. Pharmacokinetics, Metabolism and Excretion of an Adenosine A1 Receptor Antagonist, Tonapofylline, in Healthy Human Subjects

Zhaoyang Li, Liyu Yang, Liang-Shang Gan, Lewis Klunk, Aaron Deykin and Prakash Chandra

Dmpk, BiogenIdec, Cambridge, MA, USA

Tonapofylline [BG9928:((3-(4-(5,7-dioxo-4,6-dipropyl-4,5,6,7-tetrahydro-1H-benzo[d]imidazol-2-yl)bicyclo-[2.2.2]octan-1-yl) propanoic acid)), a potent adenosine A1 receptor antagonist, is being developed as both acute and chronic treatments of heart failure. The objectives of the present study were to determine mass balance, routes of excretion, pharmacokinetics and the metabolic profiles of BG9928 in healthy human subjects after oral adminis-tration of a single 50 mg dose of [14C]BG9928. In the six male subjects, the radioactive dose was rapidly absorbed, with T

max in the range of 2 to 4 hours. There appeared to be an absorption lag time of o.5 to 1.0 hour. The plasma

96

elimination T1/2

of the total radioactivity and BG9928 was 18.3 and 21.7 hours, respectively. The ratio of blood to plasma concentration of the total radioactivity was time-independent and mostly fell in the range of 0.5 to 0.8, indi-cating no considerable binding of the drug-derived radioactive species to red blood cells. Consistent with preclinical species, the majority of the administered radioactivity was excreted in the feces (71.9% of the dose) suggesting that fecal/biliary excretion was the primary route of excretion. Urinary excretion accounted for 21.3% of the dose. BG9928 is extensively metabolized, and the primary metabolic pathways included hydroxylation, β-oxidation, and conjugation with glucuronic acid and glycine. Unchanged 14C-BG9928 was the predominant source of circulating radioactivity, accounting for 64% of the circulating 14C-radioactivity (AUC

0-24). The predominant metabolites in

plasma were glycine conjugates of BG9928 and deyhdrogenated BG9928 (combined), BG9928 acyl glucuronide (BG9982-AG) and boxidized BG9928, with %AUC relative to BG9928 at 18%, 8% and 7%, respectively. The prominent metabolites in excreta were the glycine conjugates of BG9928 and deyhdrogenated BG9928 (combined, 9% of the dose, in urine), β-oxidized BG9928 (2.1% of the dose, in urine), dehydrogenated BG9928 (11.9% of the dose, in feces) and hydroxy-BG9928/BG9928 acid (combined, 13.6% of the dose, in feces). The total radioactivity, BG9928, and BG9928-AG in plasma all exhibited multi-exponential disposition profiles. BG9928 was safe and well tolerated in this study.

P111. Preclinical Pharmacokinetics, Metabolism and Distribution of TMC207, a Novel Anti-Tuberculosis Agent

M.C. Rouan1, K. Anciaux2, M. Vermeir2, T. Verhaeghe3, F. Cuyckens2, G. Mannens2, J. Verbeeck4 and A. Raoof5

1Preclinics, Tibotec BVBA, Johnson & Johnson, Beerse, Belgium, 2Preclinics, Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium, 3BA/Dmpk, Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium, 4Preclinics, Johnson & Johnson Pharmaceutical Research and Development, Raritan, NJ, USA, 5Preclinics, Tibotec BVBA, Johnson & Johnson, Beerse, Belgium

TMC207 is the first in class diarylquinoline with a unique mode of action that targets mycobacterial ATP synthase. In vitro and in vivo studies were conducted to characterize its pharmacokinetics, metabolism and distribution in mice, rats, dogs and monkeys. In vitro, TMC207 was slowly metabolized in liver subcellular fractions and in hepatocytes of the different species tested. Mono-N-demethylation, followed by further N-demethylation, was the major metabolic pathway in all species. After single oral administration, the bioavailability amounted to 70% to 80% in mice and rats and to about 40% in dogs and monkeys. The plasma concentration-time profiles showed a multi-phasic decline with a long terminal phase half-life of 1-3 days (mice), 3-9 days (rats and monkeys) and 40 days (dogs). In agreement with the slow plasma kinetics, the dose was slowly excreted in urine and feces, most of the dose being recovered in feces. In rats, the amount of unchanged TMC207 recovered in bile was negligible (about 0.1% over 24 hours after dosing), indicating that TMC207 was mainly eliminated by metabolism. N-desmethyl TMC207 was the major circulating metabolite in all species albeit species differences in the exposure ratio N-desmethyl TMC207/TMC207 were observed. Upon repeated administration, the exposure (area under the plasma curve) to N-desmethyl TMC207 was 2 to 7 times higher than the exposure to TMC207 in mice, and similar to 2-fold lower in rats and dogs. Both compounds distributed extensively to the tissues despite their high plasma protein binding (higher than 99%). After single oral administration of 14C-TMC207 to pigmented male rats and monkeys, high tissue concentrations were associated with the adrenal gland, lung, spleen, liver, bone marrow and kidney. The distribution to the brain was low. The total concentration of radioactive substances in most of the tissues and in plasma declined in parallel, indicating that there was no undue tissue retention. Upon repeated administration to rats and dogs, plasma and tissue concentrations of TMC207 and N-desmethyl TMC207 increased dose proportionally to less than dose proportionally at doses lower than 5-10 mg/kg. At higher doses, the tissue concentrations increased more than dose proportionally, the N-desmethyl TMC207 concentrations increasing more than those of TMC207. This extensive distribution was associated with induction of phospholipidosis, the higher tissue uptake of N-desmethyl TMC207 being in agreement with its higher in vitro potential to induce phospholipidosis. In conclusion, TMC207 is well absorbed and slowly eliminated. N-desmethyl TMC207 is the main circulating metabolite in all species. The two compounds distribute extensively to tissues. At low doses corresponding to an exposure above the clinical exposure at the therapeutic dose, the plasma concentrations are dose dependent and representative of tissue uptake.

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P112. Species and Gender Related Differences in the Metabolism of a Potent Adenosine A1 Receptor Antagonist, Tonapofylline, in Rats and Monkeys

Chandra Prakash, Liyu Yang, Zhaoyang Li, Liang-Shang Gan and Lewis Klunk

Dmpk, BiogenIdec, Cambridge, MA, USA

Tonapofylline [BG9928: (3-(4-(5,7-dioxo-4,6-dipropyl-4,5,6,7-tetrahydro-1H-benzo[d]imidazol-2-yl)bicyclo-[2.2.2]octan-1-yl)propanoic acid], is a potent adenosine A

1 receptor antagonist and is being developed for the treatment of

chronic heart failure. The objective of this study was to investigate the disposition of BG9928 in rats and monkeys, used for long term safety studies, after oral and IV administration of a single dose of [14C]BG9928. The radioactive dose of BG9928 was quantitatively recovered in rats (96%) and monkeys (89%) following both oral and IV administration. The major route of the radioactivity excretion in both species was via the fecal route after oral administration and based on the separate studies in the bile duct-cannulated animals, this likely reflects excretion in the bile rather than incomplete absorption. The absorption of the BG9928 radioactivity was also rapid, as the plasma concentrations for BG9928 and total radioactivity peaked within 3 h after oral administration. Based on AUC

(0-24) values, the unchanged BG9928 accounted for

>50% of the circulating radioactivity. Whole-body autoradioluminography suggested that [14C]BG9928 radioequivalents distributed rapidly in the rat with most non-GIT tissues achieving maximal concentrations at 0.5 h and the remainder of the non-GIT tissues by 2 h post-dose. The radioactivity concentrations of most non-GIT tissues declined to BLQ by 24 h post dose. BG9928 was extensively metabolized in both rats and monkeys since <5% of the administered dose was excreted in urine as unchanged. More than 22 metabolites were detected in the HPLC-radiochromatograms. The pri-mary metabolic pathways of BG9928 included hydroxylation on the propyl groups, hydroxylation and dehydrogenation of the bicyclo-octane ring, β-oxidation, and conjugation with glucuronic acid, taurine and glycine. The major circulating and excretory metabolites in rats and monkeys were species-dependent, however, several common metabolites were observed in both species. In addition to parent compound, acyl glucuronide in monkeys and hydroxy-BG9928 in rats were the most abundant circulating metabolites. All these circulating metabolites had a much lower plasma concen-trations than the parent. The prominent metabolites excreted in urine were BG9928 glycine/deyhdroBG9928 glycine, taurine and glucuronide conjugates and β-oxidized BG9928 and in feces, dehydro-BG9928 and hydroxy-BG-9928/BG-9928 acid. In bile collected from male rats, the most prominent metabolite was the hydroxylated taurine conjugate. There were no gender related differences in the metabolism of BG9928 in monkeys. However, gender related quantitative differences in the metabolism of BG9928 were observed in rats. Taurine conjugate was identified as the major metabolite in male while beta-oxidized was the major metabolite in females.

P113. The development and utilisation of intra-articular administration in the rat for a Quantitative Whole Body Autoradiography (QWBA) application

Marco Bottacini1, Andrew Patterson1, Ross Hoey1 and David Robb2

1Metabolism and Pharmacokinetics, Charles River, Tranent, United Kingdom, 2Veterinary Services Department, Charles River, Tranent, United Kingdom

Rodents are widely used to investigate the absorption, distribution, metabolism and elimination (ADME) of novel therapeutic agents. ADME studies are typically designed to reflect the intended clinical route of administration. Intra-articular administration can be employed to administer drugs for the treatment of diseases affecting the joints such as osteoarthritis. Quantitative whole body autoradiography (QWBA) is widely used as a powerful tool in determining the relative tissue distribution of drug-related radioactivity as well as distribution to specific sites of localisation within tissues. This technique presents unique advantages for investigating intra-articular dosing as it allows a visual confirma-tion of accurate dose administration at the site, which is otherwise difficult to confirm. Furthermore the distribution of the drug-related material can be visualized in detail at the site of administration relative to the remaining tissues. Additionally the absorption and elimination of drug-related material from the site of action can be assessed. In this study, this route of drug administration in the rat was established for the first time at Charles River Edinburgh. The dosing procedure was optimized in relation to dose volumes and injection technique. Each animal received an intra-articular administration of a [14C]-labelled test item. The distribution of radioactivity at the site of administration and in the body was then determined over a period of time using QWBA techniques. The distribution of radioactivity was established

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and the affinity of the test item for the intended site of action (articular surfaces) determined. The efficacy of the dose route was established by comparing exposure of the site of action with exposure of the systemic tissues.

P114. Combined Cytotoxic Effects of Heterocyclic Aromatic Amines and Polycyclic Aromatic Hydrocarbon on a Human Intestinal Cell Line

Natsuko Takakura, Ludovic Le Hégarat and Valérie Fessard LERMVD

Unité de Toxicologie Génétique des Contaminants Alimentaires, AFSSA, Javené, France

The human body is continuously exposed to a multitude of chemical compounds through its diet. The risk of food contaminants is currently assessed for each compound individually, whereas potential interactions, such as synergistic/potentiating effects of different compounds, could lead to increased toxicity. To better understand the risk induced by exposure to a mixture of food contaminants, we investigated cytotoxicity and genotoxicity induced by binary mixtures using the human intestinal Caco-2 cell line in this study. As food contaminants, we chose three heterocyclic aromatic amines (HAAs), 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx) and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and a polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). Cell viability was determined by the MTT assay and the intracellular production of reactive oxygen species (ROS) was measured using the fluorescent probe DCFH-DA. Genotoxicity was determined by the comet assay and it was combined with formamidopyrimidine glycosylase (Fpg), a repair enzyme, to detect the oxidative DNA lesions. The toxic potential of individual compounds after 24-h treatment was evaluated first, and then the toxicities observed in the binary mixtures at several concentrations were compared with those estimated on the basis of additivity, i.e., the sum of the response to each compound. In the MTT assay, B[a]P or PhIP decreased viability of Caco-2 cells in a dose-dependent manner at the concentrations ranged from 10 to 100 µM, but IQ or MeIQx did not show cytotoxic effect at the concentrations up to 100 µM. Intracellular ROS production was induced dose-dependently by B[a]P or IQ at the concentrations from 10 to 100 µM, but not by PhIP or by MeIQx. For these two assays, the binary mixtures exhibited the toxic effects not stronger than those expected on the basis of response additivity of each compound. In the comet assay, B[a]P-induced DNA damage was observed in a dose-dependent manner at the concentrations from 2 to 50 µM. It was enhanced by Fpg treatment, showing oxidative DNA damage induced by B[a]P. However, none of three HAAs induced DNA damage up to 100 µM with or without Fpg treatment. In the binary mixtures, DNA damage without Fpg treatment was comparable to that induced by B[a]P alone. Meanwhile, B[a]P-induced DNA damage with Fpg treatment was enhanced only when combined with IQ, indicating that oxidative DNA damage could be involved in the potentiating effect of IQ on the genotoxicity of B[a]P.

P115. DNA damage in human mesenchymal stem cells and human bone marrow cells: a comparative study

Aminat Chausheva1, Victoria Nikitina1, Aliy Zhanataev2 and Nikolay Bochkov3

1Laboratory of mutagenesis, Research Centre for Medical Genetics RAMS, Moscow, Russia, 2Department of pharmacogenetics, State Zakusov′s Research Institute of Pharmacology of RAMS,, Moscow, Russia, 3Laboratory of mutagenesis, Research Centre for Medical Genetics RAMS, Moscow, Russia

Genetic stability is a key factor in maintaining human stem cells pool. Cells have evolved multiple mechanisms to protect the integrity of their genomes and to repair various types of DNA damage. The abnormal functioning of these mechanisms can lead to decrease of stem cell population or abnormal expression of genes, which in turn can lead to malignant transformations of stem cells. Comparative assessment of DNA damage in bone marrow cells and multipo-tent mesenchymal stem cells (MSC) will allow estimating changes in genetic stability of stem cells under standard cultivation in vitro. Single cell gel electrophoresis (comet assay) was used to assess DNA damage. Five samples of bone marrow taken for allografting and five MSC cultures isolated from bone marrow of healthy donors were subjected to analysis. MSC were cultured for 3-4 passages. DNA damage was assessed as percent of DNA in comet tail, which is used widely and routinely in the studies of genotoxicity of endo- and exogenic DNA damaging factors. Average rate of DNA damage for different bone marrow samples was 3.6 ± 0.8% in the comet tail. Rates of DNA damage were in 2.1

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to 6.5% range. Average rate of DNA damage for MSC cultures was 5.2 ± 0.6% of DNA in the comet tail. Rates of DNA damage were in 3.7% to 7.0% range. Differences in data obtained were not statistically significant. Therefore there is no difference in DNA damage levels of bone marrow cells and cultured MSC of bone marrow origin. This complies with data obtained from literature describing DNA damage rates for lymphocytes obtained from peripheral blood of healthy donors (2 to 9% of DNA in the comet tail).

P116. Mapping DNA damage response through RNAi screening and systems approach

Erik HJ Danen1, Bob van de Water1, Jordi Carreras Puigvert1, Louise Von Stechow1, Ram Siddappa1, Leon Mullenders2 and Harry Vrieling2

1Toxicology, LACDR, Leiden University, Leiden, Netherlands, 2Toxicogenetics, LUMC, Leiden, Netherlands

The DNA damage response (DDR) involves a complex of signaling events that participate in lesion recognition, cell cycle arrest, damage repair, and ultimately cell cycle re-entry or, if repair fails, initiation of autophagy or apoptosis. The DDR is critical for maintenance of genomic integrity and its failure is associated with aging as well as cancer. In a highly simplified model DNA-damage causes activation of the sensor kinases (ATM, ATR, DNA-PK) that acti-vate p53 and the checkpoint kinases (Chk1, Chk2), which in turn regulate effector pathways that control repair, cell cycle, and survival. In reality, the process is much more complex with many additional regulators, extensive cross-talk, and multiple positive and negative feedback loops. We have taken a systems approach to map this DDR signal transduction network using embryonic stem (ES) cells treated with cisplatin as a single experimental set up for multiple different analyses. Transcriptomics and phospho- ubiquitin- sumo-proteomics are used to indentify transcriptional, translational, and post-translational alterations that are induced by cisplatin. These approaches are combined with high-throughput functional genomics, using gene family-wide short interference (si)RNA libraries targeting transcription factors, phosphatases, kinases, (de)ubiquitinases, and sumoylases. Out of ∼3000 genes tested in primary screens using cell viability as an endpoint, ∼250 hits have entered validation steps. Validated hits are currently entering secondary screens in which DNA damage-induced foci and apoptosis are analyzed in real time using automated live cell confocal imaging and their role in DDR signaling in other cell types or triggered by other compounds is being analyzed. We are currently employing bioinformatics to integrate the datasets. For instance, identified transcription factors are linked to regulation of their target genes and identified kinases/phosphatases are linked to altered phosphorylation of their substrates. In this way, we aim to derive a comprehensive DDR signaling network. This will point to biomarkers for genotoxicity and allow us to identify potential drug targets for further testing in cancer chemotherapeutic strategies.

This work is funded by Netherlands Toxicogenomics Center

P117. Melatonin Decreases DNA Fragmantation and Oxidative STRESS PARAMETERS of Gastrointestinal Mucosa

Kazime G. Akbulut1, Hakan Akbulut2 and Nalan Akgun2

1Physiology, Gazi University School of Medicine, Ankara, Turkey, 2Medical Oncology, Ankara University School of Medicine, Ankara, Turkey

In this study we planned to investigate the effects of exogenous melatonin ( 10 mg/ kg sc 7 day) on the level of malondi-aldehyde (MDA), glutathione (GSH) and DNA fragmentation levels of gastric, colonic and small intestinal mucosa of young and aged rats. The MDA levels were significantly increased with aging (5.72 ± 0.2nmol/g vs 7.97 ± 0.5nmol/g; p=0.010), GSH levels decreased (0.52 ± 0.05µmol/g and 0.87 ± 0.05 µmol/g p= 0.010). Melatonin, at the dose of 10 mg/kg, significantly increased the GSH levels of the studied mucosal tissues of aged rats and decreased the MDA levels. DNA fragmentation levels of gastric and colonic mucosa were significantly lower in the melatonin group of young rats when compared to control group. MDA, and DNA fragmentation levels of small intestinal mucosa were not significantly different in control groups of young and melatonin groups. However melatonin significantly increased the GSH levels of aged rats. In conclusion, our results suggest that aging may be related with an increase in free radical levels and a

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decrease in the antioxidant capacity of the tissues and exogenous melatonin may delay aging by means of its action on free radicals, antioxidant defense and DNA fragmentation.

P118. Determination of the ability of a recombinant protein to vaccinate against Leishmania mexicana

Basma Doro

Microbiology and immunology, Al-Fateh University, Faculty of pharmacy, Tripoli, Libya

Leishmaniasis is a protozoan diseases which causes morbidity and mortality, and is an important public health problem in many tropic regions of the World. There are several clinical forms of leishmaniasis caused by different species of Leishmania. At present disease controls relies on drug treatment and prolonged use of a limited number of drugs has led to parasite drug resistance. Ideally a vaccine is required which can protect against infection. In this study, the ability of a recombinant protein to protect against Leishmania mexicana infection was determined. Mice were immunised with PBS (control), the recombinant protein alone, or the recombinant protein incorporated into a non-ionic surfactant delivery system. Successful vaccination was monitored pre-infection by determining specific IgG1 and IgG2a antibody levels and the ability of the antigen to induce cytokine production in in vivo stimulated splenocytes. Lesion growth and specific antibody levels were determined in control and vaccinated mice and cytokine production by in vitro stimulated splenocytes was determined at termination of the experiment. Results indicate that the protein can protect against infection but only if the carrier is used.

P119. A novel prodrug approach to combination therapy in the treatment of cardiovascular disease

Nisha Perez1, Peter Germano1, Shanelle Ko2, Wayne Schrairer3, Timothy C. Barden3, Brian M. Cali4, Jeffrey Segal5 and Robert Busby1

1AP/Dmpk, Ironwood Pharmaceuticals, Cambridge, MA, USA, 2In Vivo Pharmacology, Ironwood Pharmaceuticals, Cambridge, MA, USA, 3Medicinal Chemistry, Ironwood Pharmaceuticals, Cambridge, MA, USA, 4Program Managment, Ironwood Pharmaceuticals, Cambridge, MA, USA, 5Discovery Biology, Ironwood Pharmaceuticals, Cambridge, MA, USA

Cardiovascular disease, the leading cause of death in the United States and other developed societies, can be managed with intensive risk factor modification, including treatment of hypertension and dyslipidemia. Fenofibrate is commonly used in the treatment of dyslipidemia, lowering LDL and triglycerides while raising HDL. Nitric oxide (NO) has a broad range of physiological functions encompassing nearly every system in the human body, including beneficial effects on vascular, platelet, and renal function. A combination approach that exploits the lipid-modifying properties of fenofibrate and the numerous beneficial effects of NO on vascular function and hypertension could provide an improved therapy for reducing cardiovascular risk in certain patient populations. Fenofibrate is a prodrug comprising fenofibric acid linked to an isopropyl ester. By replacing the isopropyl ester with a nitric oxide-donating moiety linked to fenofibric acid through a butyl ester, a new chemical entity (NCE) was created with the potential for additional cardiovascular benefit (IM8008-763). As expected, this NCE was rapidly hydrolyzed in both rat and human plasma (t

1/2 ≤15min), liberating

fenofibric acid and NO. In vivo, IM8008-763 was not detected in plasma following oral dosing to rats; however, both fenofibric acid and NO were detected with a T

max estimated to be 4-6 hr for both molecules. In all experiments, the par-

ent molecule IM8008-763 was detected using LC-MS/MS. This technique was also used for the detection of fenofibric acid. The NO was monitored using a colorimetric assay to detect nitrate as an in vivo surrogate for the short-lived NO species with. The ability to orally deliver both fenofibric acid and NO in a single molecule demonstrates the promise of this approach to develop a therapeutic option for the treatment of cardiovascular disease.

P120. Antioxidant Activity of Some Amide Derivatives of Diclofenac as Prodrugs

Tunca Gul Altuntas1, Tulay Coban1 and Fatma Sener2

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1Faculty of Pharmacy, Ankara University, Ankara, Turkey, 2Social Security Institution, Sivas, Turkey

Nonsteroidal anti-inflammatory drugs (NSAIDs) including Diclofenac are widely used for reducing inflammation and as an analgesic reducing pain in conditions such as arthritis or acute injury. The exact mechanism of action is not entirely known, but it is thought that the primary mechanism responsible for its anti-inflammatory, antipyretic, and analgesic action is inhibition of prostaglandin synthesis by inhibition of cycloxygenases (COX) and it appears to inhibit DNA synthesis (1). Their most common side effect is known as gastric irritation. In order to overcome this side effect amide derivatives of Diclofenac (Ia-h) were synthesized as prodrugs. Enzymatic hydrolysis of prepared amide derivatives by liver amidases gives Diclofenac in vivo in the body. In addition the cycloxygenase inhibition as an explanation of their pharmacological action, NSAID have also been shown to be powerful free radical scavengers. Reactive oxygen species and free radical reactions are related to several pathologic conditions including inflammation. Recently, Diclofenac has also been shown to suppress the production of superoxide anion (O2-) by disrupting activation of NADPH oxidase in phagocytes (2,3). In the present study the free radical scavenging properties of synthesized produgs as amide derivatives of Diclofenac (Ia-h) were examined in vitro by determining their capacity to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical. The synthesized prodrugs derivatives of Diclofenac were not able to scavenge DPPH radical. Only compound 1h showed radical-trapping ability for O2- by 95 % inhibitions at the 0.1mM concentration. These results suggest that amide derivatives of Diclofenac as prodrugs except for 1h may not involved in oxidative/anti oxidative processes in vitro system.

References

(1).Dutta NK, Annadurai S, Mazumdar K, Dastidar SG, Kristiansen JE, Molnar J, Martins M, Amaral L. (2000). The anti-bacterial action of diclofenac shown by inhibition of DNA synthesis. Int. J. Antimicrob. Agents 14 (3): 249-51.

(2).Yuda Y, Tanaka J, Suzuki K, Igarashi K and Satoh T. (1991). Inhibitory effects of non-steroidal anti-inflammatory drugs on superoxide genera-tion. Chem Pharm Bull (Tokyo) 39, 1075-1077.

(3).Bell AL, Adamson H, Kirk F, McCaigue MD and Rotman H.(1991). Diclofenac inhibits monocyte superoxide production ex vivo in rheumatoid arthritis. Rheumatol Int 11, 27-30.

P121. Does Lipid Environment Modulate Drug Efflux Activity Of ABC Transporters?

Peter Meszaros

Cell Biology, University of Groningen, Groningen, Netherlands

Introduction

ABC (ATP Binding Casette) transporters are transmembrane proteins, which influence the drugs ADME (Absorption, Distribution, Metabolism, Extretion) properties. They were discovered because of their ability to make cancer cells multidrug resistant (MDR). ABC proteins can utilize the energy derived from ATP hydrolysis to perform a directed transmembrane movement of their substrates, including elimination of certain xenobiotics. Because these efflux transporters are expressed in many important pharmacological barriers in the body, there is great need for in vitro assays which can predict the interaction of potential new drugs and ABC transporters in the drug development process. It is therefore important to understand the mechanism of these transporter proteins. There are several factors which influence the localization and/or activity of ABC proteins. Among these are interactions with other proteins, such as actin and the direct lipid environment, e.g. localization in lipid rafts, which are enriched in sphingolipids and cholesterol (Ref: Kok JW, Klappe K, Hummel I, Kroesen B-J, Sietsma H, Meszaros P, Are lipid rafts involved in ABC transporter-mediated drug resistance of tumor cells?, Trends in glycoscience and glycotechnology, Vol 20. No. 116(2008) pp.373-397). We focus on a highly MDR relevant protein, Multidrug Resistance Protein 1 (MRP1).

Approach: Our aim is to study the activity dependence of the MRP1 transporter on sphingolipids and cholesterol. In addition to intact cells, we use cell-derived membrane vesicles containing MRP1. We modify sphingolipid levels using

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an inhibitor of biosynthesis, ISP-1, and cholesterol content using physical (cyclodextrines) or chemical (cholesterol oxidase) means of de- or repletion. The effect on the specific activity of MRP1 was measured using the fluorescent substrate CFDA for intact cells, and a vesicular transport assay and ATPase assay for the vesicle system.

Results: After sphingolipid depletion (reduced to 10 % of control), the cells were viable and the activity of MRP1 increased in both intact cells and vesicles. Cholesterol depletion to 40 % of control did not change activity of MRP1, however cholesterol uploading did increase its activity.

Conclusions: Sphingolipid depletion and cholesterol uploading both increased MRP1 activity.

Future plans: Using this study we try to prepare better vesicle system to measure and predict drug-transporter interac-tions. We want to establish the underlying mechanism of MRP1 activity increase. Is it related to direct interactions with lipids or to interactions with lipid rafts? In this context we also investigate the role of the actin cytoskeleton, which is known to interact with lipid rafts.

P122. Drug Development for Pediatric Population in Japan

Junko Komura

Office of New Drug I, Pharmaceuticals & Medical Devices Agency, Tokyo, Japan

Background: ICH* E11 is the guideline of clinical investigation of medicinal products in the pediatric population for global development (* International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use). At the guideline, the timing of initiation of pediatric studies are discussed for the types of diseases, e.g. i) diseases predominantly or exclusively affecting pediatric patients, ii) serious or life-threatening diseases, and iii) other diseases and conditions. It is still unclear that how successful plan the clinical trials for pediatric population.

Method: In this study, I extracted the pediatric data from the approved review reports of medicinal products that are open to the public at the PMDA homepage. I compared the pharmacokinetics (PK) and efficacy between adult and pediatric popula-tion, and I also compared the ethnic differences of PK and efficacy in small molecule drugs and large molecule drugs.

Results: In the fiscal year (FY) of 2008 and FY of 2007, 79 and 81 medicinal products were approved in Japan respectively. Approximately quarter of those was approved for the pediatric use. Adolescent′s PK and children′s PK was similar to those of the adult, in the case of small molecule drugs and when that elimination half-life from blood was short. On the contrary, the PK of large molecule drugs were varied. Probably the results of PK parameters depend on the sampling schedule and sample measurement methods because of the long elimination half-life of the drugs.

Conclusions: Strategic plan for pediatric studies are needed for the clinical development. The simultaneous measure-ment of PK with adult and pediatric population is preferable, especially for large molecule drugs and long elimination half-life drugs.

P123. Effective MicroPET Imaging of Brain 5-HT1A Receptors in Rat with 18F-MEFWAY by Suppression of Radioligand Defluorination

Jae Yong Choi1, Chul Hoon Kim1, Byoung-Soo Kim2, Eun Jung Kim2, Jung Young Kim2, Tae Hyun Choi2 and Young Hoon Ryu3

1Department of Pharmacology, Brain Research Institute, BK 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea, 2Radiopharmaceuticals Development Team, Korea Institute of Radiological and Medical Science, Seoul, South Korea, 3Department of Nuclear Medicine, Yonsei University College of Medicine, Gangnam Severance Hospita, Seoul, South Korea

Introduction

The serotonin-1A receptors (5-HT1A

) in the central nervous systems are strongly implicated in psychiatric disorders such as depression, schizophrenia and Alzheimer′s disease. Recently, Saigal et al. developed [18F]MEFWAY which had a high

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binding affinity to these receptor and little defluorination in nonhuman primate [1]. In microPET study, we observed that this radioligand was subjected to significant bone uptake of radioactivity in rodent due to defluorination. Tipre D. N. et al. reported that the defluorination was occurred through the activation of cytochrome P450 2E1 isozyme in liver microsome and this phenomena was suppressed using antifungal agents [2] or antialcohol drugs [3]. The object of this study was to suppress defluorination of 18F-MEFWAY in rat and to evaluate the efficacy of inhibition of defluorination of 18F-MEFWAY on PET images.

Method: For in vivo experiment, male Sprague-Dawley rat was anesthetized with 2.0 % isofurane in oxygen and placed in the gantry with its head centered in the field of view. A catheter was inserted into the tail vein and fluconazole was injected at an infusion rate for 1 h. 18F-MEFWAY (13.1-19.6 MBq) was promptly injected over 1 min to the catheter and dynamic PET scans (Siemens, Inveon PET/CT) were recorded for 120 min. Control rats were injected with 18F-MEFWAY alone with no treatment.

Result: Before intravenous administration of 18F-MEFWAY, rats were pretreated with 0, 15 or 30 mg/kg fluconazole. PET experiments indicated that the skull uptake of radioactivity was reduced with adapting fluconazole, coupled with increasing brain uptake.

Conclusion: We conclude that 18F-MEFWAY is subject to defluorination in rat in vivo and such defluorination was greatly prevented by fluconazole, probably through the inhibition of CYP2E1. This 18F-MEFWAY fluconazole-treated rat may serve as an effective tool for studying 5-HT

1A receptors in rat models of neuropychiatric condition.

Acknowledgements

This study was supported by Korea Science and Engineering Foundation (KOSEF) (20090062241) and Ministry of Science & Technology (MOST), Republic of Korea, through its National Nuclear Technology Program

References

[1] Saigal, N. et al. Synthesis and biological evaluation of a novel serotonin 5-HT1A

receptor radioligand, 18F-Mefway in rodents and imaging by PET in non-human primate. J. Nucl. Med. 2006, 47, 1697-1706.

[2] Tipre, D. N. et al. PET imaging of brain 5-HT1A

receptors in rat in vivo with 18F-FCWAY and improvement by successful inhibition of radioligand defluorination with miconazole. J. Nucl. Med. 2006, 47, 345-353.

[3] Ryu, Y. H. et al. Disulfiram inhibits defluorination of 18F-FCWAY, reduces bone radioactivity, and enhances visualization of radioligand binding to serotonin 5-HT

1A receptors in human brain, J. Nucl. Med. 2007, 48, 1154-1161.

P124. Identification of novel anti-androgens that target the BF3 site of the androgen receptor

Nathan Lack, Peyman Tavassoli, Peter Axerio, Eric LeBlanc, Emma Guns, Artem Cherkasov and Paul Rennie

Vancouver Prostate Centre, Vancouver, BC, Canada

Background: Prostate cancer is one of the most commonly diagnosed cancer in men and one of the leading causes of cancer-related death in North America. If the cancer is diagnosed early, it is frequently curable by surgery or radio-therapy. However, locally advanced, recurrent or metastatic prostate cancer is much more difficult to control and patients are commonly treated with androgen withdrawal therapy. This is designed to either target the production of androgens or their binding to the androgen receptor (AR). While initially successful, the effectiveness of this type of treatment is usually temporary and the surviving tumour cells almost always progress to a “hormone-refractory” state. The treatment options for these patients are very limited and the median patient survival is 1-2 years. While the molecular mechanisms responsible for the progression to a hormone-refractory state are largely unknown, it does not involve a loss of AR and there is strong evidence that in most cases, the inappropriate activation of the AR is linked to the recurrent growth of prostate cancer. Therefore, inhibiting the AR offers a viable therapeutic strategy to treat hormone-refractory prostate.

Hypothesis: Almost all clinically approved anti-androgens work through a similar mechanism of action, whereby the compound directly inhibits the interaction of the androgen with the ligand binding pocket of the AR. Recent studies have identified a new binding site on the AR called the binding function 3 (BF3) that when bound can allosterically inhibit

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the co-activator association with the AR. Without the co-activators the AR cannot initiate the transcription pathway. This target therefore offers a novel way to inhibit AR activation.

Results and Conclusions: In an effort to identify inhibitors that target the BF3, an in silico screen was conducted whereby a database of ∼5 million chemical substances were computationally docked into the BF3 to identify those compounds that best fit. From these results, 220 compounds from several diverse chemical classes were selected and assayed using a eGFP cell-based AR transcription assay. Of the 220 compounds tested, 17 exhibited >50% inhibition of the AR at 50µM. Based on the MTS assays and cell morphologies, most of the 17 compounds were also found to be non-cytotoxic at the concentrations tested. Using the same eGFP-based assay, 3 of these compounds had an IC

50 <3 µM. These results were

further validated using a luciferase-based transfection assays and several of the compounds were found to have similar anti-AR activity to bicalutamide, one of the most potent anti-androgen drugs currently used to treat men with metastatic prostate cancer. Importantly, all of these compounds have very distinct chemical scaffolds to the anti-androgens cur-rently used in the clinic.

P125. In vitro Reactivity and Metabolism of Irreversible Tyrosine Kinase Inhibitors

Chitra Mani, Erik Verner, Wei Chen and David Loury

Pharmacyclics, Sunnyvale, CA, USA

Several irreversible tyrosine kinase inhibitors were screened for reactivity with glutathione and metabolite formation in vitro. The compounds were pyrazolopyrimidines coupled to various cyclic spacers with either a butynamide or an acrylamide substitution. The metabolic profile of PCI-43421 (m/z 453.1), with a butynamide substitution and a 4-piperidine spacer, was compared to that of PCI-31433 (m/z 441.2), with an acrylamide substitution and a 4-piperidine spacer, in both rat and human microsomal preparations. Metabolism of both compounds was dependent on a NADPH regenerating system. Following 60-minute incubation, 70.2% and 6.99% of PCI-43421 (butynamide) and 90.8% and 60.8% of PCI-31433 (acrylamide) remained unchanged in rat and human liver microsomes, respectively. These findings suggest that the butynamide-substituted inhibitors are more susceptible to oxidative metabolism than the acrylamide-substituted inhibitors. For PCI-43421, two major metabolites were identified in both rat and human liver microsome: M1 (m/z 485.2) with two inserted oxygen molecules and M2 (m/z 469.2) with single oxygen insertion. For PCI-31433, the major metabolites identified in rat and human microsomal preparation were M1 (m/z 475.2), a dihydrodiol, and M2 (m/z 457.2), with a single oxygen insertion, were identified in both species. The rate of glutathione adduct formation was three-fold lower for PCI-43421 (butynamide when compared to PCI-31433 (acrylamide). Replacing the 4-piperidine cyclic spacer of compound PCI-43421 with 2-methylpyrrolidine (R) or 4-aminocyclohexane (cis) further reduced the rate of glutathione adduct formation by approximately 7-fold and10-fold, respectively. Replacing the 4-piperidine cyclic spacer of compound PCI-31433 with 2-methylpyrrolidine (R) reduced the rate of glutathione adduct formation by approximately 3-fold. Thus, the butynamide-substituted analog with a 4-aminocyclohexane spacer had the least glu-tathione reactivity (4.94 ng/minute) and the acrylamide-substituted analog with a 4-piperidine spacer had the greatest glutathione reactivity (171.8 ng/minute). In human liver microsomes, the butynamide-substituted analog, PCI-43421 (4-piperidine spacer), exhibited poor metabolic stability.

P126. Innovative Strategy for MIST using LC/RI/MS and nanoLC-MS to quantify the comprehensive metabolites

Zenzaburo Tozuka, Shinsuke Aoyama, Kohei Nozawa, Yasuhisa Adachi and Shin-ichi Ninomiya

ADME & Tox. Research Institute, Sekisui Medical Co., Ltd, Tokai, Ibaraki, Japan

The MIST has changed from the FDA Guidance in February 2008 to ICH M3 in June 2009. The quantification method of metabolites in human plasma is one of the critical points for successful drug development.We presented the innovative strategy for MIST using microdosing clinical study of 14C-acetaminophen measured by AMS in JSSX 2009. We have investigated the relative concentrations of metabolites to total radioactivity by the highly sensitive LC-RI/MS/MS and nanoLC MS. The plasma concentrations of metabolites derived from the pre-clinical study were calculated using LC/RI/MS/MS in the absence of chemical standards. The quantification of metabolites after microdosing needs high sensitive analytical method. Now, Our data of nanoLC MS shows thousand times high sensitive LLOQ in comparison with that

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of UPLC MS.(Method)The standard some compounds (small molecules, protein and siRNA) were dissolved and made up from 0.01ng/mL t o100 µg/mL solution. The 10 uL of solution was analyzed by UPLC-MS and nanoLC-MS. Plasma and tissue samples of the compounds were quantified by UPLC-MS and nanoLC-MS. The 14C-labelled compounds were metabolized with rat liver microsomes and administered at microdose in humann and rat. The metabolites of compounds in human and rat were analyzed quantitatively and qualitatively by UPLC-MS and nanoLC-MS.(Result)The LLOQ of compounds was 1pg/mL by nanoLC-MS (Range 1-1000 pg/mL) that is thousand times higher sensitive than 1ng/mL by UPLC-MS (Range 1-1000 ng/mL). The plasma and tissue concentrations of compounds were measured the high dose samples by UPLC-MS and the low dose samples by nanoLC-MS. The metabolites of 14C-labelled compounds in rat liver microsomes were analyzed by LC/RI/MS. The ionization efficiencies of the metabolites of compounds were calculated from their RI intensity/MS intensity. The plasma concentration of the metabolites of compounds after micro-dosing were measured by UPLC-MS and nanoLC/MS and calculated using their ionization efficiencies. We solved many problems such as matrix effects and the different factors between mass spectrometers. NanoLC-MS is useful for quantitative and qualitative analysis after microdosing study.

P127. Mass Balance Studies using LC-MS with Integrated UV Detection

Carmai Seto and Takeo Sakuma

Product Application Lab, AB Sciex, Concord, ON, Canada

Introduction Forced degradation of drugs plays an essential role in drug development. These studies facilitate the design of formulations and gives insight into the stability of the drug. Mass balance is critical to forced degradation studies. HPLC-UV is often used, but sometimes mass balance cannot be attained due to the formation degradates without a UV chromophore or coelution of degradates with the parent. As a result, LC-MS combined with UV is used to identify these unknown degradates as well as for the determination of mass balance. By integrating UV and MS detection with a powerful software such as LightSight® software, a more complete picture of the degradation pathway is possible and data analysis and reporting can be easier to manage.

Methods: Methanolic solutions of nifedipine were placed in a clear vial and irradiated by an artificial sunlight (UV) source and UV/VIS light (ie daylight) for a period ranging from 0 to 360mins (n=3). Aliquots of the solutions were taken at different time points to determine mass balance. The degradates of nifedipine were identified and characterized using a 3200 QTRAP® system with a DAD detector. An LC-MS method, with integrated UV detection, was generated by LightSight® software and used to monitor potential drug degradates of nifedipine. The mobile phase was 0.1% formic acid in water and 0.1% formic in acetonitrile using a Betasil C18 column.

Preliminary Data: This work will focus on the photo-degradation of nifedipine as the model drug. One major problem with existing photostability studies of nifedipine is that mass balance is not determined and that not all degradation products were accounted for. These studies used HPLC-UV as the method of detection. By integrating LC-MS with UV, it is possible to detect if there are any co-eluting degradants or if the UV chromophore of the degradants have been drastically modified. Some of the degradates identified and characterized were the nitroso-, dehydro-, nitro- derivatives. The MS and UV data were automatically correlated using LightSight® software, version 2.2. The software was used to identify, confirm and quantitate degradates of nifedipine in using both data sets. This allows for both structural elucidation based on the MS/MS data and estimation of mass balance based in the UV and the MS. Quantitation of the degradates with out a chromophore is still possible because the MS data can be used to estimate the amount present in the sample.

P128. Abstract Withdrawn.

P129. Microcrystals prepared by in situ micronization: pharmacokinetic studies in rats

Roya Talari1, Jaleh Varshosaz2, Abolfazl Mostafavi2 and Ali Nokhodchi3

1Pharmaceutics, Food and Drug Laboratories Research Center, Tehran, Iran, 2Pharmaceutics, Pharmacy Faculty, Isfahan University of Medical Sciences, Isfahan, Iran, 3Pharmaceutics, Medway School of Pharmacy, The Universities of Kent and Greenwich, Kent, England

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The low water-solubility of gliclazide (GL) leads to a low dissolution rate and variable bioavailability. The aim of this study was to investigate the effect of micronization on the absorption and pharmacokinetics of GL after oral administration in rats. GL microcrystals were prepared using pH-shift method. Glucose lowering effect, C

max and AUC were compared

after administration to diabetic and normal rats. Glucose lowering effect in administration of 40 mg/kg GL microcrystals (d

mean: 3.09-5.3 µm) in comparison to untreated GL (d

mean: 290.00 ± 36.0 µm) was increased 21.04% and 24.33% in diabetic

and normal rats, respectively. AUC and Cmax

were increased in both groups of animals.

P130. New Prodrugs of the Antiprotozoal Drug Pentamidine

Joscha Kotthaus, Björn Cohrs, Jürke Kotthaus, Dennis Schade, Helen Hungeling and Bernd Clement

Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University, Kiel, Germany

Pentamidine is an effective antimicrobial agent and an approved drug in the treatment of African trypanosomiasis. Due to the strong basic amidine functionalities pentamidine suffers from poor oral bioavailability. Furthermore, it lacks in CNS delivery that leads to the fact that pentamidine is only useful for i.v. or i.m. treatment of the first state of African trypanosomiasis, which is called the haemolymphatic state. In order to improve the oral and CNS bioavailabilty of pentamidine many attempts have been made recently to overcome this substantial problem by using several prodrug principles.[1,2] Previous studies deal with the conversion of the amidine functions into less basic amidoximes resulting in an uncharged and thus a more permeable molecule. Further modifications resulted in the O-acetylamidoxime prodrug, the diacetyldiamidoximeester, which has greatly improved lipophilicity compared to the amidoxime-prodrug and showed oral bioavailability in a study with rats.[1] However, all prodrugs of pentamidine described to date suffer from poor solubility and CNS bioavailability and justify further efforts to develop new prodrugs with enhanced properties. This work deals with the development and comparison of new prodrugs of pentamidine. For this reason, numerous prodrugs using different prodrug principles were synthesized and characterized in in vitro and in vivo studies. Some prodrug principles, such as the O-alkylation with an carboxymethyl-residue or the conversion into amidoximes had been published earlier,[3] whereas other prodrug principles were developed more recently and are described for ami-dines within this work for the first time. In contrast to original principles these methods are not developed to increase lipophilicity and oral bioavailability via passive uptake (diffusion) from the intestine. These principles increase the affinity to transporters and mediate an active uptake via carrier systems by conjugation of amidoximes with solubil-ity improving compounds. The variety of the prodrug principles chosen resulted in several prodrugs of pentamidine bearing various advantages. The objective of this investigation was the systematic characterization and evaluation of all pentamidine prodrugs in order to discover the most appropriate pentamidine prodrug. For this reason, all prodrugs were examined concerning solubility, stability, enzymatic activation, distribution, CNS delivery, and oral bioavail-ability. In summary, this work allows reliable conclusions regarding the best prodrug principle for the antiprotozoal drug pentamidine.

References

[1] B. Clement et al., Diacetyldiamidoximeester of pentamidine, a prodrug for treatment of protozoal diseases: synthesis, in vitro and in vivo biotransformation. ChemMedChem 2006, 1 (11), 1260-7.

[2] B. Clement et al., Reduction of amidoxime derivatives to pentamidine in vivo. Arch Pharm 1992, 325 (1), 61-2. [3] D. Schade et al., The peptidylglycine alpha-amidating monooxygenase (PAM): a novel prodrug strategy for amidoximes and N-hydroxyguanidines?

ChemMedChem 2009, 4 (10), 1595-9.

P131. Abstract withdrawn

P132. Novel reduction mechanism of Azo Pro-Drugs into anti-Inflammatory Arylamines by Azoreductases from Pseudomonas Aeruginosa

Laurieri Nicola1, Ryan Ali1, Isaac Westwood1, Chan-Ju Wang1, Ed Lowe2 and Edith Sim3

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1Pharmacology, University of Oxford, Oxford, United Kingdom, 2Biochemistry, University of Oxford, Oxford, United Kingdom, 3Pharmacology, Univ of Oxford, Oxford, United Kingdom

Azoreductase enzymes are found in a wide range of prokaryotic and eukaryotic organisms, including within human gastroenteric micro flora. Azoreductases are important due to their ability to detoxify azo dyes, generating arylamines. They also activate anti-inflammatory azo pro-drugs, such as balsalazide to generate the arylamine 5-amino salicylic acid (5-ASA), which is the active therapeutic moiety in azo pro-drug treatment for Inflammatory Bowel Disease (IBD). The therapeutically active arylamine is then metabolised and inactivated by N-acetylation and hence the intestinal azoreductase activity and N-acetyltransferase activities act in concert to determine the concentration of the active metabolite available in IBD. The aim of the work is to understand the dynamic between the two enzymic activities, azoreductases and N-acetyltransferases and we have undertaken an analysis of the azoreductase enzymes present in the bacterium Pseudomonas aeruginosa which has been reported to be present in intestinal flora. Three genes encoding azoreductases have been identified in P. aeruginosa. We describe here a comparison of the three enzymes following cloning the egenes and producing pure recombinant proteins. The proteins are extremely soluble with up to 80mg/litre of culture being produced. The pure recombinant proteins each have a distinct substrate specificity profile against a range of azo substrates. Using the structure of paAzoR1 (P. aeruginosa azoreductase 1) (1) and homology models of paAzoR2 and paAzoR3, residues important for substrate specificity have been identified. We have carried out site-directed mutatgenesis to investigate the role of the individual residues including tyrosine 131, and have demonstrated that this residue does not provide the reducing proton (2). We have defined a novel flavin mononucleotide binding cradle which is a recurrent motif in many flavodoxin-like proteins. A novel structure of paAzoR1 with the azo pro-drug balsalazide bound within the active site was determined by X-ray crystallography. This structure together with the site-directed mutagenesis studies identified a novel catalytic mechanism for azoreduction. The azo substrate is present in the active site as a hydrazone tautomer and the azoreductase generates the arylamine products, the anti-inflammatory 5-ASA, via the formation of iminoquinone intermediates. These studies pave the way for the possibility of generating improved azo prodrugs for IBD.

References

1. Wang, C.J., Hagemeier, C., Rahman, N., Lowe, E., Noble, M., Coughtrie, M., Sim, E & Westwood, I. (2007) J. Mol Biol. 373, 1213-1228.2. Wang, C-J., Laurieri, N., Abuhammad, A., Lowe, E., Westwood, I, Ryan, A. & Sim, E. (2010) Acta Crystallographica F66, 2-7.

P133. Optimisation of Isoenzyme-Specific Reagents: Organic Synthesis and Biological Characterisation of Naphthoquinones as Selective Inhibitors of Human n-Acetyltransferase 1 (hnat1) and Mouse nat2 (mnat2)

Nicola Laurieri1, Cyrille Thinnes1, Isaac Westwood1, Ballester Pedro2, Peter Seden2, Stephen G. Davies2, Angela Russell1 and Edith Sim1

1Department of Pharmacology, Univ of Oxford, Oxford, United Kingdom, 2Chemistry, Univ of Oxford, Oxford, United Kingdom

Human NAT1 is one of the ten most highly overexpressed genes in oestrogen-receptor-positive (ER+) breast cancers and its overexpression is strongly related to tumour grade. With the exact endogeneous role of hNAT1 still unknown, the association with ER levels may implicate a role in cancer progression, making it an attractive potential biomarker for ER+ breast cancers and/or a novel therapeutic target. Mouse offers a good animal model for investigations on human NATs: hNAT1 and mNat2 are orthologue genes and the corresponding proteins, hNAT1 and mNat2, are homologous on the basis of C-terminus identity, substrate specificity and expression profile. We have therefore sought inhibitors which are specific for hNAT1 and mNAT2 with the aim of using these inhibitors to aid in determining their in vivo function (1). Naphthoquinone 1, with IC50 = 1.65 µM for hNAT1 and 1.86 µM for mNAT2, was identified as a selective competitive inhibitor with one binding site for hNAT1 and mNAT2 from a high-throughput screen and displays a distinctive colour change from red (?max = 484 nm) to blue (?max = 610 nm) upon reversible binding to both hNAT1 and mNAT2, but not the other human and murine isoenzymes. It has been suggested the colour change is related

108

to deprotonation of the sulphonamide moiety within the active site of the enzymes (2). With the goal of improving both inhibitory potency and colorimetric properties of the hit-compound, a set of analogues varying at the R1, R2 and R3 positions was chemically synthesised. The resulting substitutions altered inhibitory activity, range of colour change, extinction coefficient and acidity of the naphtoquinones. To confirm the hypothesis of colour change upon sulphonamide deprotonation, a series of pH titrations with both nucleophilic and non-nucleophilic bases was carried out. In addition, analogues possessing no acidic sulphonamide proton and an 15N-labelled molecule for NMR studies were synthesised in order to explore their acid-base behaviour. Besides allowing to elucidate the endogenous role of hNAT1 and mNAT2 via Chemical Genetics as a specific inhibitor, a naphtoquinone 1 analogue may also be useful as a coloured biosensor for detecting hNAT1 in breast cancer tissues. We have also used naphtoquinone 1 for ultrafast shape recognition to open exploration of all purchasable chemical space to help identify novel classes of inhibitor(3).

References

1. Russell, A.J,. et al., (2009) Bioorgan. Med. Chem. 905-918. 2. Laurieri N. et al., (2010) J. Amer. Chem. Soc. 132, 3238-3239. 3. Ballester, P.J. et al., (2009) J. Roy. Soc. Interface doi. 10.1098/rsif.2009.0170.

P134. Pharmacokinetics, distribution, metabolism, excretion and mass balance of radioactivity in rats following a single subcutaneous bolus injection of [35S]-ISIS 388626

Richard Harrison1, Colin Webber1, Rosie Yu2 and Richard Geary2

1Drug Metabolism, Huntingdon Life Sciences, Huntingdon, United Kingdom, 2Isis Pharmaceuticals Inc, Carlsbad, CA, USA

ISIS 388626 is a 2′Omethoxyethylmodified (2′MOE) phosphorothioate oligonucleotide with 12 nucleotides in length (12-mer) currently under development for use in the treatment of Type 2 diabetes mellitusIt is an antisense drug designed to inhibit the expression of human sodium-dependent glucose cotransporter 2 (SGLT2), a key component in controlling glucose re-absorption in the kidneyThe study described was conducted to obtain information on the pharmacokinetics, distribution, metabolism, excretion and mass balance of [35S]ISIS 388626 in the ratMale and female Sprague-Dawley CD rats each received a single subcutaneous administration of [35S]ISIS 388626 as a solution in phos-phate buffered saline (PBS) at target dose levels of 3 or 10 mg/kgIn addition, one male rat received a single subcutaneous administration of PBS onlyLevels of radioactivity were determined in plasma, tissues and excreta by liquid scintillation analysisThe distribution of radioactivity was also investigated using quantitative whole body radioluminography and in kidney by microautoradiographyRadioactive components present in urine, faeces and kidney were separated and quantified using HPLC coupled with radioactivity detection or mass spectrometryThe results demonstrated: 1) rapid absorption, with maximum concentrations of radioactivity in blood and plasma attained 1 h post-dose, followed by a multi-phasic, protracted decline, consistent with slow elimination from tissues; 2) [35S]ISIS 388626 related radioactiv-ity was cleared rapidly from blood, predominantly due to distribution into tissues, with the kidneys being the major tissue of distribution (approximately 45% of the administered dose at 24 h post-dose), with low levels also observed in the axillary and mesenteric lymph nodes, skin, liver and thyroid (more than 20 fold lower than in the kidneys); 3) the presence of radioactivity in the tubular epithelium, with a relatively uniform distribution throughout the kidney cortex and little radioactivity scattered in the kidney medulla; 4) slow clearance from tissues, with an elimination halflife typi-cally between one to three weeks; 5) the majority of the administered radioactivity (approximately 91.4% of dose) was eliminated by 28 d postdose, with approximately two thirds in urine and the remainder in faeces; 6) slow metabolism in tissues, principally via nuclease mediated cleavage, which was followed by elimination in urine and faeces.

P135. EVALUATION OF ANTIDEPRESSANT-LIKE EFFECTS OF MELATONIN IN FEMALE RAT IN FORCED SWIM TEST

el Mrabet fatima Zahra, Ali Ouichou and Abdehalim Mesfioui

Departemnt of Biology, Ibn Tofail University, Faculty of Sciences, Kenitra, Morocco

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The depression is a behavioral disorder which affects a wide population worldwide. This disease involves a dysfunction of the central monoaminergic neurotransmission (noradrenergic and serotoninergic). The main action of most known antidepressants passes by the regulation of the changes arising at the level of these systems. Currently, numerous studies are interested in the melatonin, as molecule susceptible to have antidepressive benefits. The aim of this study was to explore the effect of the melatonin associated with the hydroxyzin (H1 antihistaminic and anticholinergic) on the levels of depression in the female rats. Control group received NaCl 9 ‰, the treated group received 4 mg/Kg of melatonin or 10 mg/Kg of hydroxyzine or both substances. All the injections were done chronically. The depression level was evaluated by forced swim test. Then, the rats were sacrificed and their adrenal glands were removed and weighed. Statistical analysis was realized by ANOVA and” T” test. The obtained results had shown that only the mela-tonin had decreased highly significantly the immobility time (Tim) and increased significantly Exhaust time. However, the injection of hydroxyzin didn′t show any significant effect compared to control results. Also, any potentialisation between both molecules was registered. Furthermore, injection of Mel didn′t show any significant effect on the weight of adrenal glands, and this may be related to the duration of treatment that was not sufficient to reduce the volume of adrenal glands.

P136. Preclinical Pharmacokinetic Assessment of GDC-0152, a Selective Antagonist of the Inhibitor of Apoptosis (IAP) Proteins

Hank La1, Jason S. Halladay1, Young Shin1, Susan Wong1, Emile Plise1, O. Helen Chan1, John Flyg-are2, Wayne Fairbrother3 and Harvey Wong1

1Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA, USA, 2Medicinal Chemistry, Genentech, South San Francisco, CA, USA, 3Protein Engineering, Genentech, South San Francisco, CA, USA

Inhibitor of apoptosis (IAP) proteins are believed to suppress apoptosis and are overexpressed in a variety of cancers. GDC0152 is a potent and selective antagonist of the IAP proteins that was developed as a potential treatment of tumors that are resistant to chemotherapies or radiotherapy. The preclinical pharmacokinetics of GDC-0152 investigated in mouse, rat, dog, and cynomolgus monkeys. Briefly, the compound was dosed intravenously (IV) at 1 mg/kg in mouse, rat, dog and monkey. In mouse, rat and monkey, plasma clearance was high (>80% of hepatic blood flow) at 72.0, 66.7, and 47.1 mL/min/kg, respectively. In dog, GDC-0152 had a moderate plasma clearance (52% of hepatic blood flow) of 16.2 mL/min/kg. In dose ranging studies, clearance was nonlinear in mouse and rat, but linear in dog and cynomolgus monkey. Estimates of volume of distribution were moderate in all species tested, ranging from 1.04 L/kg in dogs to 2.01 L/kg in rats. Mean half-life ranged from 0.427 hours in mice to 0.933 hours in dogs. Plasma protein binding was moderate (75%-92% bound) across species, except in rabbit where it was approximately 95% bound. Blood-plasma partition ratios ranged from 0.579 to 1.08 in all species tested suggesting that GDC-0152 does not pref-erentially distribute into red blood cells. Prediction of clearance in humans was performed using allometry and in vitro scaling using human hepatocytes. Both methodologies resulted in similar predicted human clearance values of 9.4 (allometry adjusted for maximum life potential) and 10 mL/min/kg (in vitro scaling using hepatocytes). Volume of distribution was predicted to be moderate at 1.2 L/kg in humans using allometry. Predicted half-life in humans was approximately 1.5 hours. Based upon the predicted pharmacokinetic profile in humans, this compound was advanced to human phase 1 clinical trials.

P137. Preparation and Characterization of Clarithromycin Nanoparticles by Antisolvent Precipitation

Mansour Mansouri1, Hamid Reza Pouretedal2 and Mohammad Ali Hassan Ali1

1Islamic Azad University, Shahreza Branch, Isfahan, Iran, 2Department of chemistry, Malek-e-ashtar University of Technology,Shahin-shahr, Isfahan, Iran

Hypothesis: The oral bioavailability (about 55%) of clarithromycin in water solution is low because the low solubility and slow dissolution rate1. Nanoparticles were thus prepared by antisolvent precipitation method in this work for increasing accelerate dissolution of this kind of poorly water-soluble drugs2.

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Methods used: Clarithromycin nanoparticles were produced by solvent/antisolvent precipitation technique. A certain amount of drug was completely dissolved in water-miscible solvent (acetone). The obtained drug solution was then injected into the antisolvent (water), containing the (TEA / HPMC) as stabilizer under stirring at 3000 rpm. Precipitation of solid drug particles occurred approximate 10 min during mixing. The suspension was centrifuged at 15,300 rpm for 20 min and washed twice with purified water. Supporting data: All chemicals used were of ana-lytical grade or of the highest purity available, Clarithromycin was purchased from SANDOZ Pharmaceutical Co., Hydroxypropyl methyl cellulose (HPMC) was provided by JRS Pharmaceutical Co. TEA (triethanolamine), acetone and other solvents, such as HPLC grade acetonitrile and monobasic potassium phosphate were from Merck and Purified Water.

Results and conclusions: Clarithromycin nanoparticles were prepared by antisolvent precipitation in this work. For this technique, the high supersaturated drug solution (35 mg/ml) was prepared in acetone solvent because nucleation rate is fast in this concentration and produces a large number of nuclei, which reduces the solute mass for subsequent growth. Nanoparticles can be arrested by the stabilizer through steric or electrostatic mechanism. For hydrophobic drugs like clarithromycin, water is most commonly used as the antisolvent. In terms of the solvent, it is beneficial if it can solubilize the drug in large amount and possesses a fast diffusion rate to the antisolvent water, while the stabilizer needs to have good affinity for drug particles and possess a fast diffusion rate and effective adsorption onto the drug particle surface in the water–solvent mixture. The dissolution rate of clarithromycin nanoparticles is 3.663 times that of raw drugs. The accelerated dissolution for clarithromycin nanoparticles could thus be mainly ascribed to their greater surface area in comparison with raw drug. This indicates that the acceleration of drug dissolution for nanoparticles occurs mostly in the first 30 min, after that, the dissolution has no significant difference from raw drugs. Antisolvent precipitation technique was employed to producing nanoparticles of clarithromycin, a poorly water-soluble drug, for the enhancement of solubility and dissolution velocity. The precipitated clarithromycin nanoparticles had the same chemical structure with raw drug, but the particle size was reduced to 234.5 nm. Clarithromycin nanoparticles exhibited significantly faster drug dissolution compared with raw drug. The dissolution rate of clarithromycin nanoparticles is 3.663 times that of raw drugs. Antisolvent precipitation can thus be a simple and effective approach to produce submi-cron particles of poor water soluble drugs.

References

[1] SeanSweetman, Pharmaceutical Press,Clarythromycin, ISBN: 8495993201, 2008. [2] Nagy, Z. K.; Fujiwara, M.; Braatz, R. D. A Process Analytical Technology Based Investigation of the Polymorphic Transformations during the

Antisolvent Crystallization of Sodium Benzoate from IPA/Water Mixture, J. Process Control,18, 856–864, 2008.

P138. Preparation and Characterization of Erythromycin Ethylsuccinate Nanoparticles by Solvent/Anti-solvent Precipitation

Mohammad Ali Hassan Ali1, Hamid Reza Pouretedal2 and Mansour Mansouri1

1Islamic Azad University, Shahreza Branch, Isfahan, Iran, 2Department of chemistry, Malek-e-ashtar University of Technology,Shahin-shahr, Isfahan, Iran

Hypothesis: Erythromycin is a macrolide antibacterial with a wide spectrum of activity, that has been used in the treatment of a wide variety of infections caused by susceptible organisms. Erythromycin Ethylsuccinate have about 55% oral bioavailability in water due to its low solubility and slow dissolution rate speed1. The prepared nanoparticles by sol-vent / anti-solvent method accelerate dissolution rate of poorly water-soluble drugs like erythromycin Ethylsuccinate2. Reducing drug particles size is an effective and widely used approach to speed up dissolution by enlarging the effective surface area. According to Noyes–Whitney equation, the dissolution rate is proportional to the surface area exposed to the dissolution medium.3

Methods used: Erythromycin Ethylsuccinate nanoparticles were prepared by solvent/antisolvent precipitation method. We solved a certain amount of raw drug completely in water-miscible solvent (Acetone). Then the obtained drug solu-tion was injected into the anti-solvent (water), containing the (Triton X100 / HPMC) as stabilizer under stirring at 3000 rpm. Precipitation of solid drug particles occurred approximate 10 min within mixing time. The suspension was centrifuged at 15,300 rpm for 25 min and washed twice with purified water. Supporting data: Erythromycin ethylsuccsinat

111

was purchased from SANDOZ Pharmaceutical Co., Hydroxypropyl methyl cellulose (HPMC) was provided by JRS Pharmaceutical Co.. Triton X100 and acetone were from Merck.

Results and conclusions: We prepared Erythromycin Ethylsuccinate nanoparticles by solvent / anti-solvent precipita-tion method. In this method, the high supersaturated drug solution (250 mg/ml) was prepared in acetone, because nucleation rate is fast in this concentration and produces a large number of nuclei. For Erythromycin ethylsuccsi-nat, was used the water as the anti-solvent. The dissolution rate of Erythromycin ethylsuccinat nanoparticles is 2.1 times of raw drugs. The accelerated dissolution for Erythromycin ethylsuccinat nanoparticles could thus be mainly ascribed to their greater surface area in comparison with raw drug. Solvent / anti-solvent precipitation method was developed to producing nanoparticles of Erythromycin ethylsuccsinat. The precipitated Erythromycin ethylsuccsinat nanoparticles in comparison with raw drug, show the same chemical structure, but the crystallinity was reduced. The solubility of Erythromycin ethylsuccsinat nanoparticles was increased in comparison to raw drug, but the particle size was reduced to 99.5 nm. The dissolution rate of erythromycin ethylsuccsinat nanoparticles is 2.1 times of raw drug. Solvent /anti-solvent precipitation can thus be a simple and effective approach to produce nanoparticles of poor water soluble drugs.

References

[1] SeanSweetman, Pharmaceutical Press,Clarythromycin, ISBN: 8495993201, 2008.[2] Merisko-Liversidge, E., Liversidge, G.G., Cooper, E.R., Nanosizing: a formulation approach for poorly- water-soluble-compounds. Eur. J. Pharm.

Sci. 18, 113– 120, 2003.[3] Kesisoglou, F., Panmai, S., Wu, Y., Nanosizing-oral formulation development and biopharmaceutical evaluation. Adv. Drug Deliv. Rev. 59,

631–644, 2007.

P139. Structural Studies on the Mycobacterial Arylamine N-Acetyltransferases

Areej M. Abuhammad, Edward D. Lowe, Elizabeth Fullam, Martin Noble, Judith Schweichler, Elspeth F. Garman and Edith Sim

Department of Pharmacology, Univ of Oxford, Oxford, United Kingdom

Treatment of latent tuberculosis infection remains an important goal of global TB eradication. To this end, targets which are essential for intracellular survival of M. tuberculosis are particularly attractive. Arylamine N-acetyltransferase (NAT) represents such a target along with the enzymes encoded by the associated gene cluster involved in cholesterol degradation. Cholesterol is likely to be the fuel for M. tuberculosis inside macrophages and NAT can use the cholesterol catabolite n-propionyl-CoA, as well as acetyl-CoA (1), whilst the other gene products in the same operon catabolise sterol ring degradation. These gene products are essential for mycobacterial survival inside macrophages. Deleting the nat gene and inhibiting the NAT enzyme prevents survival of the microorganism in macrophages and induces cell wall alterations, rendering the mycobacterium sensitive to antibiotics to which it is normally resistant (2). Solving the 3D structure is considered essential starting step towards understanding the biological function. The insolubility of recom-binant NAT form M. tuberculosis (TBNAT) has been a major limitation towards solving the 3D structure of this enzyme. A comparison of the enzyme from M. tuberculosis with the more readily expressed protein from Mycobacterium marinum has demonstrated that the proteins are very similar (3,4). It is also essential to determine the structure of the enzyme with a ligand bound to aid structure based design of inhibitors. The aim of the work is to determine the structure of the NAT enzyme from M. marinum with a ligand bound and also to improve the production of soluble recombinant NAT from M. tuberculosis. We have improved of the production of soluble TBNAT by subcloning the his-tagged gene into pVLT31 expression vector. The new expression system generates sufficient soluble enzyme (up to 16 mg/L bacterial culture) which was purified to a level suitable for crystallisation trails. We also present a parallel structural studies on the active site of MMNAT. We chose the best substrate from a range of hydrazines and arylamines. The bext substrate was hydralazine and we have solved the structure of the co-crystallised complex to 2.1 Å resolution. From these stud-ies a novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl CoA to the heterocyclic aromatic N atom of hydralazine rather than the hydrazine N atom. This reaction mechanism explains why only the cyclic acetylated metabolite N-methyltriazolophthalazine is produced when hydralazine is acetylated.

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References

1.Lack, N., Kamamura, A., Fullam, E., Laurieri, N., Beard, S., Russell, A., Evangelopoulos, D., Westwood, I & Sim, E. (2009) Biochem J, 418, 369-378.

2. Westwood, I., Bhakta, S., Russell, A., Fullam, E., Anderton, M.C., Kawamura, A., Mulvaney, A.W., Vickers, R., Mulvaney, A., Bhowruth, V., Besra, G.S., Lalvani, A., Davies, S., Sim, E. (2010) Protein & Cell DOI 10.1007/s13238-010-0006

3. Fullam, E., Kawamura, A., Wilkinson, H., Abuhammad, A., Westwood, I., Sim, E. (2009) Protein J., 28, 281-293. 4. Sikora, A.L., Frankel, B.A., Blanchard, J.S. (2008) Biochemistry, 47, 10781-10789.

P140. Synthesis and Evaluation of 4-Nitro- Substituted 1,3-Diaryltriazenes as a Novel Class of Potent Antitumor Agents

Tamara Čimbora-Zovko1, Anamaria Brozović1, Krešimir Molčanov2, Biserka Kojić-Prodić2, Ivo Piantanida3, Gerhard Fritz4, Andrej Virag5, Branko Alič5, Marijan Kočevar5, Slovenko Polanc5 and Maja Osmak1

1Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia, 2Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia, 3Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia, 4Department of Toxicology, University of Mainz, Mainz, Germany, 5Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

Various triazenes have an important application in the development of anticancer molecules. In order to improve the antitumor efficacy of diaryltriazenes, we synthesized and examined cytotoxic activity of a new class of 1,3-diaryltriazenes, namely 4-nitro-substituted 1,3-diaryltriazenes. Structure-activity-relationship analysis reveals that 1,3-diaryltriazenes can be modified from not active to highly cytotoxic compounds by the introduction of two nitro groups at the para positions of benzene rings and two additional electron-withdrawing groups (bromo, chloro, trifluoromethyl or fluoro substituents) at their ortho position. Generally, the trifluoromethyl substituent has the strongest influence on antiproliferative activity, while fluoro substituted triazenes were the least active. Acetylation of the ortho substituted bis(4-nitrophenyl)triazenes increased the cytotoxicity of these coumpounds; an introduction of other acyl groups influenced the cytotoxicity of these compounds as well. To examine in more details the activity of one of the compounds having IC

50 < 1 μM, we selected for

further studies 3-acetyl-bis(2-chloro-4-nitrophenyl)triazene (8b). This compound was highly cytotoxic against different tumor cells, whereas its antiproliferative activity depended on the origin of tumor cell lines. It turned out that 8b was significantly more cytotoxic to tumor cells than to normal cells. Therapeutic index (T.I.), calculated from the ratio between cytotoxicity (IC

50) on tumor cells and cytotoxicity (IC

50) on normal fibroblasts was the lowest for metastatic SW620 colon

carcinoma cells (0.032) and increased in the following order: primary colorectal carcinoma SW480 cells and pancreatic carcinoma MIA PaCa-2 cells < cervical HeLa and laryngeal carcinoma HEp-2 cells <urinary bladder carcinoma RT-112 cells (0.514). Importantly, triazene 8b was similarly cytotoxic to parental laryngeal carcinoma HEp-2 cells as to their cisplatin-resistant CA3

ST subline. 8b compound arrested HeLa cells in the G0/G1 phase of the cell cycle. X-ray structure

analysis revealed non-planar conformation of 8b, which hampered intercalation into double stranded DNA. Furthermore, DNA binding analysis suggests that 8b does not bind into the minor groove of DNA, either. Instead, it induces reactive oxygen species that could provoke endoplasmic reticulum (ER) stress, finally leading to apoptosis. Our data suggest that 4-nitro-substituted 1,3-diaryltriazenes are a new class of anticancer molecules which preferentially target malignant cells. Therefore we propose these novel derivatives as potential antitumor agents.

P141. Understanding retention of inhaled therapies in the lung

Sebastien Ronseaux, Ruth Lock and Bindi Sohal

Novartis Horsham Research Centre, Horsham, United Kingdom

It is anticipated that in 2020 out of 68 million deaths worldwide, 11.9 million will be caused by lung diseases 1. For treatment of respiratory diseases, the lung has several key properties which make inhaled dosing an attractive option for drug delivery over standard oral therapies (e.g., target site, large surface area for absorption, highly perfused with blood). However these same properties also make it very difficult for the pre-clinical scientist to design low dose small

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molecule inhaled therapies that will be retained by the lung at their site of action. The relationship between physico-chemical properties and oral absorption of drug substances is fairly well established. Lipinski (1999) demonstrated that approximately 90% of orally active drugs all had a MWT ≤500, a logP ≤5, ≤5H-bond donors and ≤10H-bond acceptors. Trend analyses have also been carried out to assess the evolution of synthetic oral compound properties 3. In contrast to oral compounds, inhaled compounds have been less well studied with only one study that has tried to rationalise physicochemical properties of inhaled compounds. The study carried out by Ritchie et al looked at 81 currently marketed respiratory compounds. Among these compounds only 39 compounds were administered via inhaled (33 compounds) and/or intranasal (6 compounds) routes. The analysis revealed that inhaled/intranasal compounds had significantly higher hydrogen bonding and polar surface area when compared with oral respiratory compounds 4. Further work has been initiated to understand which properties of inhaled drugs can be manipulated to improve retention of drug within the lung. A comprehensive literature search has been conducted and an Excel database of 66 inhaled compounds has been populated with physicochemical properties (55 descriptors), in vitro (11 descriptors), pre-clinical PK and clinical PK (62 descriptors). Similar data from a database of oral compounds have also been added for comparison and hypoth-esis testing 2. Physicochemical descriptors were generated from the collected data using a variety of commercial software packages such as ACD/LAB, Focus (Molsoft ChemistPro V3.6) and in house in silico profiling tools. The initial findings show that: the rule of five is respected by 80% of inhaled drugs on the market; among the more common chemical parameters only the H-bond acceptor is significantly lower in inhaled drugs. Early work will be presented that implies permeability is inversely proportional to the percentage of dose left at 30 min in rat lungs.

References

1 www.lunguk.org. 2 R. S. Obach, F. Lombardo, and N. J. Waters, “Trend analysis of a database of intravenous pharmacokinetic parameters in humans for 670 drug

compounds,” Drug Metab Dispos. 36(7), 1385 (2008). 3 J. R. Proudfoot, “The evolution of synthetic oral drug properties,” Bioorg. Med. Chem. Lett. 15(4), 1087 (2005). 4 T. J. Ritchie, C. N. Luscombe, and S. J. Macdonald, “Analysis of the calculated physicochemical properties of respiratory drugs: can we design for

inhaled drugs yet?,” J. Chem. Inf. Model. 49(4), 1025 (2009).

P142. Veliparib (ABT-888): a Novel, Potent PARP Inhibitor with Favorable ADME Properties

Richard Voorman1, Xiaofeng Li1, Alexander Shoemaker2, Thomas Penning2, Vincent Giranda3, Juergen Delzer4, Sonia de Morais1 and Yanbin Lao1

1Drug Metabolism, Abbott Laboratories, Abbott Park, IL, USA, 2Cancer Research, Abbott Laboratories, Abbott Park, IL, USA, 3Oncology Development, Abbott Laboratories, Abbott Park, IL, USA, 4Dmpk, Abbott Laboratories, Ludwigshafen, Germany

Poly (ADP-ribose) polymerases (PARP-1 and -2) are key enzymes involved in repair of DNA single-strand break through base excision repair pathway. Accordingly, PARP inhibition could enhance the efficacy of DNA-damaging chemotherapeutics. Veliparib is a potent PARP inhibitor with excellent in vitro potency and in vivo efficacy in tumor models when combined cytotoxic agents, and currently being investigated in clinical trials for treatment of various human cancers. Veliparib demonstrated favorable absorption, distribution, metabolism and excretion (ADME) prop-erties in vitro and in vivo. It is water soluble and orally bioavailable in multiple preclinical pecies. In the Caco-2 cells, veliparib showed high permeability (Papp: 12.2 x 10−6 cm/sec). In vitro studies demonstrated minimal metabolism of [3H]veliparib in hepatic systems from various species (<5% turnover). Following oral or intravenous administration of [3H]veliparib to rats and dogs, radioactivity was well absorbed (>75% of the dose), and rapidly eliminated with >80% of dose recovered within 24-hour post dose. Radioactivity was primarily eliminated via renal route (80% of the dose) with minimal elimination in bile and feces (each route < 5.0% of the dose). Parent was the major component in the urine (50% of the dose). Biotransformation of veliparib proceeded via hydroxylation, lactam formation on pyrrolidine, hydrolysis of lactam, and a combination of oxidation and glucuronidation. M8, the lactam metabolite, was the major metabolite in excreta (16-24 % of dose). In addition, M3, the amino acid metabolite, represented 12% of the dose in dogs. In both species, parent drug was the major circulating component. M2, a mono-hydroxylation product, and M8 were two major metabolites in rat plasma, each representing 12-29% of the total plasma radioactivity. In dog plasma, M2, M3 and M8 were three major metabolites with estimated AUC representing up to 27%, 39%, and

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48% of parent AUC, respectively. Altogether, these data was consistent with the Phase 0 clinical observations that veliparib was well absorbed, and largely recovered as unchanged parent drug in human urine. In vitro metabolism data also indicate a low potential for clinically significant pharmacokinetic drug-drug interactions. Veliparib was not a potent inhibitor of major human CYPs, and did not significantly induce activities of CYP1A2, 2B6, 2C9 and 3A4. Reaction phenotyping study with human recombinant CYPs suggested potential involvement of multiple isoforms in the metabolism of veliparib, including CYP1A1, 2D6, 3A4, and 2C19. Velparib was a weak substrate of human P-glycoprotein (Pgp) (efflux ratio 1.8 in MDR1-MDCK cells) and did not inhibit Pgp-mediated efflux of digoxin up to 100 mM, altogether indicating limited influence of Pgp in its disposition. High permeability and minimal Pgp involvement in veliparib disposition suggest low potential for brain penetration impairment and drug resistance mediated by efflux transporters, which accordingly could benefit its pharmacological effects.

P143. Application of Different In Vitro Systems for Prediction of In Vivo Induction Drug-Drug Interactions

Ian E. Templeton, J. Brian Houston and Aleksandra Galetin

School of Pharmacy & Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom

Rifampin is commonly used as a calibrator for quantifying inducers of CYP3A4. However, the in vitro induction param-eter estimates for rifampin are highly variable both within and between different in vitro systems. Our preliminary predictions resulted in differential success and in some instances pronounced over-estimation of in vivo drug-drug interactions (DDI) [1]. In order to investigate this further, we have compiled a database of published E

max and EC

50 esti-

mates, based on either CYP3A4 mRNA or metabolic activity as induction endpoint. Parameter estimates from different in vitro systems were collated, namely hepatocytes (primary and cryopreserved), HepG2, Fa2N-4 and HepaRG cell lines. In vitro studies were analysed both by the cell line and length of incubation with rifampin. A subset of our in vivo database (n=12) in which victim drug was administered i.v. (hepatic induction only) was used to compare the predictive utility of these in vitro systems. In general, predictions based on CYP3A4 activity were more accurate than those based on CYP3A4 mRNA data. Average E

max and EC

50 estimated from activity data in primary hepatocytes incubated for 48h

and 72h with rifampin predicted only 8% and 17% within the 2-fold range, respectively; the remaining DDIs were over-predicted. Induction data generated in Fa2N-4 cells after 72h incubation resulted in the best prediction success (100 % within 2-fold). E

max was identified as the most variable parameter in the prediction model and optimization of hepatic

Emax

was performed in MATLAB 7.8® across the different in vitro systems and using i.v. DDIs. The optimized Emax

was then applied for the prediction of 25 DDIs with orally-administered victim drugs using a model with incorporated intestinal induction. This dataset contained 15 victim drugs subject to varying extent of intestinal first-pass metabolism and displaying variable contribution of CYP3A4-mediated metabolism to overall elimination. Incorporation of optimized E

max into the model resulted in 40% to 48% of the in vivo induction DDIs predicted within the 2-fold range, regardless

of the in vitro system used. Optimisation improved the prediction by primary hepatocyte activity data (from 36% to 48% within 2-fold), but reduced the percentage of oral induction DDIs predicted within 2-fold by Fa2N-4 from 60% to 48%. In conclusion, Fa2N-4 activity data generated E

max and EC

50 estimates which predicted the greatest percentage of

induction DDIs within 2-fold. Optimisation of Emax

based on the dataset of DDIs with i.v. administered drugs improved the prediction outcome; implications of these findings and further prediction success criteria are discussed.

References

1. Galetin A, Gertz M and Houston JB (2010) Contribution of intestinal cytochrome P450-mediated metabolism to drug-drug inhibition and induc-tion interactions. Drug Metab Pharmacokinet 25:28-47.

P144. Effect of metronidazole on the pharmacokinetics of fexofenadine in healthsy subjects

Kyoung-Ah Kim and Ji-Young Park

Department. of Clinical Pharmacology & Toxicology, Anam Hospital, Korea University College of Medicine, Seoul, South Korea

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Metronidazole has been reported to cause a number of drug interactions with coadministered drugs clinically and it is suggested that metrondizole might be an inhibitor of P-glycoprotein (P-gp). The aim of the present study was to assess the possible inhibitory effect of metronidazole on the P-gp mediated drug disposition using fexofenadine as a P-gp substrate in healthy subjects. Twelve healthy subjects were enrolled for the study and treated 500-mg metronidazole or placebo three times daily for 7 days. On day 7, a single dose of 120-mg fexofenadine was treated orally. Plasma levels of fexofenadine were measured and its pharmacokinetics was assessed. Metronidazole treatment did not affect pharmacokinetics of fexofenadine. The area under the time versus concentration curve (AUC) of fexofenadine in met-ronidazole phase (2075.7 ng*hr/mL) was similar to that of placebo phase (1999.2 ng*hr/mL) (P=0.356). Additionally, there was no difference in the maximum plasma levels of fexofenadine (304.4 ng/mL for placebo phase versus 293.2 ng/mL for metronidazole phase) (P=0.423). The elimination half-life and oral clearance of fexofenadine were not affected by metronidazole treatment. The results of the present study showed that metronidazole did not have any inhibitory effect on the pharmacokinetics of fexofenadine and thus provided the evidence that metronidazole do not act as an inhibitor of P-gp mediated disposition in humans.

P145. Herb-Drug interaction: effect of isopeulustrin on the metabolism and pharmacokinetics of tetramethylpyrazine by in vivo and in vitro methods

Xin He, Shan Feng, Yuefei Wang, Chao Li, Jipeng Hou, O. N. K. Martey and Changxiao Liu Tianjin

Key laboratory of chemical and analysis of Chinese materia medica, Tianjin University of Traditional Chinese Medicine, Tianjin, China

Drug-drug or herb-drug interaction may sensitively affect the therapeutics and toxicity. As we know, herbs contain a diverse array of active constituents, and each with the potential to modulate the activity of specific cytochrome P450 enzymes. Rhizome chuanxiong is a kind of Traditional Chinese medicine. According to TCM theory, is used for stagnation of the blood and qi; Bi-syndrome of wind and dampness, but also used for treatment of headache together with Angelica dahurica to enhance its effect. The purpose of this study is to scientifically investigate the rationale behind the clinical combination of Angelica dahurica and Rhizome chuanxiong, by preliminary screening the active ingredients of Angelica dahurica ( isopeulustrin and peulustrin) and Rhizome chuanxiong (tetramethylpyrazine, TMP) for potential inhibition of five main metabolic enzymes(CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in vitro and to compare the pharmacokinetic alterations of oral administration of isopeulustrin to co-administration of isopeulustrin and TMP in rat (in vivo). In the preliminary screening study, isopeulustrin and peulustrin displayed strong inhibition on four main metabolic enzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4), with IC50 of 0.101, 7.768, 15.426, 1.215μM respectively for peulustrin, and 9.248, 8.94, 2.515, 1.528μM respectively for isopeulustrin; there is no inhibition of TMP on any of the enzymes. The pharmacokinetic parameters of oral administration of TMP were markedly enhanced when co-administered with isopeulustrin showing an increase of AUC from 0.65 mg/ml*h. to 4.96mg/ml*h, with the elimination time of co-administration of TMP with isopeulustrin prolonged four times (24h) than TMP administered alone (6h) even though the rate of absorption parameter (Ka) was insignificantly difference. From the result, isopeulustrin may increase TMP stagnation time by inhibiting its metabolic enzyme, result in long active effect in body. In conclusion, herb-drug interaction indeed exist which result from metabolic enzymes, patients should pay more attention on the herb-drug interaction when they take Angelica dahurica with other drugs. (Supported in part by grants of 2009ZX09304-002, No.200807051, No.07JCYBJC18800 and NSFC grant No.30772778)

P146. In vitro inhibition and induction of human liver cytochrome P450 enzymes by NTBC and its metabolism in human liver microsomes

Jason N. Neat1, Andrea Wolff1, Faraz Kazmi1, Pilar Prentiss1, David Buckley1, Elizabeth M. Wilson2, John Bial2, Cedo M. Bagi3, Markus Grompe2 and Andrew Parkinson1

1XenoTech, LLC, Lenexa, KS, USA, 2Yecuris Corporation, Portland, OR, USA, 3Global Science & Technology, Pfizer, Inc., Groton, CT, USA

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2-(2-Nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC, also known as nitisinone and marketed as Orfadin) is an inhibitor of 4-hydroxyphenylpyruvate dioxgenase (HPPD) that is used to prevent the liver and kidney toxicity associated with tyrosinemia type 1, a metabolic disorder in the tyrosine catabolism caused by fumarylacetoacetate hydrolase (FAH) deficiency. Genetically modified mice deficient in FAH [Fah-/-/II2rg-/-Rag2-/- (FRG) mouse strain] can be repopulated with human hepatocytes to support, among other applications, studies of drug metabolism and disposition. To sustain mouse hepatocellular function prior to (and during) the repopulation with human hepatocytes, FRG mice are treated with NTBC to inhibit the formation of hepatotoxic levels of fumarylacetoacetate (FAA). In the present study, we investigated the metabolism of NTBC in human liver microsomes (HLM) and the potential for NTBC to inhibit or induce human cytochrome P450 (CYP) enzymes. NTBC (1, 10 and 100 µM) was incubated with multiple concentrations of NADPH-fortified pooled HLM (0.5, 1 and 2 mg /mL) for multiple incubation times (0, 30, 60, 120 and 240 min). Little-to-no loss of NTBC was observed, suggesting that NTBC undergoes little or no oxidative metabolism by human liver CYP enzymes and little-to-no ketone reduction by microsomal carbonyl reductase. These results are consistent with the long clinical plasma half-life (t

1/2 ∼ 52 –54h) reported for NTBC. In CYP inhibition experiments, performed with pooled HLM (0.1 mg/

mL), NTBC caused direct inhibition of CYP2C9 (IC50

11 µM). NTBC caused no direct inhibition of CYP1A2, 2B6, CYP2C8, 2C19, 2D6 and 3A4/5. Furthermore, NTBC caused no metabolismdependent inhibition of any of the CYP enzymes evaluated. To evaluate CYP induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for three consecutive days with NTBC (1, 10, and 100 µM), after which microsomal CYP activities and mRNA expression were measured. NTBC (100 µM), had a negligible effects (< twofold) on CYP2B6 and 3A4/5 activity but caused a 7.60-fold increase in CYP1A2 activity. However, as a CYP1A2 inducer, NTBC (100 µM) was only 9% as effective as omeprazole. In summary, NTBC has no capacity to inhibit or induce human CYP enzymes which suggests that repopulated FRG mice undergoing NTBC treatment are suitable for studies of drug metabolism involving human CYP enzymes.

P147. Nano-silver Particle “Mineral Supplement” Effects On Drug Metabolism

Michael R. Franklin1, Laura B Hathaway2, John G Lamb2 and Judy Raucy3

1Department. of Pharmacology & Toxicology, University of Utah, Salt Lake City, UT, USA, 2Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, USA, 3Puracyp, Inc, Carlsbad, CA, USA

Solutions containing nano-silver particles (95% 5-15 nanometers in size) comprising an interior of elemental silver and an exterior of ionic silver oxide are commercially available as mineral supplements and are promoted for immune support. Recommended adult dosing for the 30 ppm preparation evaluated is 150 micrograms of silver, orally, three times per day (4.2 micromoles silver/day). The biodistribution of ingested nano-silver particles is not known, but orally ingested, they likely first impact cells in the GI tract, especially intestinal cells, and then the liver. The effects of these nano-silver particles on the ability of reporter-engineered (human epithelial colorectal adenocarcinoma) Caco-2 cells (MDR1.C) and (human hepatocellular carcinoma) HepG2 (DPX2) cells (Puracyp Inc.) in culture to respond to xenobiotic inducing agents has been investigated. Also direct effects on human microsomal cytochrome P450 activities in vitro have been evaluated. For reporter-engineered Caco-2 and HepG2 cells in culture, the 24-hour LD50 values of nano-silver particles were slightly lower than for silver carbonate (65 vs. 95 uM) for HepG2 cells and similar for the two forms of silver for Caco-2 cells (95 uM). When the ability of the variously engineered cells to respond to inducing agents was investigated at cell-medium silver concentrations lower than the LD10, (1-10 uM), the 24-hour PXR-driven 5-fold induction response of MDR1.C cells to 50 uM omeprazole was unaffected by either form of silver. However silver toxicity appeared greater in the presence of inducer. The 24-hour PXR-driven 7-fold induction response of DPX2 cells to 10 uM rifampicin was reduced by 50% by both silver agents. In the absence of drugs, neither silver carbonate nor nano-silver particles elicited any induction. Silver inhibition of human microsomal CYP-selective activities was proportional to the silver:protein ratio. However when examined at 0.5 mg protein/ml, the CYP-selective activities showed some differences in sensitivity. Coumarin 7-hydroxylation exhibited the highest IC50 (38 uM), and S-mephenytoin 4-hydroxylation the lowest (4 uM). Other IC50 values (lowest to highest) were testosterone 6-beta hydroxylation, 7-benzyloxyquinoline debenzylation and diclofenac 4-hydroxylation (8 uM), chlo-rzoxazone 6-hydroxylation (14 uM), MAMC [7-methoxy-4-(aminomethyl) coumarin] O-demethylation (20 uM), and 7-methoxyresorufin O-demethylation and 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (28-29 uM). Silver

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carbonate IC50 values were almost identical to those of the nano-silver particles. Microsomal NADPH cytochrome c reductase activity was inhibited <10 % at 70 uM by the nano-silver preparation so in contrast to mercurials, electron flow is not the major locus of the inhibition. Silver, whether in the form of silver-silver oxide nano-particles or ionic, is far from innocuous, and can have effects on drug metabolizing enzymes systems in vitro that could lead to inhibi-tion- and induction-related drug-drug interactions in vivo.

P148. Pharmacokinetic Interaction Between Ivabradine and Carbamazepine in Healthy Volunteers

Laurian Vlase1, Adina Popa2, Maria Neag2, Dana Muntean1 and Sorin Leucuta1

1Pharmaceutical Technology and Biopharmaceutics, University of Medicine and Pharmacy Iuliu Hatieganu, Cluj-Napoca, Romania, 2Clinical Pharmacy, University of Medicine and Pharmacy Iuliu Hatieganu, Cluj-Napoca, Romania

Objectives: the evaluation of pharmacokinetic interaction between ivabradine and carbamazepine in healthy volunteers.

Methods: A single dose of 10 mg ivabradine was administered alone or in combination with 400 mg carbamazepine to 18 healthy volunteers in a two treatment study design, separated by 15 days in which the carbamazepine alone was administrated as a single p.o. dose daily. Plasma concentrations of ivabradine were determined during a 12 hours period following drug administration, using a high-throughput liquid chromatography coupled with mass spectrom-etry analytical method. Pharmacokinetic parameters of ivabradine administered in each treatment were calculated using non-compartmental and compartmental analysis and compared to determine if the differences were statistically significant.

Results: In the two periods of treatments, the mean peak plasma concentrations (Cmax) were 16.25 ng/ml (ivabradine alone) and 3.69 ng/ml (ivabradine after pre-treatment with carbamazepine). The times taken to reach Cmax, tmax, were 0.97 hr and 1.14 hr respectively and the total areas under the curve (AUC0-inf) were 52.49 ng.hr/ml and 10.33 ng.hr/ml, respectively. Statistically significant differences have been observed for Cmax and AUC0-inf of ivabradine when administered alone or with carbamazepine, whereas for tmax, half-life and mean residence time the differences were non-significant.

Conclusion: The experimental data demonstrate the pharmacokinetic interaction between ivabradine and car-bamazepine and the observed interaction may be clinically significant.

P149. Physiologically-based pharmacokinetic modelling approach to assess the uptake transporter-mediated drug-drug interaction potential of cyclosporine

Michael Gertz1, J. Brian Houston2 and Aleksandra Galetin2

1School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom, 2School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom

In addition to drug-drug interactions (DDI) mediated by inhibition or induction of cytochrome P450 enzymes, recent studies have indicated that inhibition of OATP-transporters in the liver may contribute considerably to the magnitude of DDI observed with certain victim drugs (e.g. statins, repaglinide). The largest proportion of these transporter-mediated DDIs includes interactions with cyclosporine, a potent inhibitor of human OATP1B1 and OATP1B3 in vitro. The avail-ability of tissue distribution data across different dose regimens in rat [1, 2] allowed the assessment of cyclosporine DDI potential using a whole body physiologically-based pharmacokinetic (PBPK) modelling approach in MATLAB 7.8. Concentration dependent tissue and blood binding of cyclosporine [2] were accounted for by the model. The extent of reduction in transporter activity (1/R) was assessed as 1/(1+[I]/IC

50), where [I] is expressed as a function of

time; considering the location of the OATP transporters, cyclosporine unbound blood concentration was considered as [I] in the current analysis. Cyclosporine DDI potential at the level of the uptake transporter was initially assessed in rat (i.v. and oral) and consequently the analysis was extended to human. In rat, single i.v. dose of cyclosporine reduced the hepatic transporter activity by 26 – 91% (1.2 and 30mg/kg, respectively). For an intermediate cyclosporine

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dose of 6mg/kg, the remaining transporter activity was approximately 37%. Following this dosage regimen, >50% of transporter activity was recovered within 30min after infusion due mainly to distribution of cyclosporine. In contrast, recovery of 50% transporter activity took >10h at the high (30mg/kg) dose level due to the relatively slow metabolism of cyclosporine. The PBPK model could recover the oral PK profiles at different dose levels; however, a tendency to over-estimate cyclosporine C

max was observed, in particular at the highest dose. The reduction in transporter activity

exceeded 50% only at the highest oral dose in rat (100mg/kg) and displayed a slow recovery (similar to results from high i.v. dose). Scaling to human i.v. and oral data was performed successfully and the impact of variability in cyclosporine fu

p in human (1.5–8%) on the assessment of the transporter interaction potential is discussed. Further work will focus

on the assessment of cyclosporine net DDI potential considering also inhibition of metabolic enzymes and efflux transporters.

P150. Predicting Transporter-Mediated Drug-Drug Interactions Based on the B-CLEAR®-Rapid Transport Screen in Sandwich-Cultured Hepatocytes

Jin Kyung Lee1, Cassandra H. Perry2, Chris Parsons2, Robert L. St. Claire III2 and Kenneth R. Brouwer2

1Research & Development, Qualyst Inc, Durham, NC, USA,2Research and Development, Qualyst, Inc., Durham, NC, USA

It is widely recognized that hepatic transport proteins play an important role in drug-drug interactions. B-CLEAR® is a novel, proprietary, in vitro technology which characterizes transporter-based hepatic interactions of drug candidates and metabolites using sandwich-cultured hepatocytes (SCH). The present study was designed to use B-CLEAR® as a rapid transport screening tool to determine the effect of drug candidates on the hepatic uptake and efflux of clinically-relevant probe substrates. Four compounds were selected as probe substrates for relevant hepatic uptake and efflux transporters in rat SCH [d

8-taurocholate for Na+-dependent taurocholate co-transporting polypeptide (Ntcp)/bile salt

export pump (Bsep); digoxin for organic anion-transporting polypeptide (Oatp)/p-glycoprotein (P-gp); rosuvastatin for Oatp/multidrug resistance-associated protein 2 (Mrp2); and nitrofurantoin for breast cancer resistance protein (Bcrp)]. To identify a positive control inhibitor for the transport of each probe substrate, erythromycin estolate (EE, 100 µM), bromosulfophthalein (BSP, 100 µM), troglitazone (TGZ, 50 µM), rifamycin SV (RIF, 100 µM), and cyclosporin A (CYA, 50 µM) were evaluated with each probe substrate. Ntcp/Bsep-mediated transport of taurocholate was decreased to less than 20% of control by EE and TGZ. P-gp-mediated biliary excretion of digoxin was markedly decreased by EE and CYA, but Oatp-mediated uptake of digoxin was only marginally affected by EE (75% of control). Oatp-mediated rosuvastatin uptake was markedly decreased by all inhibitors to less than 40% of control; however, Mrp2-mediated rosuvastatin efflux was inhibited only by CYA (55% of control). Although the mechanism of hepatic uptake of nitrofurantoin remains to be elucidated, EE and BSP decreased the nitrofurantoin uptake (63% and 75% of control, respectively), while CYA decreased Bcrp-mediated nitrofurantoin biliary excretion to 75% of control. In conclusion, the B-CLEAR®-Rapid Transport Screen, using clinically-relevant probe substrates and inhibitors for hepatic transport proteins (Ntcp, Oatp, Bsep, P-gp, Mrp2, and Bcrp), can be used to examine the potential for relevant in vivo transporter-based drug-drug interactions.

P151. Predictions of metabolic Drug Drug Interactions after Intravenous administration: impact of hepatic extraction ratio

Cristina Lopez1, Patrice Dehanne1, Estelle Rapine1, Olivier Barberan1 and Xavier Boulenc2

1Aureus Pharma, Paris, France, 2Drug Safety and Animal Research platform, Drug Disposition Domain, Sanofi-Aventis, Montpellier, France

Nowadays drug-drug interactions (DDIs) represent one of the greatest concerns in terms of drug discovery for phar-maceutical industry. From an industrial point of view, drugs participating in the interaction can be considered as a victim and/or as a perpetrator. These interactions can cause increased or decreased drug exposures. In spite of this, most studies have been attempted for drugs administered by oral route, while prediction of DDIs after intravenous

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administration of the victim remains poorly reported in the literature. Nevertheless, predictions involving high and low clearance drugs were addressed by Ito1. For high clearance drug (E

H=1), Cl

h is rate limited by the hepatic blood

flow rate. If the hepatic clearance in presence of the perpetrator (ClHi

) is still rate limited (EH′=1), AUC ratio is equal to

unit indicating no change in AUC and therefore no drug drug interaction. For low clearance drugs (EH′0), Cl

H and Cl

Hi

are described by the unbound intrinsic clearance of the victim without and with perpetrator, and interactions can be predicted with equations developed for oral administration. The aim of this study is to predict DDI′s after intravenous administration of drugs which hepatic extraction ratio values lay between 0 and 1 and compare predicted AUC ratio to actual in vivo. Consequently, a model was developed to predict the change in AUC after intravenous administration based on the following parameters: the fraction of hepatic blood clearance in the total blood clearance of the victim drug (f

h), the fraction of affected drug cleared by each enzyme (f

m), the intrinsic clearance values for each enzyme affected

or not affected by perpetrator respectively and the hepatic extraction ratio of the victim drug (EH

).

Reference

1 Ito K, Iwatsubo T, Kanamitsu S, Ueda K, Suzuki H, Sugiyama Y (1998) Prediction of pharmacokinetic alterations caused by drug–drug interac-tions: metabolic interaction in the liver. Pharmacol Rev 50(3):387–412

P152. Seizurogenic Effects of Low-dose Naloxone in Tramadol Overdose

Esmaeil Farzaneh1, Babak Mostafazadeh2, Farnaz nasl Seraji1 and Iraj sayad Rezai1

1medical Toxicology and Forensic Medicine, Ardebil University of Medical Sciences, Ardabil, Iran, Ardabil, Iran, 2Medical Toxicology & Forensic Medicine, Shaheed Beheshty University Of Medical Sciences, Tehran, Iran

Objective: Tramadol is used in treatment of moderate to severe pain. Nowadays tramadol overdose is one of the com-mon emergencies. Naloxone is an antagonist which is used as a first step of treatment in these patients. This study was designed to evaluate the seizurogenic effects of naloxone in tramadol overdose.

Methods: 124 patients with the diagnosis of tramadol overdose were divided to receive low-doses of intravenous naloxone (0.8 mg, case group) or just supportive cares (control group). All patients in case and control groups were observed by a single emergency resident and fallowed for 1.5 hours to document the happening of seizures.

Results: In the naloxone group, incidence of seizure was higher than in control group. The possibility of seizure occur-rence was significantly higher in naloxone group than the control group (P < 0.05).

Conclusion: Naloxone induced a seizurogenic effect in patients with tramadol overdose. This finding could be consid-ered in the management of patients with tramadol overdose.

Table 1. Demographic characteristics of 124 patients with tramadol overdose

Variable Case (N = 62) Control (N = 62) p-value

Age(year) 26.33 29.46 0.5

Male 59 55 0.18

Female 3 7 —

Single 35 27 0.15

Married 27 35

· P-value less than 0.05 considered significant

Table 2. Comparison of past medical history of the patients with tramadol overdose between the two studied groups (N = 124).

Case (N = 62) Control (N = 62) p-value

Former drug overdose 24 34 0.07

Former tramadol overdose 8 7 0.78

History of seizure 3 5 0.71

CNS* disease 3 5 038

Alcoholism 0 1 1.00

Cigarette smoking 52 45 0.12

* Central Nervous System

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References

1. Rehni AK, Singh I, Kumar M. Tramadol-induced seizurogenic effect: a possible role of opioid-dependent aminobutyric acid pathway. Basic Clin Pharmacol Toxicol 2008; 103: 262-66.

2. Ashish K. Rehni, Thakur Gurjeet Singh, Nirmal Singh2 and Sandeep Arora1 Tramadol-induced seizurogenic effect: a possible role of opioid-dependent histamine (H

1) receptor activation-linked mechanism. Naunyn Schmiedebergs Arch Pharmacol 2009 DOI: 10.1007/s00210-009-

0476-y 3. Yang L, Li F, Ge W, Mi C, Wang R, Ruopeng Sun R. Protective effects of naloxone in two-hit seizure model. Epilepsia 2009 (In Press).

P153. Sequential Arabinosylcytosin with or without Fludarabine in Paracmastic Patients of Acute Myeloid Leukemia

Ai-Bin Liang1, Zhang Hong1, Yu Fang, Ying Li, De-Qiu Zhu1, Fang-Yuan Chen2, Hui-Na Lu1, Jian-Fei Fu1, Lan-Jun Bo1

1Clinical Pharmacology, Tongji Hospital of Tongji University, Shanghai, China,2Renji Hospital, Shanghai Jiaotong University, Shanghai, China

Objective: The synergistic action between Fludarabine(Fa) and Arabinosylcytosin(Ara-C) has been well reported. However, this phenomena doesn′t always happen in clinical, and little has been studied on how to decide whether Ara-C can be combined with Fa or not in clinical. The purpose of this study is to assess what the difference of Ara-C′s behavior is with or without Fa.

Methods: The plasma, cerebrospinal and urinal samples from two self-control groups (Group A with Ara-C only, Group B with Fa + Ara-C) were withdrawn at specific time points, and analyzed with HPLC.

Results: The AUC0-4

of Group B and Group A was 12.245 ± 3.863 and 5.131 ± 0.936 respectively, which there existed statistics difference (P=0.016). The Ara-C dose in Group B was 2g/m2°Á2, but the AUC

0-4 was over double than that

of Group A (3g/m2°Á2); It suggests that there might exist difference in the ratio of CAra-u

and CAra-C

and Tmax

of the two concentrations′ ratio between Group A and B; Fa might conduce to the increasing of the concentration of C

Ara-C in

cerebrospinal fluid; The ratio of C 6b-hydroxycortisol

/C cortisol

(a noninvasive marker for CYP3A enzymes activity) was below 1.5 in the two groups, which was obviously lower than that of healthy subjects.

Conclusions: It is indicated that there existed synergistic action between Fa and Ara-C, in which Fa conduces to increasing the concentration and AUC of Ara-C in plasma, and even increasing the Ara-C concentration in cerebros-pinal fluid. The obvious change in T

max of the ratio or the ratio data of C

Ara-u and C

Ara-C might be an indicator to decide

if Ara-C combined with Fa or not. However, there was an exception, and these 5 self-control cases are of limited numbers, so the large number of cases and further study should be needed to support and prove this hypothesis. The two study groups are all under the condition of the complete remission (CR) process, but the ADR and bone inhibition of Group B were no more serious than that of Group A although Group B demonstrates higher AUC

0-4 with lower doses

of Ara-C(2g/m2°Á2).

P154. The impact of PBPK modeling on prediction of drug drug interaction potential of a candidate drug with non linear pharmacokinetics

Jan Snoeys1, Jos Van Houdt2, Vikash Sinha3, Geert Mannens1, Patrick Sterkens2, Nancy Van Osse-laer4 and Mario Monshouwer1

1Drug Metabolism and Pharmacokinetics, Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., Beerse, Belgium, 2Drug Metabolism and Pharmacokinetics, Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., Beerse, Belgium, 3Global Clinical Pharmacology, Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., Beerse, Belgium, 4Neuroscience Development, Johnson & Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., Beerse, Belgium

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There is a fast growing interest within pharmaceutical industry in using Physiologically based pharmacokinet-ics (PBPK) modeling to predict and mechanistically understand human pharmacokinetics (PK) of potential drug candidates. The scope of this study was to evaluate whether PBPK could positively impact further drug develop-ment of a drug candidate with non-linear human PK in function of both dose and time. In vitro experiments were performed to mechanistically characterize the human non-hepatic and hepatic clearance of this BCS Class I com-pound, which can be further characterized as a basic lipophilic amine with a pKa of 8.2. Hepatic unbound intrinsic clearance, the contribution of several metabolic pathways and enzymes in the hepatic clearance and the interplay between hepatic uptake and metabolism was determined at several concentrations of the compound based on experiments with human hepatocytes, human liver microsomes and recombinant enzymes. The time-dependent CYP inhibition properties were characterized by determination of the inactivation kinetics in vitro in human liver microsomes. Finally, all experimental data on intrinsic clearance and time-dependent CYP inhibition together with physicochemical data and preclinical data on absorption and distribution were integrated in a PBPK model using Simcyp® ADME Simulator v.9.3 (Simcyp Ltd., UK). This PBPK model very nicely simulated the observed non-linear PK (both dose and time) of our candidate drug and allowed us to predict (1) drug drug interactions with several perpetrators at various dose strengths and various dosing regimens and (2) the inter-individual variability in PK in healthy volunteers and special populations. Clinical trial results indicate that the performed simulations were very predictive at both low and high doses of our candidate drug. In summary, PBPK modeling had a significant impact on the drug development of our candidate drug because it helped us to mechanistically understand the non-linear PK, but even more importantly allowed us to optimally design clinical drug drug interaction studies at different doses with several perpetrators.

P155. The Pharmacokinetic Interaction Between JBP485 And Cephalexin In Vivo And In Vitro

Kexin Liu1, Jian Zhang1, Changyuan Wang1, Qi Liu1, Qiang Meng1, Xinjin Guo1, Jian Cang1 and Taiichi Kaku2

1Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, China, 2Bioproducts Industry Co. Ltd., Tokyo, Japan

Objective: The purpose of this study was to investigate the pharmacokinetic mechanism of interaction between JBP485 (cyclo-trans-4-L-Hydroxyprolyl-L-serine), a synethesized hydrosoluble dipeptide (Kexin L et al., 2000) and Cephalexin, an oral b-lactam antibiotic.Methods: Oral co-administration, everted small intestine in vitro, in situ jejunal perfusions, urinary excretion, kid-ney slices and cell uptake studies were performed to clarify the combined pharmacokinetic characters of JBP485 and Cephalexin.

Results: It showed that the plasma concentrations of JBP485 and Cephalexin were both decreased significantly after co-administered orally. But little differences were observed after simultaneous intravenous administration of the two agents. These data suggested that the interaction between the two most likely happened in intestine during absorp-tion process. The uptake of JBP485 and Cephalexin in everted intestinal sacs and absorption in jejunal perfusions (Qinghao Zhang et al., 2009) were dramatically reduced after combination. When co-administered, both JBP485 and Cephalexin exhibited a decrease in accumulative renal excretion rates (81.9% to 64.9% of JBP485 versus 91.8% to 74.5% of Cephalexin) and in renal clearance (2.89 to 1.87 ml/min/kg of JBP485 versus 2.23 to 1.58 ml/min/kg of Cephalexin), suggesting that other transporter(s) are involved in the process of excretion except PEPT2. We also found that probenecid (a typical substrate of organic anion transporters, OATs) could reduce renal excretion of JBP485 and Cephalexin in vivo. This could partially explained by an inhibition of the renal uptake via OATs, which leads to a decreased uptake of JBP485 when co-cultured with probenecid, PAH or PCG in kidney slices. Our data also indicates that the accumulation of JBP485 in hOAT1-/hOAT3-HEK293 cells was greater than that in vector-HEK293 cells, and the uptake could be inhibited by probenecid.

Conclusion: These findings further confirmed that the pharmacokinetic mechanism of drug-drug interaction between JBP485 and Cephalexin could be explained by their inhibition of the same transporters in the intestinal mucosa (PEPT1) and kidneys (PEPT2 and OATs). Hence, we are the first to report that JBP485 is not only a substrate of PEPTs but also is excreted through OATs.

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References

1. Kexin Liu,Yukio K,Kaku T I, et al. (2000) Hydroxypmlylserine defivatives JBP923 and JBP485 exhibit the antihepatitis activities after gastrointes-tinal absorption in rats£®J Pharmaeol Exp Ther 294: 5l0-5l5.

2. Qinghao Zhang, Qi Liu, Jingjing Wu, Changyuan Wang, Jinyong Peng, Xiaochi Ma and Kexin Liu (2009) PEPT1 involved in the uptake and tran-sepithelial transport of cefditoren in vivo and in vitro. Eur J Ph 612: 9-24.

P156. The POSSIBLE Interaction BETWEEN SLC TRANSPORTER Mediated UPTAKE and FDA Recommended Potent CYP Inhibitors

Yasuhisa Adachi1, Miki Fujishima1, Takami Sarashina1, Teruhiko Yanagisawa1, Shinsuke Aoyama1, Yoshihiro Ohzone2 and Shin-ichi Ninomiya3

1ADME & Tox. research institute, Sekisui Medical Co., Ltd., Ibaraki, Japan, 2ADME & Tox. Research Institute, Sekisui Medical Co., Ltd., Ibaraki, Japan, 3ADME & Tox Research Institute, Sekisui Medical Co.,Ltd., Tokai, Japan

To date, drug-drug interactions related to hepatic clearance have been mainly reported as a drug metabolism enzyme facilitated interaction, primarily involving CYP isoforms. The U.S. Food and Drug Administration (FDA) in vivo in vitro (1)In March 2010, a concept paper on transporter studies in new drug development was published by the International Transporter Consortium (ITC). This paper focused on recommended in vitro assay methods and criteria for the inter-pretation of assay results for several uptake transporters.(2) evaluated the effect of FDA recommended CYP inhibitors on transporter mediated uptake. We evaluated the effect of each CYP inhibitor on the uptake of typical substrates of the major uptake transporters (OATP1B1, OATP1B3, OCT2, OAT1 and OAT3 etc.). The effect of sulfaphenzole, a potential inhibitor of CYP2C9, on OATP1B1 and OATP1B3 mediated uptake was investigated using each transporter expressing HEK293 cells and S2 cells. The IC50 values of sulfaphenazole on the OATP1B1 and OATP1B3 mediated 3H-E

217bG 16

µM and over 300 µM, respectively. This result suggests that sulfaphenazole inhibits OATP1B1 mediated-transport, whereas OATP1B3 was not inhibited. The reported in vitro Ki value of sulfaphenazole on CYP2C9 mediated metabolism, 0.3 µM, is approximately 50 times smaller than the IC50 value of OATP1B1 mediated uptake. A previously reported in vivo study showed the the maximum unbound concentration at the inlet to the liver (Iu,max) of sulfaphenzole to be 27.2 µM in response to 500 mg/man of sulfaphenazole oral administrtion, suggesting that sulfaphenazole may cause a OATP1B1 mediated drug-drug interaction(3). The effect on the transporter mediated uptake of each CYP inhibitor will be evaluated and reported in this presentation.

References

1) Guidance for Industry Drug Interaction Studies -Study Design, Data Analysis, and Implications for Dosing and Labeling. U.S. Department of Health and Human Services Food and Drug Administration (September 2006)

2) The International Transpoter consortium, Nature Reviews Drug Discovery, 9, 215-236 (March 2010) 3) Komatsu K et. al, Drug Metabolism and Disposition, 28, 475-481 (2000)

P157. Determination of Benzene, Toluene, Ethyl Benzene and Xylenes in urine of exposed and non-exposed residents of Ankara

Emrah Dural, Erhan Boran, Betul Isiner, Görkem Mergen and Tülin Söylemezoglu

Institute of Forensic Medicine, Ankara University, Ankara, Turkey

Benzene, Toluene, Ethyl Benzene and Xylenes (BTEX) are in a class of chemicals known as volatile organic compounds (VOCs). These chemicals have toxic health effects depending on duration and levels of exposure, even at microgram per cubic meter concentrations. Toluene, ethylbenzene and xylenes have harmful effects on the central nervous system. Benzene in particular is a well-known genotoxic carcinogen and has caused great concern historically as an occupational health hazard. BTEX exposure is basically known to be originated from industrial sources highlighting occupational exposure; on the other hand, recent studies showed that non-occupational exposure is becoming a growing risk for

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the city dwellers. In this study, quantitative analysis of BTEX group of organic solvents in urine was performed and the method validated; occupational and non-occupational levels were detected. For observing occupational chronic exposure, urine samples of 50 furniture staining workers from Siteler - Ankara, which is one of the largest furniture production centers of Turkey and Middle Eastern countries, and urine samples of 50 control subjects who live in Ankara city, have been collected. Analysis was performed by Head Space GC-FID. The developed method shows high selectiv-ity, sensitivity, repeatability, recovery and linearity (correlation coefficients >0.998), providing a useful alternative to assess human exposure to BTEX compounds due to occupational reasons or eventual exposure to organic solvents. For the urine samples of the group who were exposed to BTEX group of organic solvents, results for Benzene, Toluene, Ethyl Benzene, m-Xylene, p-Xylene and o-Xylene are 1.4 ± 2.3 ng/mL, 28.8 ± 65 ng/mL, 4.6 ± 9.3 ng/mL, 0.72 ± 1.2 ng/mL, 1.55 ± 1.64 ng/mL and 2.55 ± 2.77 ng/mL, respectively. For the urine samples of the group who were not exposed to BTEX group of organic solvents, results are 0.54 ± 0.71 ng/mL, 0.20 ± 0.72 ng/mL, 2.9 ± 4.3 ng/mL, not detected, 1.33 ± 0.58 ng/mL and ng/mL. The BTEX concentrations in exposed workers were markedly higher than the values of control group. In addition, the existence of even very low levels of organic solvents in control group shows that there is an inevitable exposure for BTEX for city dwellers.

P158.NDetermination of Six Fungicides Using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) in Combination with LC in Food

Gül Özhan1 and Buket Alpertunga2

1Department of Pharmaceutical Toxicology, Istanbul University, Istanbul, Turkey, 2Pharmaceutical Toxicology, Istanbul University, Istanbul, Turkey

Pesticides are used on an increasingly wider scale throughout the world. Besides, the misuse of pesticides by concerned individuals and lack of or weak national controlling plans are behind the outbreak of adverse effects in developing countries. Procymidone, Prochloraz, Tebuconazole, Triadimefon, Triadimenol and Vinclozolin are six pesticides widely used in agriculture for the control of fungal diseases in a variety of vegetables and fruits such as carrot, apple, strawberry, green pepper, tomato, cucumber. Even low-level exposures may be associated with adverse health effects such as endocrine disruption, chemical sensitivity, and cancer. The purpose of the study was to evaluate the residual concentration of the selected pesticides in various foods and to estimate the potential health risk associated with each of selected pesticides. They were determined using quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction method followed by liquid chromatography (LC) with photodiode array (PDA) detection for quantification. In this way, the simultaneously analysis for the selected pesticides was successfully applied in various vegetables and fruits, using instrumentation accessible for almost every laboratory equipped for carrying out routine controls. With the analysis of health risk estimates, we also believed that the obtained data would help in assessing the human exposure to toxic pesticides.

P159. Dioxin-induced Disorders in Fetal Steroidogenesis and Gonadotropin Formation: Minor Contribution of Oxidative Stress to the Damages and the Protective Effect of a Vitamin, a-Lipoic Acid

Takayuki Koga, Tomoki Takeda, Takumi Ishida, Yuji Ishii and Hideyuki Yamada

Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a representative environmental pollutant, causes a number of toxic effects in laboratory animals. In particular, it is of much concern that TCDD causes reproductive and developmental toxic effects in pups by maternal exposure at low doses which are little toxic to the dams. Many studies have been conducted to clarify the mechanisms of these toxic effects. However, although the mechanisms suggested so far and their combi-nations may partially explain the reproductive and developmental toxicity caused by TCDD, it has not yet been fully understood how these mechanisms contribute to the toxicity. Our previous studies have demonstrated that TCDD reduces the fetal expression of gonadal steroidogenic proteins, such as steroidogenic acute-regulatory protein (StAR) and cytochrome P450 17 (CYP17). The pieces of evidence provided in this laboratory have also suggested that the above defects produced by TCDD are triggered by suppression of the expression of fetal pituitary gonadotropins. However, it has not yet been known how TCDD reduces the expression of fetal pituitary gonadotropins. Dioxins are known to

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enhance oxidative stress in animals. Therefore, the present study focused on whether oxidative stress plays a role in the TCDD-produced damages to steroidogenesis and gonadotropin formation, and we examined whether anti-oxidants can attenuate the disorders. We used three anti-oxidants, α-Lipoic acid (α-LA), 3-tert-butyl-4-hydroxyanisole and ascorbic acid. These anti-oxidants (i.v.) were co-administered with TCDD (1 µg/kg, p.o.) to pregnant rats at gestational day 15. Following treatment with an anti-oxidant for 5 consecutivedays, the fetuses were removed to determine their expression levels of gonadal steroidogenic proteins and gonadotorpins as well as those mRNAs. TCDD reduced the fetal expression of steroidogenic proteins including StAR and CYP17. While α-LA restored the reduction to the control level, the other anti-oxidants did not show any alleviative effect. A similar picture was also seen in the fetal gonadotropins; that is, a reduction in fetal luteinizing hormone and follicle-stimulating hormone by TCDD was restored only by co-treatment with α-LA, but the other anti-oxidants lacked the protective effect. Thus, α-LA was revealed to effectively restore a reduction in the fetal expression of gonadotropins and steroidogenetic proteins by TCDD. The data obtained suggest that 1) oxidative stress poorly contributes to the TCDD-induced disorder of fetal steroidogenesis, and 2) α-LA exhibits a restorative effect via its characteristic as a cofactor for intermediary metabolism rather than as an anti-oxidant.

P160. Do multiple cytochrome P450 isoforms contribute to the activation of the insecticide carbosul-fan in human?

Khaled Abass1, Petri Reponen2, Miia Turpeinen1, Sampo Mattila2 and Olavi Pelkonen1

1Pharmacology and Toxicology, Institute of Biomedicine, University of Oulu, Oulu, Finland, 2Department of Chemistry, University of Oulu, Oulu, Finland

This study aims to characterize individual CYP enzymes involved in the in vitro metabolism of the carbamate insecticide carbosulfan [2,3-dihydro-2,2-dimethylbenzofuran-7-yl(dibutylaminothio)methyl carbamate] in human. Metabolites were identified by LC-TOF (Micromass LCT) and quantified by triple quadrupole mass spectrometry (Micromass Quattro II). Seven phase I metabolites were detected by LC-MS in pooled human hepatic microsomes. The primary metabolic pathways were detoxification by the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulphur oxidation pathway′) and activation via cleavage of the nitrogen sulfur bond (N-S) to give carbofuran and dibutylamine (‘carbofuran path-way′). Carbofuran was further hydroxylated to 3-hydroxycarbofuran, which was further oxidized to 3-ketocarbofuran or 3-hydroxy-7-phenolcarbofuran, and finally to 3-keto-7-phenolcarbofuran. CYP recombinant enzymes, relative contributions, correlation studies and a selective inhibitor were used to identify CYP enzymes catalyzing carbosul-fan metabolism in human. CYP3A5 and CYP2B6 had the greatest efficiency to form carbosulfan sulfinamide, while CYP3A4 and CYP3A5 were the most efficient in the generation of the carbofuran metabolic pathway. Based on average abundances of CYP enzymes in human liver, CYP3A4 contributed to 98% of carbosulfan activation, while CYP3A4 and CYP2B6 contributed 57 and 37% to detoxification, respectively. Significant correlations between carbosulfan activation and CYP marker activities were seen with CYP3A4 (omeprazole sulfoxidation), CYP2C19 (omeprazole 5-hydroxylation) and CYP3A4 (midazolam 1′-hydroxylation), displaying r2 = 0.96, 0.87 and 0.82, respectively. Activation and detoxifica-tion pathways were inhibited by ketoconazole, a specific CYP3A4 inhibitor, by 90-97% and 47–94%, respectively. Our studies, although restricted to in vitro metabolic data from human hepatic preparations, provide valuable quantitative carbosulfan-specific data for risk assessment. These results suggest that the carbosulfan activation pathway is more important than the detoxification pathway, and that carbosulfan activation is predominantly catalyzed in humans by CYP3A4.

P161. EFFECT OF HIGH DOSE OF ALUMINUM NITRATE ON CHOLINERGIC SYSTEM IN WISTAR RAT

Fatima-Zahra Azzaoui and Ahmed Ahami

Biology, Unit of Clinic and Cognitive Neuroscience and Health, Laboratory of Biology and Health, Faculty of Science, Kenitra, Morocco

Aluminum is the 3rd abundant metallic element in the nature after oxygen and silicon and is extensively used in modern daily life (food, drugs, cosmetics, vaccines, household materials, and water). It was considered, for long time, as non toxic element and completely excreted out of the body by renal way. However, currently several studies had found that aluminum was involved in pathology of nervous system, but its pathway is still discussed. The aim of this study was to

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investigate the toxicological effect of high dose of aluminum nitrate administered once by gavage on various organs and on the cholinergic system of Wistar male rat. This toxicological effect was evaluated in the brain, liver, spleen, and kidney by external gross examination, weighting organs and in the cholinergic system by measuring the rat′s brain acetylcholinesterase (AchE) and acetylcholine (Ach) levels by colorimetery. The results have shown that the acute high dose of aluminum caused a significant decrease in spleen weigh (p<0.05) with visible dark coloration of this organ, but no statistical difference in the weight of brain, liver and kidney were registered in the intoxicated rats compared to the control ones. Also, high significant decrease in the level of AchE was registered in the intoxicated rats′ brain (p<0.01), but no significant difference was shown in the Ach brain levels.

P162. Embryotoxic and Teratogenic Effects of Roundup® Max on Rat Development

Ayþe, Cansýn. Aslý, Dürdane Geriþlioglu and Dürdane Kolankaya Sr.

Biology, Hacettepe University, Ankara, Turkey

Glyphosate is the active ingredient of Roundup® Max, commercially available as a non-selective, organophosphorated agrochemical and broad-spectrum herbicide. It is widely used in many countries, including Turkey to control weeds in emerged grasses, broad-leaf weeds, rice, corn and soy plantations which acts after the sprout in a systemic way. In recent study, to evaluate the possible developmental effects of Glyphosate, female Wistar rats were treated with Roundup® Max containing 78.5% Glyphosate by oral gavage during pregnancy. There were three groups of each containing five pregnant rats. We administrated 10% and 20% doses of Roundup® Max, which is the commercial form of Glyphosate with LD

50 dose 5600 mg/kg. Rats in Group I, II and III were fed with standard diet, 560 mg/kg Roundup and 1120 mg/kg

Roundup during gestational days (GD 0-20) respectively. We assessed foetal body lengths and weights, organ weights and also made morphometric examination of placenta and umbilical cord. There were a significant decrease in foetal body weights and foetal liver weights in treatment groups (P≤0.05). The placental weights of Group II and Group III were found to be increased statistically. Placental lengths were increased in treatment groups. The umbilical cord lengths in Group II showed a significant increase when compared to Group I. It was evaluated that implantation ratios were decreased and resorption ratios were increased in treatment groups. According to skeletal stainings of foetuses, calsification lengths of humerus (4.9%, 19.8%), radius (14.2%, 22.6%), ulna (14.7%, 21%), femur (2%, 30%), tibia (3.9%, 29.1%) and fibula (4.3%, 13.5%) were statistically decreased in 10% and 20% Roundup treatment groups respectively. These results suggest that prenatal development of rats during gestation is sensitive to Roundup® Max exposure.

P163. Experimental Study on the Effect of Roasting, Boiling and Microwaving Cooking Method on Enrofloxacin Residues in Edible Tissues of Poultry

Afshin Javadi, Seied Amin Khatibi and Hamid Mirzaie

Food hygiene Dep., Veterinary science faculty, Islamic Azad University, Tabriz Branch,Tabriz, Iran, Tabriz, Iran

The purpose of this study was to determine the effects of different cooking processes like boiling, roasting and microwaving on enrofloxacin residues in chicken muscle, liver and gizzard tissues of broiler chickens. Each of chicks was fed by water and food with %0.05 of enrofloxacine in their drinking water for consecutive 5 days. Then; three locations were sampled aseptically from each carcass: breast muscle; liver and gizzard. Enrofloxacin residue was analyzed using microbial method by plates seeded with Escherichia coli. After doing different phases of the test on raw samples, the positive raw samples cooked by various cooking procedures and we surveyed cooked samples with similar method again for present of residue. The results show reduction in concentration of enrofloxacin residue after different cooking processes. The most reduction of the residue in cooked meat and gizzard samples related to boiling process and roasting process for cooked liver samples and the highest detectable amount of residue belonged to microwaving process in all cooked samples. Regarding to the results of this study, we can conclude that cooking processes can′t annihilate total amounts of these drug and it can only decrease their amounts and the most of residue in boiling process excreted from tissue to cooking fluid. The purpose of this study was to determine the effects of different cooking processes like boiling, roasting and microwaving on enrofloxacin residues in chicken muscle, liver and gizzard tissues of broiler chickens. Each of chicks was fed by water and food with %0.05 of enrofloxacine in their drinking water for consecutive 5 days. Then; three locations were sampled aseptically from

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each carcass: breast muscle; liver and gizzard. Enrofloxacin residue was analyzed using microbial method by plates seeded with Escherichia coli. After doing different phases of the test on raw samples, the positive raw samples cooked by various cooking procedures and we surveyed cooked samples with similar method again for present of residue. The results show reduction in concentration of enrofloxacin residue after different cooking processes. The most reduction of the residue in cooked meat and gizzard samples related to boiling process and roasting process for cooked liver samples and the highest detectable amount of residue belonged to microwaving process in all cooked samples. Regarding to the results of this study, we can conclude that cooking processes can′t annihilate total amounts of these drug and it can only decrease their amounts and the most of residue in boiling process excreted from tissue to cooking fluid.

P164. Highly Elevated 7-Ethoxyresorufin O-deethylase (EROD) Activities in Flathead Mullet (Mugil cephalus) and Striped Red Mullet (Mullus surmuletus) from Zonguldak Harbour

Azra Bozcaarmutlu1, Canan Sapmaz1, Sema Gulen1 and Emel Arinç2

1Department of Chemistry, Abant Izzet Baysal University, Bolu, Turkey, 2Graduate Programme in Biochemistry and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

Industrial areas, rivers, coastal cities have tendency to create local pollution in the Black Sea. Zonguldak is a highly urbanized and industrialized city in the West Black Sea Region of Turkey. It is an important coal mining area in Turkey. The harbour of this city is polluted especially with coal processing wastes, shipping activities, uncontrolled discharges from industrial and domestic wastes. The studies concerning the effects of pollutants on the organisms are relatively few in the Black Sea. Organisms are often exposed to complex mixtures of pollutants, including polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polyaromatic hydrocarbons (PAHs), alkyltin compounds, and metals. Induction of CYP1A and associated EROD activity are the best characterized and used biomarker of exposure of fish to organic pollutants such as PCBs, PAHs and dioxins. Its induction also acts as an early warning sign for potential harmful effects of many organic molecules. In this study, the degree of PCBs, PAHs and dioxins type pollution was determined in Zonguldak Harbour and relatively clean area Amasra using CYP1A associated 7-ethoxyresorufin O-deethylase (EROD) activity in two feral fish species: flathead mullet (Mugil cephalus) as a pelagic and striped red mullet (Mullus surmuletus) as a benthic fish. Flathead mullet samples were caught from Zonguldak Harbour and Amasra in 2008 and 2009. Striped red mullet samples were caught from the same sites in 2009. Fish were killed by decapitation and livers were removed immediately and flash frozen in liquid nitrogen. Liver microsomes were prepared by differential centrifugation. EROD activities of fish liver microsomes were determined by spectrofluorometric assay. EROD activities of flathead mullet caught from Zonguldak Harbour were 3114 ± 399 pmol/min/mg protein (n=12) in 2008 and 4780 ± 487 pmol/min/mg protein (n=11) in 2009. EROD activities of flathead mullet caught from Amasra were 613 ± 13 pmol/min/mg protein (n=3) in 2008 and 702 ± 131 pmol/min/mg protein (n=3) in 2009. Flathead mullet caught from Zonguldak Harbour displayed 5-7 fold higher EROD activity than Amasra. CYP1A protein levels were also determined in flathead mullet liver microsomes using the polyclonal antibodies produced against leaping mullet (Liza saliens) CYP1A by western blot analysis. The CYP1A bands of flathead mullets caught from Zonguldak Harbour were stained more intensely than those obtained from Amasra. CYP1A protein level measurements were highly correlated with EROD activity measurements. Highly elevated EROD activities were also measured in striped red mullet caught from Zonguldak Harbour (268 ± 25 pmol/min/mg protein (n=24)) than that of striped red mullet caught from Amasra (93 ± 13 pmol/min/mg protein (n=20)). The elevation in EROD activity in two fish species indicates that sea water and sediment of Zonguldak Harbour are highly contaminated with PAHs/PCBs and dioxins type carcinogenic pollutants. Flathead mullet and striped red mullet are economically important and highly consumed fish species in Turkey. It is clear that the level of pollutants in this region is a threat to both fish species and human health.

Acknowledgement: This study was supported by a grant from TUBITAK (Project ID: 104Y083)

P165. In vivo Genotoxicity Assessment of Esbiothrin in Fish (Cyprinus carpio L., 1758) Using the Micronucleus Test and Comet Assay

Mahmut Selvi1, Tolga Çavas2, A.Caglan Karasu Benli3, Burcu Kocak Memmi4, Nilüfer Cinkilic2, Aylin Sepici Dincel5, Ozgur Vatan2, Dilek Yilmaz2, Rabia Sarikaya6, Tolga Zorlu2 and Figen Erkoç7

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1Department of Science Education, Gazi University, Ankara, Turkey, 2Department of Biology, Uludag University, Bursa, Turkey, 3Department of Environmental Sciences, Gazi University, Ankara, Turkey, 4Insecticide Test Laboratory, Hacettepe University, Ankara, Turkey, 5Faculty of Medicine, Gazi University, Ankara, Turkey, 6Department of Primary School Education, Gazi University, Ankara, Turkey, 7Department of Biology Education, Gazi University, Ankara, Turkey

Esbiothrin (CAS No: 584792) is a synthetic pyrethroid with quick activity against household pest insects. It is also widely used in public health against mosquitoes, houseflies and cockroaches. Despite its widespread use, data on the genotoxic effects of esbiothrin are extremely scarce. Thus, in the present study, we aimed to evaluate the genotoxic potential of esbiothrin on a model fish species Cyprinus carpio L., 1758 (Pisces: Cyprinidae, koi) using the micronu-cleus test and the comet assay in peripheral blood erythrocytes. Furthermore, histological changes in gill tissue were examined. The plasma total antioxidant status (TAS μmol/L) were also analyzed using commercial kits. The 96 h LC

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of esbiothrin was determined as 32 µg/L for common carp (http://cfpub.epa.gov/ecotox/quick_query.htm) and based on this value two test concentrations (5 μg/L and 10 μg/L) were selected. Ethyl methane sulphonate (EMS) at a single dose of 5 mg/L was used as positive control. Five fish were used for each dose/duration group. Fish were exposed to the selected test concentrations of esbiothrin and EMS for 24, 48 and 72 h under controlled laboratory conditions. The fish showed behavioral changes at the higher dose. As a result it was observed that plasma TAS levels increased sig-nificantly at the 48th and 72nd hours in both esbiothrin dose groups. Our results further revealed significant increases in the frequencies of micronuclei, as well as in the levels of DNA strand breaks following exposure to esbiothrin and thus demonstrated the genotoxic potential of this pesticide on fish, a non-target organism of the aquatic ecosystem. To our knowledge this is the first study to report observable genotoxic effects of esbiothrin on fish.

P166. Preventive effect of magnesium on nickel hepatotoxicity and nephrotoxicity in albino (Wistar) rats

Zine Kechrid

Department of Biochemistry, University of Annaba, Annaba, Algeria, Annaba, Algeria

We studied the effect of intraperitonial magnesium treatment on nickel sulphate-induced hepatotoxicity and nephro-toxicity in Wistar strain male albino rats. Liver and kidney dysfunction parameters represented by aspartate transami-nase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), blood glucose, serum total protein, serum urea, serum creatinine, and serum belurebin were estimated. Liver glutathione level, catalase and GPx activities were also determined in liver as indicators of oxidative damage. Nickel treatment was led to high serum glucose concentration and produced hepatotoxicity and nephrotoxicity characterized by increasing GPT, GOT and alkaline phosphatase activities, serum total protein, serum urea, serum creatinine and serum belurebin concentrations. In addition liver glutathione level, catalase and GSH-Px activities were diminished due to high lipid peroxidation. The simultaneous administration of magnesium with nickel sulphate resulted in a remarkable improvement of the previous parameters comparison with rats treated with nickel alone. Normal control and magnesium treated animals revealed normal histology of liver. On the other hand, nickel treated animals showed alterations in normal hepatic histoarchitecture which comprise of vacuolization of the hepatocytes and dilatation of sinusoids. Administration of magnesium to nickel treated rats resulted in marked improvement in the structure of hepatocytes, thus emphasizing the protective potential of magnesium in restoring the altered hepatic histoarchitecture. In conclusion, nickel sulphate led to liver and kidney dysfunctions and hepatic lipid peroxidation in animals, but simultaneous treatment with magnesium offers a relative protection against nickel induced hepatotoxicity, nephrotoxicity and lipid peroxidation.

P167. Protective role of ascorbic acid on lambdacyhalothrin-induced oxidative stress and alterations of acetylcholinesterase activity in the cerebellum of adult rats

Hamadi Fetoui and Najiba Zeghal

Life sciences, Laboratory of Animal Physiology, Sfax, Tunisia

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The wide use and wide-spectrum toxicity of synthetic pyrethroids (SPs) insecticides make them an emerging ecotoxi-cological concern. The objective of the current study was to investigate the involvement of oxidative stress on lambda-cyhalothrin (LTC)-induced cerebellum damages in adult rats, and to evaluate the possible protective effect of vitamin C (Vit. C) as antioxidant. Exposure rats to lambda-cyhalothrin during 3 weeks, caused a significant (P < 0.01) increase in the levels of LPO, nitric oxide (NO) and protein carbonyls (PCO) along with significant (P < 0.01) decrease in the levels of reduced glutathione (GSH) and the activities of AChE, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and gluthione-S-transeferase, (P < 0.05) compared to control group. Oral administration of vita-min C (200 mg/kg/day) to LTC-treated rats significantly (P < 0.01) reduced the levels of LPO, nitric oxide and protein carbonyls and increased the activities of GSH and antioxidant enzyme activities. Our results indicated that vitamin C attenuated the lipid peroxidation, protein oxidation and impaired antioxidant enzyme activities in LTC exposed rats. Thus suggested that ascorbic acid protected the brain against toxic effects of synthetic pyrethroids.

P168.NStudy of occupationnal hazards on human infertility

Bensoltane Samira1, Abdelaziz Amina2, Djekoun Mohamed3, Loucif Wahida1 and Berkat Faical1

1Medecine, Faculty of medecine, Annaba, Algeria, 2Biology, University of Badji Mokhtar, Faculty of sciences, Annaba, Algeria, 3Biology, Faculty of sciences and ingeniery, 08 mai 1945 University, Guelma, Algeria

Research into occupational exposures and effects on reproductive systems has made important scientific contributions in the past few decades. In the workplace, sources of exposure to these xenobiotics are numerous because they are very different products (pesticides, solvents, metals....), and toxicity is mainly long term because the absorption is at small doses over long periods. This study was conducted in the laboratory of Cytogenetics-Ibn Sina Annaba, (ALGERIA), had a goal of highlighting the impact of pesticides, metals, organic solvents and construction materials, in male fertility. On 2350 spermiograms, over a period of 05 years, we have identified a target population of 270 workers in atmospheres chemically polluted. The results show a slur of all subjects in six groups: workers in masonry, painting, carpentry, agri-culture, plumbing, mechanics and employees ISPAT (major steel manufacturing plant in the north east of Algeria).

P169. Abstract withdrawn

P170. The Epidemiologyic Study of Suicide in North West Of Iran

Esmaeil Farzaneh1, Iraj Sayadrezai2, Babak Mostafazadeh3 and Farnaz nasl Seraji1

1Medical Toxicology and Forensic Medicine, Ardebil University of Medical Sciences, Ardabil, Iran, Ardabil, Iran, 2Department of medical toxicology & forensic Medicine, Ardabil university of medical sciences, Ardabil, Iran, 3Medical Toxicology & Forensic Medicine, Shaheed Beheshty University Of Medical Sciences, Tehran, Iran

The Epidemiologyic Study of Suicide in Ardabil Province BetweenYear 2003-2009. Objective: Suicide is an important problem for social safety and health. Recognizing some factors of risk forecasting after epidemiologic studies on people whom attempted to suicide could prepare and present outlines and proper guides for preventing by health and social planners. this research is purposed on suicide epidemiologic study to obtain full and enough data about deceased people by suicide has been accomplished in Ardebil province. Methods: The accomplished research is a descriptive and analytic sectional research which is based on all deceased people by suicide during 6 years, which their statistics have been registered in Bualy, Fatemi & Alavy hospitals and forensic medicine of Ardebil province. The used tool was created researcher form that included epidemiologic particular of deceased people. The data were analyzed using SPSS software version 13. Results: The highest rate of suicide had been belonged to age rank of 15-24 years(43.8%) and had occurred in female (62.5, %), married people (57.8%) urban society (65.6%) and patient without past chronic physical illness or psychiatric history and without pre-attempt suicide. Self poisoning by drugs and toxins was the most common used method in this case (90.6%). Orderly among drugs and toxins organo-phosphorate toxin, tricyclic antidepressants and aluminum phosphid had been most used. Conclusion: This research is indicating various risk factors such as sex, age, marriage status, residing place of society and easy access to drugs and toxins. According to obtained results, it is

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better or proper to accomplished related education and training of preventation and interference of mental health in second and third decades of life with allocation of much time for females (especially the married people of this group). It is suggested that must be considered more attention to education, preventation, diagnosis and treatment of poisoning especially poisoning by most common Drugs and toxins.

References

1) Sadock B, Sadock V. Kaplan and Sadocks Pocket Hand Book of Clinical Psychiatry, Vol l; 4thed; Lippincott Willams and Wilkins; 2005: 289-304.

2) Levin A. The startling increase in the number of suicides by members of the U.S. Army in the last few years prompts a major collaborative study of risk and protective factors. Psychiatric News. Aug 2009, 44(16):1.

3) Sadock B, Psychiatric Emergencies in Sadock B, Sadock V, Ruiz P. Kaplan and Sadocks Comprehensive Textbook of Psychiatry. Vol 2; 9thed; Lippincott Williams & Wilkins. 2009: 2717-2745.

P171. The impact of Propiconazole on the fertility in domestic rabbits

Mallem Leila1 and Boulakoud Mohamed Salah2

1Medecine, Faculty of medecine, university of Annaba, Annaba, Algeria, 2Biology, University Badji Mokhtar- Annaba, Faculty of science, Annaba, Algeria

The main objective of this work was to study the effect of a widely used fungicide Propiconazole on fertility in the rab-bits Oryctologus cuniculus. The treatment was made in food with two doses 1 and 5 mg/ml/kg of the food /day for 15 days. Summarizing the finding, it can be established that the administration of Propiconazole in the treated groups as compared with control groups caused a reduction in the weight of testes with increase in the weight of the liver. The present experiment reported a very significant reduction in the number, vitality and mobility in the treated spermatozoa. In conclusion, Administration of propiconazole affects the biology of sperms.

P172. The mechanism of defects in sexual behavior by maternal exposure to dioxin: Focusing on gene expression in the pituitary and hypothalamus

Tomoki Takeda1, Misaki Fujii1, Junki Taura1, Midori Yamamoto2, Masaru Himeno2, Yuji Ishii1, Takumi Ishida1 and Hideyuki Yamada1

1Graduate School of Pharmaceutical sciences, Kyushu University, Fukuoka, Japan, 2Pharmaceutical Sciences, Nagasaki International University, Sasebo, Japan

Many forms of reproductive toxicity, such as defects in sexual behavior caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) occur in pups whose mother is exposed to the lower doses of this substance. However, the mechanism underly-ing these defects remains to be clarified in spite of many researches conducted so far. Our previous studies have provided evidence that the administration of TCDD (1 μg/kg, orally) to pregnant Wistar rats at gestational day 15 (GD15) causes a reduction in gonadotropin biosynthesis in the fetal pituitary, resulting in the attenuated expression of steroidogenic pro-teins in the fetal testes. Such attenuation occurred during a short period ranging from GD20 to postnatal day 0 (PND0). The direct supplementation of equine chorionic gonadotropin into the fetuses exposed to TCDD at GD15 restored not only the reduced expression of gonadal steroidogenic proteins but also defects in sexual behavior. This observation strongly supports the view that defects in gonadotropin formation trigger the impaired expression of steroidogenic proteins. To further clarify the mechanism of TCDD effect on fetal gonadotropin biosynthesis, we investigated whether TCDD can directly affect the fetal pituitary to reduce gonadotropin biosynthesis, using cultured pituitary. When fetal pituitary was cultured in the presence of gonadotropin-releasing hormone (GnRH), TCDD interfered with the GnRH-induced expression of gonadotropin β-subunit. Such an interference was fetus-specific because cultured postnatal pituitary (PND7) was insensitive to TCDD treatment. These observations suggest that TCDD reduces gonadotropin biosynthesis via, at least partially, its direct action on the fetal pituitary. We then performed DNA microarray analysis to identify the target genes linked to a reduction in gonadotropin β-subunit (fetus) and to defects in sexual behavior (adult). In fetal pituitary, a number of genes, such as transcription factors and apoptosis-related factors, as well as

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gonadotropin β-subunit were affected by maternal exposure to TCDD at GD15 (1.3-fold up: 86 genes, 0.7-fold down: 59 genes). The effect of TCDD was also observed in fetal hypothalamus, the regulatory organ of the pituitary. For example, the expression of some growth factors and signal transduction factors was reduced by TCDD. These results suggest that TCDD damages the gene regulation of fetal pituitary and hypothalamus, and this is a possible mechanism whereby the attenuation in gonadotropin β-subunit takes place. More importantly, change in gene expression was observed in adult hypothalamus, even although the animals were exposed to TCDD at fetal age (GD15). The genes reduced by TCDD include GnRH which plays a role in the expression of sexual behavior. This observation suggests that TCDD imprints the abnormality of gene expression during the developmental processes, and an adult age-specific reduction in GnRH is one of the mechanisms causing defects in sexual behavior.

P173. The Role of CYP51 in Global Health

Michael R. Waterman1 and Galina I. Lepeshesheva2

1Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN, USA, 2Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN,

Infectious protozoa threaten the lives of several hundred million people throughout the world, being responsible for large numbers of death globally. Pathogenic protozoa are transmitted to mammalian species (including humans) by insect vectors. An excellent potential drug target for treatment of infections by Trypanosoma brucei (African sleeping sickness), Trypanosoma cruzi (American trypanosome causing Chagas disease), and Leshmania spp. is CYP51 (sterol 14α-demethylase). This is the most widely distributed P450 found in all biological kingdoms. From one kingdom to another the primary amino acid sequence identity ranges between 20-30%.Yet all metabolize very closely related substrates and catalyze the removal of the 14α methyl group by the same three step reaction. The current drugs used for treatment of protozoa infections are decades old, having low efficiency and being toxic. CYP51 is such a promising drug target for treatment of infection by Trypanosomatidae because it is essential for biosynthesis of sterols and as a result is essential for the synthesis of membranes. Without normal membranes, the protozoa cannot survive. We have determined the high resolution structure of CYP51 from the different infectious protozoa and are using these structures to design novel inhibi-tors for each monooxygenase. A key property of these inhibitors is that they will be specific for CYP51 from pathogenic protozoa, thereby not inhibiting the enzyme in the host (human). Here we present the role of conserved amino acids in all sterol 14α-demethylases and how they serve as a base on which inhibitors can be designed. We have now identified several inhibitors for CYP51 (azoles and substrate-based) which have as much as 200-fold greater specificity for inhibition of Trypanosomatidae 14α-demethylases compared to the human enzyme. These molecules can serve as scaffolds from which even more efficient and specific inhibitors will be synthesized. Infected rodents are being used in these studies as the beginning of determination of the efficiency and toxicity of novel 14α-demethylase inhibitors. The wide distribution of protozoa infections clearly establishes the role that inhibition of CYP51 can play in global health. We are confident that our studies will lead to novel drugs which can be used to cure infections by protozoa around the world.

P174. Toxicities of selected insecticides on the survival rate of Drosophila melanogaster (mwh x flr3) larvae

Burcu Kocak Memmi1, Rabia Sarıkaya2, Sibel Sümer3 and Figen Erkoç4

1Insecticide Test Laboratory Beytepe Campus, Hacettepe University, Ankara, Turkey, 2Department of Primary School Education Teknikokullar, Gazi University, Ankara, Turkey, 3Faculty of Science, Department of Biology, Beytepe Campus, Hacettepe University, Ankara, Turkey, 4Department of Biology Education Teknikokullar, Gazi University, Ankara, Turkey

In this study, the effects of some widely used insecticides (fenitrothion, deltametrin, imidacloprid, thiamethoxam, bifentrin, propoxur) on the survival rates of Drosophila melanogaster larvae were investigated. For this purpose, mwh-flr3 strains was crossed; third-instar larvae were chronically fed with the Drosophila instant medium to which five ml test solutions were added. Feeding ended with pupation of the surviving larvae. Lethal doses and survival rates of insecticides

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used in the experiments were determined. Two solvent control groups were used: acetone control and ethanol control. The survival rates of the control and experimental groups were compared by using the Chi-square test. Deltamethrin experimental group at 25 mg/L dose was found statistically significant (p<0.001). Imidacloprid experimental groups were found significant at p<0.01 level for 5 mg/L dose and significant at p<0.001 level for 10, 20 and 40 mg/L doses. Thiamethoxam experimental groups were found significant at p<0.01 level for 2.5 mg/L dose and significant at p<0.001 level for 5 and 10 mg/L doses. Bifenthrin experimental group was found statistically significant (p<0.01) at 15 mg/L dose. Fenithrothion was less toxic at 15.68 mg/L with 66% survival rate. In conclusion, thiamethoxam was the most toxic of the insecticides tested, with 21% survival rate at 10 mg/L dose; bifenthrin was the least toxic with 83% survival rate at 7.5 mg/L. The study indicates that insecticides tested were all toxic to early life stage of D. melanogaster, a non-target species in the ecosystem; therefore further risk assessment should be carried out.

P175. Characterization of polyphenol oxidase from quince (Cydonia oblonga) leaves

Gulnur Arabaci and Ayse Usluoglu

Chemistry, Sakarya University, Sakarya, Turkey

Polyphenoloxidase (PPO); [EC 1.14.18.1], a copper-containing enzyme, catalyzes oxidation of endogenous monophenols to ortho-dihydroxyaryl compounds and of ortho-dihydroxyaryl compounds to ortho-quinones. Subsequent nucleophilic addition reactions of phenols, amino acids, and proteins with the electrophilic ortho-quinones form brown-, black-, or red-colored secondary products associated with the undesired discoloration of fruit, vegetables and animal tissues. Thus, extensive research has been carried out on PPO activities and PPO has been partially purified from many fruits, including grape, apple, peach, banana, pear, kiwi, strawberry, plum, cherry and pineapple [1]. Quince leaves (Cydonia oblonga) have been known and used for bronchitis, diarrhea in local pollutions. n this work, Quince leaves (Cydonia oblonga) were used PPO characterization with some specific substrates. In this work, Quince leaves were harvested fresh from the region of Sakarya, in Turkey. Quince leaves were homogenized in 0.1 M of phosphate buffer (pH 7.0) contain-ing 10 mM ascorbic acid, 4% triton X-100 and 0.5% polyvinylpyrrolidone. The homogenate was filtered and then the filtered material was centrifuged at 14,000 rpm for 15 min at 4°C. PPO activity was determined by measuring the initial rate of quinone formation as indicated by an increase in absorbance at 420 nm. PPO activity, as a function of pH, was determined in a pH range of 4.5–5.5 in 50 mM acetate buffer, 6.5–7.5 in 50 mM phosphate buffer and 8.5–9.5 in 50 mM Tris–HCl and Tris-Base buffer. PPO activity was assayed using catechol, 4-methylcatechol, caffeic acid as diphenolic substrates and L-tyrosine as a monophenolic substrate, gallic acid and pyrogallol as three phenolic substrates. The optimum pH, temperature obtained for all substrates was used for determining substrate specificities [2]. For each substrate, the kinetic data were plotted as reciprocals of activities versus substrate concentrations. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) were determined as the reciprocal absolute values of the intercepts on the x- and y-axes, respectively, of the linear regression curve. Substrate specificity (Vmax, Km) was calculated by using the data obtained on a Lineweaver-Burk plot. he results showed that the apparent substrate specificity of quince leaf PPO was established from values of Km and Vmax as: caffeic acid > gallic acid >pyrogallol> L- Tyrosine> 4-methyl catechol >catechol. For each substrate, optimum pH and temperature values were caffeic acid pH: 4 and 40°C, gallic acid pH: 4 and 20°, pyrogallol and L-tyrosine pH: 5 and 20°C, 4-methyl catechol pH: 7 and 20°C, catechol pH:7.5 and 30°C.

References

[1] Dziki, U. G., Szymanowska, U., Baraniak, B., Characterization of polyphenol oxidase from broccoli (Brassica oleracea var. botrytis italica) florets. Food Chemistry 105, 1047–1053, 2007.

[2] Erat, M., Sakiroglu, H., & Kufrevioglu, O. I., Purification and characterization of polyphenol oxidase from Ferula sp. Food Chemistry, 95, 503–508, 2006.)

P176. Whole-Cell Biotransformation With Recombinant Fission Yeast Expressing Natural Occurring Variants Of Human CYP3A Enzymes Generated By Directed Evolution: Biotechnicological Use Of Gene-Polymorphisms With Elevated Enzyme Activity

Ina Neunzig1, Frank Peters2, Hans Maurer3 and Matthias Bureik1

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1PomBioTech GmbH, Saarbrücken, Germany, 2University Hospital Jena, Institute for Forensic Medicine, Jena, Germany, 3Department. of Experimental and Clinical Toxicology, Uniklinik Homburg, Homburg, Germany

Cytochromes P450 3A are the major drug-metabolizing P450s in the human liver. This P450 subfamily comprises the four isoforms CYP3A4, CYP3A5, CYP3A7, and CYP3A43, respectively, of which the first three have been shown to be relevant for endogenous and xenobiotic metabolism. These enzymes exhibit similar substrate specificity but different levels of activity and in some cases even different stereospecificity. For human CYP3A enzymes there are two known SNPs (CYP3A4.18 and CYP3A7.2) which have been shown to cause elevated enzyme activity towards certain substrates. As the four isoforms show at least 85 % AA-sequence identity among each other, a conformational change and altered activity caused by a SNP naturally occurring in one isoform could also influence the activity of other isoforms. We cloned fission yeast strains that express the natural enzyme variants as well as combinations of them in order to test them for increased biotransforma-tion activity. The quantification of the resulting metabolites was performed by HPLC/UV-vis detection. We show that the resulting strains display elevated activity towards the standard substrates testosterone and midazolam, respectively. These strains can now be used in whole-cell biotransformations for the biotechnological production of CYP3A metabolites.

P177. Assessment of Cytochrome P450 (1A2, 2B6, 2C9 and 3A4) Induction in Cryopreserved Human Hepatocytes Cultured in 48-well Plates Using the Cocktail Strategy

Brigitte Gerin, Sylvie Dell′Aiera, Hilmar Boekens, Steven Smith and Hugues Chanteux

UCB Pharma SA, Braine-L′Alleud, Belgium

Increasing the throughput of in vitro P450 induction studies has always been a challenge, regarding the availability and the cost of human hepatocytes. In order to reduce the number of hepatocytes needed and increase the throughput of the assay, the cocktail strategy (concomitant incubation of all probe substrates) using in situ incubation has already been proposed. However, the use of the cocktail strategy versus standard approach has never been fully investigated. This study aimed at validating, in the frame of the CYP induction assay, the cocktail strategy as an alternative to standard protocol. To this aim, we used cultures of cryopreserved human hepatocytes. Monolayers were cultured for 72h with prototypical CYP inducers (β-napthoflavone 25µM for CYP1A2, phenobarbital 1000µM for CYP2B6 and CYP2C9 and rifampicin 10µM for CYP3A4 and CYP2C9). Media were renewed every 24h. Preliminary checks were performed to select the appropriate incubation time with respect to time linearity. At the end of the induction period, kinetic parameters (Km, Vmax) for each probe (phenacetin for CYP1A2, bupropion for CYP2B6, diclofenac for CYP2C9 and midazolam for CYP3A4) were determined in control and in induced hepatocytes to allow the selection of an adequate substrate concentration that will be used in the cocktail. The lowest concentration as practically possible in terms of analytical sensitivity was selected to reduce the risk of interference when using the cocktail. In the second part of the study, we investigated the use of the cocktail strategy compared to the classical approach. To this aim, 3 different human donors were used to check for the inter-donor vari-ability. After the induction period, in situ incubations were performed with each probe substrate either alone or in cocktail at a fixed concentration, i.e. 10µM for phenacetin, 250µM for bupropion, 10µM for diclofenac and 3µM for midazolam. In addition, the effect of salicylamide, usually used as phase II inhibitor, was also investigated. The results showed that, in the conditions of this assay, there was no significant effect of the use of the cocktail strategy on the absolute CYP activities and on the fold induction parameter regardless of the human donors. However, the use of salicylamide dramatically decreased the apparent CYP1A2 activity whereas it increases the apparent CYP3A4 activity. Salicylamide had no significant effect on CYP2B6 and CYP2C9 activity and no significant effect on CYP1A2, 2B6, 2C9 and 3A4 when the results were expressed as fold induction. In summary, the study showed that the cocktail approach as defined in our assay can be used for CYP induction assay whereas the concomitant use of salicylamide in the incubation is not necessary and can generate some drug interactions. For this reason, we recommend to not use salicylamide in CYP induction assay.

P178. Automated Triplexed Hepatocyte-Based Viability and CYP1A and CYP3A Induction Assays in 96- and 384-well Microplates

Bradley R. Larson1, Peter Banks2, Timothy A. Moeller3 and James J. Cali4

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1Applications, BioTek Instruments, Winooski, VT, USA, 2Marketing and Sales, BioTek Instruments, Winooski, VT, USA, 3Celsis In Vitro Technologies, Baltimore, MD, USA, 4Department of R & D, Promega Corp, Madison, WI, USA

The ability to monitor CYP induction, inhibition, and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workloads and increase data confidence. Here we demonstrate the ability to monitor CYP1A and -3A induction, combined with a cytotoxicity measurement, from a single well using cryopreserved human hepatocytes. The assay procedure was automated in 96- and 384-well formats, including cell manipulations, compound titration and transfer, and reagent dispensing, using simple, yet robust robotic instrumentation. Eight compounds were tested to validate the assay; four known CYP1A inducers, and four known CYP3A inducers. A combination of luminescent and fluorescent chemistries was used to monitor the combined effects from each compound in the well. Fold induction, and EC50 values were derived for each compound, using an 11-point titration curve. Induction and toxicological responses in the triplex system were validated based on consistency with conventional single parameter assays, as well as comparison to FDA guidelines. Validation and pharmacology data confirm that automated, multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate the data needed from today′s ADME/Tox laboratory.

P179. Efavirenz induces hepatic and peripheral but not intestinal target genes of CAR

Stefan Oswald1, Henriette Meyer zu Schwabedissen2, Ali Nassif1, Christiane Modess1, Jette Peters1, Dieter Luetjohann3, Zeruesenay Desta4, Heyo K. Kroemer5 and Werner Siegmund1

1Department of Clinical Pharmacology, University of Greifswald, Greifswald, Germany, 2Department of Pharmacology, University of Greifswald, Greifswald, Germany, 3Department of Clinical Chemistry and Pharmacology, University of Bonn, Bonn, Germany, 4Department of Med/Div of Clin Pharmacol, Indiana Univ Sch of Med, Indianapolis, IN, 5Department of Pharmacology, University of Greifswald, Greifswald, Germany

Background: Efavirenz (EFA) is a non-nucleoside reverse transcriptase inhibitor, which is frequently used in the highly active antiretroviral therapy (HAART) in HIV-infected patients. The drug is known to be a potent activator of the nuclear constitutive androstane receptor (CAR) thereby inducing several metabolizing enzymes and transporter proteins in vitro. In this study, we measured the influence of EFA on the expression of several CAR target genes in intestinal tissue and peripheral blood mononuclear cells (PBMCs) of healthy volunteers pre-treated with EFA. In parallel, disposition of the lipid-lowering ezetimibe (EZE), a substrate of the CAR-regulated genes ABCB1, ABCC2 and UGT1A1, was studied before and after pre-treatment with EFA to conclude on functional changes.

Methods: 12 healthy male subjects (age 20-36, body mass index 19.9-27.2) received orally 400 mg EFA for 6 days. Genome wide expression analysis was performed in duodenal biopsies and in isolated PBMCs taken before and after EFA treat-ment. Subsequently, the results were verified on mRNA (real-time PCR) and protein level (Western blot). Before and after repeated administration of EFA, pharmacokinetics of EZE was evaluated at steady-state (10 mg, po). EZE and EZE glucuronide (GLUC) in serum, urine and feces were quantified by LC-MS/MS. 4-ßOH-cholesterol was determined as a surrogate for hepatic CYP3A4 activity.

Results: Intestinal expression of the CAR target genes ABCB1, ABCC1, ABCC2, ABCG2, CYP3A4, CYP2B6, UGT1A1, UGT2B7 and UGT2B15 was not affected by chronic EFA pre-treatment. In well agreement to this, EFA had no effect on the pharmacokinetics of EZE. The EZE glucuronide exposure was significantly decreased (411 ± 195 vs 466 ± 223 ng×h/ml, p=0.019) as caused by inhibition of UGT1A1 after EFA pre-medication (in vitro data). Strikingly, chronic treatment with EFA significantly up-regulated ABCB1 (p=0.0007) and CYP2B6 (p=0.0158) but decreased expres-sion of UGT1A1 (p=0.035) and UGT2B15 (p=0.0198) in PBMCs of our subjects. Moreover, serum concentrations of 4-ßOH-cholesterol were significantly increased compared to baseline values (100 ± 33.8 ng/ml vs. 64.0 ± 24.2 ng/ml, p<0.05).

Conclusions: Chronic treatment with EFA did not significantly influence intestinal target genes of the nuclear CAR but up-regulated hepatic CYP3A4 as well as ABCB1 and CYP2B6 in PBMCs. Consequently, disposition of EZE was altered by changes of glucuronidation rather than of intestinal ABCB1 and ABCC2.

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P180. Effect of Diet on Developmental Changes of Cytochrome P450 in Rat and Mouse

Kazumi Sugihara1, Hanaka Sako2, Miyuki Arai2, Seigo Sanoh2, Shigeyuki Kitamura3 and Shigeru Ohta2

1Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Japan, 2Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan, 3Nihon Pharmaceutical University, Saitama, Japan

The developmental changes of drug metabolizing enzymes, cytochrome P450 (CYP) and others, and the effect of diet on the activities of the enzymes in rat and mouse liver were examined. Wistar/ST rats and C57BL/6J mice were used. A regular cereal-based diet (MF; Oriental Yeast Co. Japan) or a purified diet (AIN-93G; Clea Japan, Inc.), according to the recommendations of the American Society of Nutrition, was fed to rats and mice before mating. The offspring of rats were sacrificed at PND 14, 21, 28, 35, and the liver microsomal CYP activities were assayed. The activity of CYPs were measured using the specific substrates of CYP isozymes, e.g. resorufin ethyl ether for CYP1A1, resorufin methyl ether for CYP1A2, resorufin pentoxy ether for CYP2B, 7-methoxy-4-trifluoromethylcoumarin for CYP2C9 and 7-benzoxy-4-trifluoromethylcoumarin for CYP3A. The expression of CYP isoforms was determined by Western blotting and mRNA was measured by realtime-RT-PCR. Little activity of CYPs was found in the liver of rat neonates (PND 1, 3, and 5). Their activities increased with increasing age and reached adults levels at 5 - 7 weeks of age. As CYP1As activities were significantly increased around weaning, we thought that the development of CYP activity could be affected by diet. Therefore, the comparative effects of two diets on the ontogeny of hepatic CYP in neonatal rats and mice were investi-gated. The neonatal body weight gain in the pure diet (AIN-93G) group was slightly larger than that of the normal diet group. The CYP activities were extremely low at PND 14 and PND 21 in both diet groups. In the regular diet (MF) group, the activities were remarkably elevated at PND28, whereas the pure diet (AIN-93G) group showed lower CYP activities than those of the normal diet group and still showed low activities at PND35. The developmental changes of the CYP activities of mice showed similar differences between two diets. These results suggest that regular cereal-based diets contain inducers of CYP. The ingredients of the diet were corn, soybean, wheat, alfalfa and others. Flavonoids and other components seemed to induce CYP.

P181. In situ evaluation of CYP1A2, CYP2B6 and CYP3A4/5 induction in cultured human hepatocytes with a cocktail of probe substrates: A comparison of percent positive control values

Kevin Lyon, Immaculate Amunom, Jason N. Neat, Jeff Holsapple, David Buckley and Andrew Parkinson

XenoTech, LLC, Lenexa, KS, USA

It has previously been demonstrated that a cocktail of probe substrates can be effectively used to screen simultane-ously for induction of CYP1A2, CYP2B6 and CYP3A4/5 with in situ (cell-based) incubations based on the magnitude of induction relative to appropriate positive controls (Amunom et al, 2009). Industry guidelines recommend that a compound be considered an inducer in vitro and therefore warrant further in vivo investigation if it elicits an increase in cytochrome P450 (CYP) enzyme activity that is equal to or greater than 40% of that caused by a suitable enzyme inducer (percent positive control). The objective of this study was to determine the percent positive control value of known CYP inducers (at varying concentrations) compared with the FDA-preferred positive controls, omeprazole (100 µM, CYP1A2), phenobarbital (750 µM, CYP2B6) and rifampin (10 µM, CYP3A4/5) based on in situ incubations of primary cultures of human hepatocytes with three marker substrates (namely, 100 µM phenacetin, 500 µM bupropion, and 100 µM midazolam) either individually or as a cocktail. Freshly isolated human hepatocytes (n=3) were cultured in a sandwich configuration and treated for three days with vehicle control (dimethyl sulfoxide, DMSO; 0.1%, v/v), the aforementioned positive controls or one of the following enzyme inducers: b-naphthoflavone (1-100 µM), lansoprazole (1-100 µM), dexamethasone (1-100 µM), phenytoin (1-100 µM), carbamazepine (1-100 µM), and efavirenz (0.25-25 µM). CYP induction was measured by LC/MS/MS based on phenacetin O-dealkylation (CYP1A2), bupropion hydroxylation (CYP2B6) or midazolam 1′-hydroxylation (CYP3A4/5). The results indicate that, when calculated as percent of the applicable positive control, the cocktail approach is equal to individual substrate approach in terms of assessing the enzyme-inducing potential of drugs. When CYP inducers exceeded the 40% threshold relative to the appropriate positive

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control, they did so in both the individual and cocktail substrate incubations at similar concentrations. These results further support the use of a cocktail of probe substrates to screen simultaneously for the induction of CYP1A2, CYP2B6 and CYP3A4 in cultured human hepatocytes.

References

Amunom I., Brawner S., Simpson J., Sawi J., Campbell R., Yerino P., Holsapple J., Neat J. and Parkinson A. (2009) In Situ Evaluation of CYP1A2, CYP2B6 and CYP3A4/5 Induction in Human Hepatocytes with a Cocktail of Probe Substrates. Drug Metabolism Reviews 41: 89.

P182. Abstract Withdrawn.

P183. Intact Glucosinolates Modulate Hepatic Cytochrome P450 and Phase II Conjugation Activities and can Contribute Directly to the Chemopreventive Activity of Cruciferous Vegetables

Ahmad Faizal Abdull Razis1, Manuela Bagatta2, Gina Rosalinda De Nicola2, Renato Iori2 and Cos-tas Ioannides1

1Molecular Toxicology Group, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom, 2Research Center for Industrial Crops, Agricultural Research Council (CRA-CIN), Bologna, Italy

The widely documented chemopreventive activity of cruciferous vegetables in epidemiological studies is believed to be due to the presence of glucosinolates, a class of sulphur-containing glycosides, which are present at substantial amounts in these vegetables. The currently accepted view is that the chemopreventive activity of glucosinolates is exclusively mediated by their degradation products, such as isothiocyanates, formed by the action of largely plant myrosinase. Although the mechanism of action of isothiocyanates appears to be multifactorial, a principal mechanism of action underpinning their chemopreventive activity is modulation of carcinogen-metabolising enzyme studies so as to suppress the levels of genotoxic metabolites and limit interaction with DNA. Isothiocyanates have been shown to suppress generation of genotoxic metabolites by suppressing the cytochrome P450 families catalysing their for-mation, and to stimulate their detoxification by up-regulating enzymes such as quinone reductase and glutathione S-transferases. In the present study, evidence is presented for the first time that intact glucosinolates can per se modulate these carcinogen-metabolising enzyme systems in rat liver. The glucosinolates glucoraphanin and glucoerucin were isolated and purified from cruciferous vegetables and incubated (1-25 μM) with precision-cut rat liver slices for 24 hours; at the end of this period, slices were removed from the incubation medium, homogenised, microsomal and cytosolic fractions were prepared, and the various activities determined. At concentrations as low as 1 μM, both glucoerucin and glucoraphanin elevated the O-dealkylations of methoxy- and ethoxyresorufin, markers for CYP1 activity; supple-mentation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished these effects. Moreover, both glucoerucin and glucoraphanin increased the apoprotein levels of microsomal CYP1A1, CYP1A2 and CYPIB1, determined immunologically. At higher concentrations (10-25 μM), both glucosinolates enhanced glutathione S-transferase and quinone reductase activities in rat liver slices; in this instance, however, supplementation of the incubation medium with myrosinase exacerbated the inductive effect. Finally, both glucosinolates increased modestly cytosolic quinone reductase, GSTα and GSTμ protein levels, which became more pronounced when myrosinase was added to the incubations with the glucosinolate. The current observations allow us to infer that intact glucosinolates, like their degradation products isothiocyanates, can modulate the activity of hepatic carcinogen-metabolising enzyme systems and are likely to directly impact on the chemopreventive activity linked to cruciferous vegetable consumption.

P184. Nuclear Receptors Mediated Induction of CYP3A4 by Some Traditional Chinese Medicines

Chunna Yu, Y. Liu, SS Ye, HY Sun, LB Gao, SQ Chen and S. Zeng

Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang University, Hangzhou, China

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Herb-drug combination use is very popular in China. However, herb-drug interactions and adverse effects have not been well examined. Induction of CYP3A4 mediated by pregnane X receptor (PXR) represents an important mechanism for herb-drug metabolic interactions. This work attempted to evaluate the inducible potential of CYP3A4 by some Traditional Chinese Medicines (TCMs) and their constituents. First, we established a cell-based reporter gene assay and then 29 TCMs were examined. The results showed that ginkgo biloba, Danshen, Chinese Angelica and Schisandra chinensis could induce CYP3A4 through PXR pathway. We further examined trans-activation activities of the compounds from ginkgo biloba, Danshen, Chinese Angelica and Schisandra chinensis on CYP3A4 reporter. Ligustilide from Chinese Angelica, schisandrin A, schisandrin B, schisandrol A and schisantherin A from Schisandra chinensis, ginkgolide A from ginkgo biloba, trans-resveratrol, berberine were identified as PXR agonists. Constitutive androstane receptor (CAR) and glucocorticoid receptor (GR) were also involved in tanshinones mediated CYP3A4 induction. Based on the docking model, schisantherin A was found to interact with polar residue Ser247 and other 12 hydrophobic residues lining in the ligand binding pocket of PXR.

P185. Oxidative stress and xenobiotic ligand-activated transcription factors affect the alternative splicing of the UGT1A gene in human cell lines

Étienne Audet-Walsh, Judith Bellemare, Kim Journault and Chantal Guillemette

Pharmacogenomics Laboratory, CHUQ Research Center and Faculty of Pharmacy, Laval University, Quebec, QC, Canada

The nuclear factor erythroid-2 related factor 2 (Nrf-2) and the aryl hydrocarbon receptor (AhR) are transcription fac-tors controlling genes encoding antioxidants, drug pumps and xenobiotic detoxification enzymes including human UDP-glucuronosyltransferases (UGTs). Recently, we reported novel spliced products derived from the human UGT1A gene, so-called UGT1A_i2 isoforms, which exhibit a dominant negative function upon glucuronidation activity. Here, we tested the hypothesis that activators of Nrf-2 and AhR may affect alternative splicing at the UGT1A locus. Cellular models derived from human liver (HepG2), kidney (ACHN) and mammary gland (MCF-7) were exposed for 24h and 72h to specific inducers of the AhR pathway (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD; 10nM], 3-methylcholentrene [3-MC; 10uM] and benzo[a]pyrene [BaP; 10uM]) and the Nrf-2 pathway (tert-Butylhydroquinone [tBHQ; 80uM]). Given bidirectional interactions of these pathways, an inducer affecting both factors, the dietary flavone chrysin (20uM), was also tested. Induction was investigated by quantitative PCR to study individual UGT1As (1A1, 1A3, 1A4, 1A6, 1A9 and 1A10) and pools of spliced variants, UGT1A_i1′s or UGT1A_i2′s. Expression of i1 and i2 spliced products was induced to similar levels by the AhR specific inducers, TCDD, 3-MC and BaP, in HepG2 and MCF-7 cells, but not in ACHN cells. In contrast, treatment of these cells with the potent Nrf2 activator tBHQ resulted in a significantly different induction of i1 species compared to i2 isoforms in HepG2 and MCF-7 cells. Similarly, chrysin treatment led to a specific induction of i1 species while i2 levels remained unchanged. The expression of UGT1A1 and UGT1A4 in HepG2 cells, and UGT1A1 and UGT1A10 in MCF-7 cells was particularly modulated by chrysin exposure. tBHQ specifically induced UGT1A6 in HepG2 cells while TCDD, 3-MC and BaP were much less specific and induced most of the UGT1As. Additional studies are required to decipher the molecular mechanisms underlying the differential induction of UGT1A alternative splice products by these biological sensors. Hence, the regulation of alternative spliced products may represent a novel mechanism for the control of cellular protection and defence against oxidative and xenobiotic stresses.

P186. The mechanism of sex-dependent induction of CYP 3A by zolmitriptan in rat

Lushan Yu, Sijie Lu and Su Zeng

Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

In our preliminary enzyme activity and midazolam (MDZ) pharmacokinetics studies, we observed zolmitriptan (ZOL) treatment led to induction of CYP3A in male not female rats. As determined by immunoblotting and real-time PCR, we found this sex-dependent ZOL inducibility of CYP3A is largely due to male-selective induction of CYP3A2 rather than CYP3A1. Interestingly, no changes in both genes were detected in primary cultured rat hepatocytes. Since growth hormone (GH) is known as the major mechanistic determinant of sexually-dimorphic gene expression like CYP3A2 in rat liver, the impacts of ZOL on both plasma GH levels in non monosodium glutamate (MSG) treated rats and CYP3A2

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expression in GH depleted MSG-treated rats were studied. ZOL was shown to partially suppress GH levels in both genders. Furthermore, CYP3A2 protein and mRNA level declined in male not female MSG-treated rats. These findings demonstrated CYP3A2 inducibility by ZOL was sex-selective and GH may play an important role in CYP3A2 induction. In order to study the possible molecular events involved in the depression of GH and sex-selective induction on rat CYP3A2 by ZOL, the mRNA and protein level (whole protein and nuclear protein) of PXR, CAR, Stat5b and HNF4a were investigated. Nuclear accumulation of HNF4a and CAR were observed in the normal male not female rat liver tissue following ZOL treatment. However, this kind of nuclear translocation didn′t occur in rat hepatocytes and MSG-treated rats. In addition, immunoblot analysis showed that the expression of PXR (whole protein and nuclear protein) in normal rats, rat hepatocytes and MSG-treated rats was not obviously altered after the treatment with ZOL though real-time PCR showed that ZOL can sex-selective induction the mRNA level of PXR. The results demonstrated that this sex-dependent ZOL inducibility of CYP3A is largely due to male-selective induction of CYP3A2 rather than CYP3A1. GH and HNF4a may play an important role in CYP3A2 induction.

P187. The Natural Chemopreventive Phytochemical R-Sulforaphane is a Far More Potent Inducer of the Carcinogen-Detoxifying Enzyme Systems in Rat Liver and Lung than the S-Isomer

Ahmad Faizal Abdull Razis1, Renato Iori2 and Costas Ioannides1

1Molecular Toxicology Group, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom, 2Research Center for Industrial Crops, Agricultural Research Council (CRA-CIN), Bologna, Italy

Epidemiological studies have repeatedly revealed an inverse relationship between dietary intake of cruciferous vegeta-bles and cancer incidence at a number of sites, including lung. This has been attributed to the presence of phytochemi-cals, such as sulforaphane [(-)1-isothiocyanato-4R-(methylsulfinyl)-butane] that is encountered in substantial amounts in these vegetables as a glucosinolate (glucoraphanin), which is broken down largely by the action of plant myrosinase. The chemopreventive activity of sulforaphane has also been demonstrated in animal models of cancer using the race-mate, despite the fact that humans are exposed only to the naturally-occurring R-enantiomer through the diet. Since a principal mechanism of the chemopreventive activity of sulforaphane is modulation of the carcinogen-metabolising enzyme systems, in such a way as to limit the generation of the genotoxic metabolites of chemical carcinogens, a study was conducted in precision-cut rat liver and lung slices comparing the ability of R- and S-sulforaphane to modulate these enzyme systems. Slices were incubated with the a range of concentrations of each isomer for 24 hours; at the end of this period slices were removed from the incubation medium, homogenised, microsomal and cytosolic fractions were prepared, and the various activities determined. R-sulforaphane elevated hepatic glutathione S-transferase and quinone reductase, two pivotal enzymes in the detoxification of the reactive intermediates of chemical carcinogens, whereas the S-enantiomer had no effect; moreover, the R-enantiomer was more effective in up-regulating GSTα, GSTμ and quinone reductase protein levels determined immunologically. In the lung, both enantiomers increased the same enzyme activities with the R-enantiomer being more potent; in addition, the R-enantiomer was more effective in up-regulating GSTα and quinone reductase protein levels. Both isomers increased glutathione levels in both tissues, with R-sulforaphane being more potent. Finally, R-sulforaphane was the more effective of the two isomers in up-regulating CYP1A1/1B1 apoprotein levels in both liver and lung, and CYP1A2 in the liver. These studies demonstrate clearly the superiority of R-sulforaphane, when compared with the S-enantiomer, in stimulating detoxification enzymes such as quinone reductase and glutathione S-transferases, and raises the possibility that the animal studies that employed the racemate may have underestimated the chemopreventive activity of this isothiocyanate. Moreover, these studies advise caution when extrapolating experimental data emanating from studies using synthetic R,S-sulforaphane to humans who are only exposed to the R-enantiomer through the diet.

P188. Cadmium Alters the Biotransformation of Carcinogenic Aromatic Amines by Xenobiotic-Metabolizing Enzymes Arylamine N-Acetyltransferases

Nilusha Ragunathan, Julien Dairou, Elodie Sanfins, Florent Busi, Christophe Noll, Nathalie Janel, Jean-Marie Dupret and Fernando Rodrigues-Lima

Sciences du Vivant, Universite Paris Diderot, Paris, France

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Cadmium (Cd) is a carcinogenic heavy metal of great environmental concern. Exposure to both Cd and carcinogenic organic compounds such as polycyclic aromatic hydrocarbons or aromatic amines (AA) is a common environmental problem. Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes (XME) that play a key role in the biotransformation of AA carcinogens such as 4-aminobiphenyl (4-ABP) and 2-aminofluorene (2-AF). Changes in human NAT activity has long been associated with variations of susceptibility to different cancers in relation with exposure to certain AA. We found that Cd at biologically-relevant concentrations impairs the NAT-dependent acetyla-tion of carcinogenic AA such as 2-AF or 4-ABP in lung epithelial cells. Moreover, NAT activity was found to be strongly impaired in the tissues of mice exposed to Cd. Mice exposed to Cd and 2-AF displayed altered in vivo pharmacokinetics with a significant decrease (∼50%) in acetylated-2-AF in plasma thus confirming the functional impairment of NAT enzymes by Cd. Further kinetic and molecular experiments using recombinant enzymes showed that NAT1 was rapidly and irreversibly inhibited by Cd (IC50Å75 nM, ki=5.104 M-1. s-1) through the formation of Cd adduct with the active-site cysteine residue of the enzyme. Similar results were found for the human NAT2 isoform, although this isoform was found to be less sensitive to inactivation (IC50Å1 _M, ki=1.104 M-1. s-1). Overall, our data suggest that Cd can alter the metabolism of carcinogenic aromatic amines through the impairment of the NAT-dependent pathway which may have important toxicological consequences.

P189. Cofactor Metal Levels and Enzyme Activities in Cisplatin-Treated Rats: Effect of Antioxidant Intervention

Suna Sabuncuoglu1, Ayse Eken2, Hilal Ozgunes3, Ahmet Aydin4 and Hilmi Orhan5

1Department of Pharmaceutical Toxicology, Hacettepe University, Faculty of Pharmacy, Ankara, Turkey, 2Department of Toxicology, Gulhane Military Medical Academy, Ankara, Turkey, 3Hacettepe University, Faculty of Pharmacy, Ankara, Turkey, 4Department of Toxicology, Yeditepe University, Faculty of Pharmacy, Istanbul, Turkey, 5Department of Toxicology, Ege University Faculty of Pharmacy, Izmir, Turkey

In the present study, we explored the association between antioxidant enzyme (Se-glutathione peroxidase; GPx-, glutathione S-transferase; GST-, catalase; CAT-, and Cu,Zn-superoxide dismutase; SOD-) activities and their cor-responding metal concentrations in plasma, erythrocyte, liver and kidney of the same rats. The potential modulation effect of various antioxidants such as aminoguanidine (AMG), vitamin E, vitamin C, and their combination (EC) was also investigated. The liver platin (Pt) concentration was three fold higher than the kidney concentration. Liver GPx activity was found to be statistically significantly decreased in CP-treated rats compared to the control, while kidney Se-GPx activity was not different in the same group. Erythrocyte GST activity was significantly diminished in CP-treated rats compared to the control. Similar significant decrease in CP treated rats was also observed in liver GST activity compared to the controls. AMG intervention before CP administration was not sufficient to reverse this decrease, while EC intervention was. We observed a different pattern in kidney GST activity; AMG and EC treat-ments caused significant increases in enzyme activity compared to the control. CP treatment, however, diminished GST activity significantly. CAT activity in erythrocytes was significantly higher only in EC group compared to the control. In liver, however, CP treatment caused a statistically significant decrease in CAT activity compared to the controls. EC intervention, but not AMG, ameliorates this decrease. When considering Pt levels were higher in liver compared to kidney, this decrease in enzyme activity can be attributed to inhibitory effect of Pt on the enzyme. CP treatment caused similar decrease in CAT activity also in kidney compared to the control. Liver zinc (Zn) and copper (Cu) levels were significantly lower in CP group compared to the control. Cu level was three fold higher than Zn level. Similar pattern was observed in kidney tissue of CP treated rats for Zn, kidney Cu level was not changed in any group. CP treatment caused significant increases in iron (Fe) level compared to the control in liver tissue. Similar pattern was observed in kidney, Fe level was higher in CP group compared to the control. When considering metal levels with corresponding enzyme activity in the same tissue, diminished Zn level was in accordance with diminished Cu,Zn-SOD in kidney of CP treated rats. AMG and EC intervention reversed both enzyme activity and Zn levels back to the control levels. CAT activity seems inversely correlated with its cofactor Fe level. Both in liver and kidney, CP treatment caused significant increases in Fe level compared to the control, while CAT activity was significantly diminished in these tissues. These observations suggest that interaction between enzyme activities and their cofactor metal levels differ significantly, probably because of conformational orientation of the metals on the protein. For instance, Cu is known sitting on the side of a deep cleft on the protein compared to completely buried

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Zn in Cu,Zn-SOD. Further studies are required to elucidate those interactions between biological metal levels and the enzymatic activities.

P190. Commonly used inhibitors of drug metabolizing enzymes: Do they also inhibit drug transporters?

Emile G. Plise, Jason S. Halladay, Jonathan Cheong, Jasleen Sodhi and Laurent Salphati

Drug Metabolism and Pharmacokinetics, Genentech Inc, South San Francisco, CA, USA

Non-specific inhibitors of cytochrome P450s (CYPs), Monoamine Oxidase (MAO), Aldehyde Oxidase (AO), Xanthine Oxidase (XO), Flavin Monooxygenase (FMO), and UDP-glucuronosyltransferase (UGT) are frequently used in vitro and in vivo as tools to elucidate metabolic pathways of compounds. Since transporters can play an important role in the disposition of compounds, unexpected transporter inhibition in metabolism studies can lead to ambiguous results. The purpose of this work is to examine the potential of these tools to inhibit transporters (MDR1, Bcrp1, and MRP2) at relevant in vitro and in vivo concentrations. MDR1 inhibition was evaluated using MDCKI-MDR1 transfected cells and the calcein-AM fluorescence based inhibition assay. Bcrp1 inhibition was tested using MDCKII cells transfected with mouse Bcrp1 and a modified version of a Hoechst 33342 inhibition assay. Multidrug resistance protein 2 (MRP2) was examined using a Solvo MRP2-PREDIVEZ™-VT fluorescence inhibition kit. Concentration ranges of the potential inhibitors were chosen based on concentrations used for in vitro and pharmacokinetics studies or where limited by aqueous solubility. The CYP inactivator 1-aminobenzotriazole (ABT) did not inhibit any of the transporters tested up to 1 mM while SKF525A (CYP inhibitor) had mean IC

50 values of 103 µM, 479 µM, and 216 µM for MDR1, Bcrp1, andMRP2,

respectively. The AO inhibitor menadione (a known inhibitor of human BCRP; IC50

7.3 µM)1 inhibited mouse Bcrp1 with an IC

50 of 48-98 µM, but did not inhibit MDR1 or MRP2. Raloxifene (AO inhibitor) has been identified as a MDR1

inhibitor and has been recently shown to inhibit BCRP2. However this may be the first reported IC50

(117.1-146 µM) for raloxifene against BCRP of any species. Raloxifene did not inhibit MRP2 up to 250 µM despite its known inhibition of MRP1. The MAO inhibitor pargyline, the AO/XO inhibitor allopurinol, and the FMO inhibitor methimazole did not inhibit any of the transporters tested up to 5 mM. The UGT inhibitor piperine did not inhibit MRP2. The results for MDR1 and Bcrp1 are inconclusive because it appears piperine inhibits the esterase responsible for hydrolysis of the calcein-AM bond in the MDR1 assay and fluoresces at the same wavelength as Hoechst 33342 in the Bcrp1 assay. These results suggest SKF525A, menadione, and raloxifene have the potential to inhibit transporters at relevant in vitro and in vivo concentrations. Studies with these tool compounds are ongoing to evaluate the potential impact and severity of transporter inhibition on metabolic results.

References

1Shukla S., Chung-Pu W., Krishnamachary N., Ambudkar S.V. The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are sub-strates of the multidrug resistance linked ATP binding cassette drug transporter ABCG

2. Mol. Cancer Ther. 6 (2007), 3279-86. 2Zhang Y., Byun Y., Ren Y.R., Liu J.O., Laterra J., Pomper M.G. Identification of Inhibitors of ABCG2 by a Bioluminescence Imaging–Based High-Throughput Assay. Cancer Res. 69(14), (2009), 5867-5875.

P191. COMT EXPRESSION and Activity AFTER Chronic Administration of Bia 9-1067 to Wistar Rats

Maria Joao Bonifacio1, Leonel Torrão1, Maria João Pinho2, Lyndon Wright1 and Patrício Soares-da-Silva1

1Department of Research & Development, BIAL, S Mamede do Coronado, Portugal, 2Instituto de Farmacologia e Terapêutica, Faculdade de Medicina, Universidade do Porto, Porto, Portugal

Catechol-O-methyltransferase (COMT; EC 2.1.1.6), responsible for the introduction of a methyl group from S-adenosyl-methionine into a catechol substrate, is one of the enzymes involved in the metabolism of catecho-lamines and it is a particularly important entity in inactivation of levodopa, the golden standard treatment in

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Parkinson′s Disease. BIA 9-1067 [2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)–4,6-dimethylpyridine 1-oxide]1 is a novel COMT inhibitor that was developed to be used as an adjunct to the levodopa/aromatic amino acid decarboxylase inhibitor therapy of Parkinsonic patients. The objective of this study was to evaluate the effect of chronic administration of BIA 9-1067 on the activity rat liver COMT and also to find out whether or not COMT gene expression and enzyme levels were affected by this treatment. For that purpose, Wistar rats were orally administered with BIA 9-1067 (3 mg/kg) everyday for up to 5 days. Sacrifices occurred every day during treatment and every other day after last administration until 7 days post-treatment. One group of animals was daily administered 30 mg/kg BIA 9-1067 for 5 days and sacrificed on the last day. Liver homogenates were prepared and used for evaluation of COMT activity and protein levels (both of the membrane-bound and soluble forms of the enzyme). COMT gene expression was quantified by real-time PCR. Daily administration, for up to 5 days, of 3 mg/kg BIA 9-1067 to rats resulted in marked inhibition of liver COMT during the first 5 days (between 76-95 % inhibition) with enzyme activity gradually recovering to control values 7 days after the last administra-tion. COMT activity was abolished in animals administered 30 mg/kg BIA 9-1067 for 5 consecutive days. Liver COMT expression levels quantified in animals treated for 3 and 5 days with BIA 9-1067 (3 and 30 mg/kg) revealed no significant alterations between treated and control animals. The corresponding protein levels, of both the soluble and membrane-bound forms, quantified by western blot analysis were also unchanged in these animals. In conclusion, chronic administration of BIA 9-1067 leads to marked but reversible inhibition of liver COMT and affects neither COMT protein nor COMT RNA levels.

References

1 Kiss LE, Ferreira HS, Torra??o L, Bonifa??cio MJ, Palma PN, Soares-da-Silva P, Learmonth DA. J Med Chem. 2010 Mar 24. [Epub ahead of print]

P192. Cytochrome P450 time-dependent inhibition assays: Considerations for the preincubation step and data processing

Charles L. Crespi, Elke S Perloff and David M Stresser BD

Biosciences Discovery Labware, Woburn, MA, USA

Recent draft FDA and EMEA guidance now advocate the in vitro determination of time-dependent inhibition of cyto-chrome P450 by drug candidates. In this assay, typically a 30 minute pre-incubation is conducted with liver microsomes, test article and NADPH and compared to either non-preincubated samples or pre-incubated samples lacking NADPH. If the inhibition response increases as a result of the preincubation step, follow-up studies are typically conducted to determine the parameters K

I and k

inact. A dilution approach for both abbreviated and definitive testing was recom-

mended in a recent consensus document from PhRMA [Grimm SW et al. (2009) conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manu-facturers of America. Drug Metab Dispos 37:1355–1370]. Data from us [Perloff, ES et al (2009) Xenobiotica, 39: 99–112] and others [e.g. Obach RS et al (2007) Drug Metab.Dispos.35: 246–255] demonstrate that the dilution method for use in IC

50 shift assays, is a highly sensitive and robust test system to screen for time-dependent inhibition. In this analysis,

we demonstrate that even highly protein bound and/or rapidly metabolized inhibitors (e.g. S-fluoxetine, ticlopidine, tienilic acid, paroxetine, ritonavir) are easily flagged for follow on analysis, whereas established reversible, non-time-dependent inhibitors (e.g. ketoconazole, quinidine, sulfaphenazole) do not show evidence of TDI. One may elect to use the nominal concentrations in the preincubation or secondary incubation to calculate IC

50 however we elected to use

those in the secondary incubation to facilitate comparison with the zero-minute preincubation samples. The choice of which absolute values to use has no influence on the IC

50 shift calculation, because it is a simple ratio of IC

50 values.

Therefore, this does not affect decision-making for follow on testing.

P193. Deoxyschizandrin Could Attenuate Cytochrome P450 3A4-Mediated Bioactivation of Gomisin A in Human Liver Microsomes

Yan-Yan Zhang and Ling Yang

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Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Dibenzocyclooctadiene lignans are discussed as the major absorbed effective ingredients of Schisandra fruit, which is widely used as an antitussive, sedative, tonic agent, and a component of dietary supplement products. Our previous investigations have revealed that biotransformation of the methylenedioxy lignans (e.g. gomisin A) by cytochrome P450 (CYP) 3A can lead to mechanism-based inactivation and reactive ortho-quinone metabolites. In the present study, to gain insight into the clinical value of this herb medicine, a detailed study on the metabolic and inhibitory properties of deoxyschizandrin, another schisandra lignan without methylenedioxy functionality, was conducted. Its influence on the bioactivation of gomisin A was also evaluated. In human liver microsomes (HLMs), deoxyschizandrin underwent C-7 monohydroxylation. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs and enzyme kinetics (K

m, 1.6 ± 0.1 μM; V

max, 623 ± 9.9 pmol/min/mg protein) indicated that its metabolite was

generated predominantly by CYP3A4. However, no GSH adduct was observed, which was quite different from gomisin A under the same incubation conditions. Deoxyschizandrin was also found to potently inhibit CYP3A in a competitive manner (K

i, 2.6 μM). IC

50 shift studies demonstrated that deoxyschizandrin was not a time and cofactor-dependent

inhibitor of CYP3A. Co incubation of deoxyschizandrin (0∼100 μM) with gomisin A (50 μM) in HLMs resulted in great reduction of time-dependent inhibitory potential of gomisin A against CYP3A (% activity remaining, 20∼80). Moreover, the production of GSH adduct demethylenated gomisin A could be strongly impaired by deoxyschizandrin in vitro. These results collectively demonstrated that deoxyschizandrin might exhibit significant modulatory effects on CYP3A4 activity, thus reducing the bioactivation potential of gomisin A. The presence of deoxyschizandrin in Schisandra fruit could increase the clinical safety of this herb medicine.

P194. Effects of Some Indole Derivates On Aldose Reductase Enzyme Activity

Erdem Kursun1, Gökçe Gurkok2, Sibel Suzen2, Net Evcimen3 and Özlem Yildirim1

1Biology, Ankara University Faculty of Science, Ankara, Turkey, 2Pharmaceutical Chemistry, Ankara University Pharmacy Faculty, Ankara, Turkey, 3Biochemistry, Ankara University Pharmacy Faculty, Ankara, Turkey

Aldose reductase is one of the most thoroughly studied of the aldo-keto reductases due to its involvement in the pathogenesis of diabetic eye disease. It catalyzes the NADPH-dependent reduction of glucose to sorbitol, the first step of the sorbitol pathway. Experimental animal studies, supports the hypothesis that enhanced metabolism of glucose through the polyol pathway results in biochemical imbalances associated with diabetic complications. Biochemical alterations appear to be derived from accelerated flux of glucose through the sorbitol pathway, AR inhibition represents an attractive strategy for prevention of diabetic complications. The beneficial effect of aldose reductase inhibitors (ARIs) in preventing or substantially delaying the onset of diabetic complications in experimental models provides strong support to this hypothesis. Indolic compounds are a broad family of substances present in microorganisms, plants and animals. They are very efficient antioxidants, protecting both lipids and proteins from peroxidation and it is known that the indole structure influences the antioxidant efficacy in biological systems. This study aimed to analyze the effect of new indole derivatives on aldose reductase enzyme activity. For this purpose, aldose reductase was purified from bovine lens and inhibitory capacities of sixteen indole derivatives were tested in vitro. These showed between 3% to 42.86 % AR inhibition. Especially inhibitor effect was observed at 5-bromo-1H-indole-3-carboxyaldehyte (4-bromophenyl) hydrazone, 5-bromo-1H-indole-3carboxyaldehyte (3,4-dichlorophenyl) hydrozone and 5-bromo-1H-indole-3-carboxyaldehyte isonicotinoile hydrazone and these inhibitor effects are respectively %25.49 ± 5.01, %23.08 ± 10.99 and %43 ± 3.49.

P195. Evaluation Of Single Point And IC50 Shift Assays For Measuring Time-Dependent Inhibition Of Drug Discovery Compounds

Katie Fox, Rosey Pearson, Philip Butler and Clive Dilworth Cyprotex,

Macclesfield, United Kingdom

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Assessment and early detection of new chemical entities (NCEs) which inhibit cytochrome (CYP) P450 enzymes is of great importance in reducing the development of compounds which can cause drug-drug interactions (DDIs). In particular, irreversible time dependent inhibition (TDI) is of concern for its inhibition results in inactivated enzyme which must be re-synthesised in order to regain activity. Therefore such compounds which cause TDI have greater potential to cause longer lasting DDIs. Unlike reversible inhibition where there are guidelines from the FDA1, current opinion on investigating TDI is quite varied with little detailed recommendations. At present there is divided opinion on experimental methods and interpretation of data, as explored by the PhRMA review (2009)2. The aim of this study is to evaluate different assay designs, and subsequent data analysis methodology for measuring the extent of TDI for known time-dependent inhibitors. In particular we have focused on CYP3A4 activity using midazolam as the probe substrate, analysing 1-hydroxymidazolam formation by LC-MS/MS. Firstly, we look to explore loss of activity with a single concentration of inhibitor, comparing the percentage inhibition measured with 30 minute pre-incubations with/without NADPH and correcting for vehicle activity. By compar-ing different equations3,4 we will analyse the impact of reversible inhibition on interpretation and accuracy of TDI data. The study then looks to correlate this single point data with IC

50 shift data, investigating the impact of

using a dilution step. Finally, we aim to evaluate the predictive qualities of single point TDI studies and IC50

shift assays on estimating potential in vivo DDIs. By assessing both of these initial drug discovery phase TDI screens we propose a reversible inhibition and TDI screening platform to cover early phase compounds, which should enable early accurate decisions to be made regarding development of those compounds which could potentially cause DDIs.

References

1 Huang SM. (2006). Draft Guidance for Industry: Drug Interaction Studies — Study Design, Data Analysis, and Implications for Dosing and Labeling.

2 Grimm SW, Einolf HJ, Hall SD, He K, Lim HK, Ling KH, Lu C, Nomeir AA, Seibert E, Skordos KW, Tonn GR, Van Horn R, Wang W, Wong YN, Yang TJ, Obach RS. (2009). The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America. Drug Metab Dispos 37 (7):1355-70

3 Atkinson A, Kenny JR, Grime K. (2005). Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis. Drug Metab Dispos 33 (11):1637-1647

4 Obach RS, Walsky RL, Venkatakrishnan K. (2007). Mechanism-Based Inactivation of Human Cytochrome P450 Enzymes and the Prediction of Drug-Drug Interactions. Drug Metab Dispos 35 (2):246-255

P196. Human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irrevers-ibly inhibited by inorganic mercury (Hg2+) and methylmercury (CH3Hg+)

Nilusha Ragunathan, Florent Busi, Jean-Marie Dupret, Fernando Rodrigues-Lima and Julien Dairou

Sciences du Vivant, Universite Paris Diderot, Paris, France

Human arylamine N-acetyltransferase 1 (NAT1) is a phase 2 xenobiotic-metabolizing enzymes (XME) that play a key role in the biotransformation of many aromatic and heterocyclic amines drugs and carcinogens. This XME has toxicologically significant functions in the detoxification of xenobiotic aromatic amines and/or bioactivation of N-arylhydroxylamines by O-acetylation. Inorganic mercury (Hg2+) and methylmercury (CH3Hg+) are major pollutants that have been reported to inhibit certain XME such as epoxide hydrolases. Here we show that biologically-relevant concentrations of Hg2+ and CH3Hg+ impair the activity of the NAT1 enzyme. Both compounds react irreversibly with the active- site cysteine of the enzyme (IC50 = 250 nM and kinact=1.4.104 M-1.s-1 for Hg2+ and IC50 = 1.4 _M and kinact=2.102 M-1.s-1 for CH3Hg+) likely through the formation of a thiol-Hg bond that leads to inhibition. Accordingly, we found that exposure of lung epithelial cells to these pollutants led to the inhibition of the endogenous NAT enzyme, Hg2+ displaying a more potent effect than CH3Hg+ (IC50 = 3 _M and 20 _M for Hg2+ and CH3Hg+, respectively). Our data suggest that mercury and its derivatives may affect the xenobiotic-metabolizing functions of NAT1 which may be of toxicological or clinical significance. 1

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P197. Identification of a novel carbamoyl glucuronide as a metabolism-dependent inhibitor of CYP2C8

Faraz Kazmi1, Brian Smith1, Mette G. Hvenegaard2, Lene Bendahl2, Amy Gipson1, David Buckley1, Brian Ogilvie1 and Andrew Parkinson1

1XenoTech, LLC, Lenexa, KS, USA, 2Department of Drug Metabolism, H. Lundbeck A/S, Copenhagen, Denmark

Glucuronidation is a major route of drug biotransformation and detoxification, whereby a drug is conjugated with glucuronic acid in a reaction catalyzed by UDPglucuronosyltransferases (UGTs). However, previous reports suggest glucuronide conjugates can be inhibitors of Phase I metabolism. Of particular note is gemfibrozil glucuronide, which is oxidized by CYP2C8, causing clinically relevant irreversible inactivation of this enzyme (Ogilvie et al., 2006). In the present study, we evaluated the ability of Lu AA34893 and its carbamoyl glucuronide to inhibit in vitro the major drug-metabolizing cytochrome P450 (CYP) enzymes. In NADPH-fortified human liver microsomes (HLM), Lu AA34893 was found to directly inhibit CYP2C19 (IC

50 = 0.20 µM), CYP2D6 (IC

50 = 6.9 µM), CYP3A4/5 (as measured by testosterone

6β-hydroxylation, IC50

= 9.7 µM; midazolam 1′-hydroxylation, IC50

= 15 µM; and nifedipine oxidation, IC50

= 15 µM), CYP2A6 (IC

50 = 17 µM), CYP2B6 (IC

50 = 17 µM), CYP2C9 (IC

50 = 20 µM), CYP2C8 (IC

50 = 24 µM), and CYP1A2 (IC

50 = 54

µM). Furthermore, Lu AA34893 was found to competitively inhibit CYP2C19 and CYP3A4/5 (testosterone) with Ki values

of 0.08 µM and 6.7 µM, respectively, and to act as a mixed inhibitor of CYP2D6 (Ki = 3.0 µM), and CYP3A4/5 (midazolam,

Ki = 6.1 µM; nifedipine, K

i = 7.8 µM). Minimal metabolism-dependent inhibition (MDI) of several CYP enzymes by Lu

AA34893 was observed. Assessment of the carbamoyl glucuronide of Lu AA34893, showed that, of the CYP enzymes evaluated, only CYP2C8 was inhibited directly (IC

50 = 71 µM). Of particular interest, when pre-incubated with NADPH-

fortified human liver microsomes for 30 minutes prior to measurement of CYP activity, the carbamoyl glucuronide was found to be a MDI of CYP2C8, with over an 8-fold shift in IC

50 (IC

50 = 8.5 µM), unlike its aglycone. Additionally,

there was some evidence of MDI of CYP2C19 (IC50

shift from >100 µM to 66 µM). These findings demonstrate another example (i.e., in addition to gemfibrozil) in which glucuronidation of a drug candidate converts the parent to a potent MDI of CYP2C8.

Reference

Ogilvie BW, Zhang D, Li W, Rodrigues AD, Gipson AE, Holsapple J, Toren P, Parkinson A. 2006. Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions. Drug Metab Dispos 34:191-197.

P198. Identification Of Time-Dependant CYP450 3A4 Inhibitors Using Cryopreserved Hepatocytes

Ralf R.H. Lotz

Drug Discovery Support, Boehringer Ingelheim Pharma GmbH&Co KG, Biberach, Germany

Typically mechanism-based inhibitors of Cyp450 3A4 are identified using human liver microsomes as an in vitro system. But due to the time dependency of the mechanism and the relatively short incubation time, depending on the values of Ki and kinact, the degree of inhibition may be rather low and therefore some inhibitors may not be identified or the results may be ambiguous. Hepatocytes provide an alternative in vitro model with the possibility to incubate for a longer time period and therefore generate a more pronounced inhibition or an inhibition at lower concentrations. We have used human cyropreserved hepatocytes in suspension to investigate the effects of known mechanism-based inhibitors upon the 3A4 substrate midazolam. Following a preincubation time of 4h a significant reduction of 1-OH-midazolam formation (mean of N=3 incubations per experiment, 3 independent experiments) was observed for clarithyomycin (0.3µM 20 ± 2%; 1µM 38 ± 6%, 3µM, 68 ± 4%), erythromycin (10µM, 76 ± 3%), diltiazem (10µM, 66 ± 5%) and verapamil (10µM, 94 ± 1%), demonstrating the feasibility of the approach and the robustness of the method. The assay has been used successfully in a discovery setting to identify time-dependant 3A4 inhibitors and to optimize lead structures. Obviously a further prolongation of the incubation time allows reduction of the inhibitor concentration to levels which are close to the anticipated levels in the clinics. To test the feasibility cells in suspension were preincubated for 24h. For

144

erythromycin (1, 3, 10µM), diltiazem (1, 3, 10 µM) and an internal compound a concentration-dependant decrease in 3A4 activity was observed, which was in excess of 80% at 10µM. Additionally plated hepatocytes were preincubated for 20h and a reduction was seen at concentrations close to those in the clinics for clarithyromycin (3µM, 75%), verapamil (1µM, 50%) and diltiazem (1µM, 50%). In summary hepatocytes can be used as an alternative in vitro system to identify time-dependant 3A4 inhibitors.

P199. Inhibition of Human Cytochrome P450 Isoforms by Mitragyna speciosa Extracts

Sabariah Ismail1, Nur Aziah Hanapi1, Juzaili Azizi1, Sharif Mahsufi Mansor1 and Roziahanim Mahmud2

1Centre for Drug Research, Universiti Sains Malaysia, Penang, Malaysia, 2School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia

Cytochrome P450s (CYPs) play a major role in the metabolism of endogenous compounds and xenobiotics which include drugs and herbal components. When herb and therapeutic drug are co-administered, herb-drug interaction may occur since component(s) in the herbal preparation can modulate the metabolism of the drug by induction or inhibition of specific CYP450 enzyme activity. Mitragyna speciosa or ketum had long been used traditionally in some states in Malaysia as a medicinal herb to boost physical stamina, as well as a folk medicine for pain and fever. The effects of Mitragyna speciosa aqueous and methanolic extracts towards human recombinant cytochrome P450s from an insect cell expression system were investigated. The abilities of these extracts to inhibit the activities of the major hepatic CYP450 enzymes, CYP3A4, CYP2D6, CYP1A2 and CYP2C19 isoforms were analyzed using a simple and sensitive luminescent assay. This assay employed luminogenic CYP450 probe substrates that are derivatives of beetle luciferin, a substrate for luciferase enzymes. The derivatives were converted by CYP450s to luciferin, which then reacted with luciferase to produce luminescence that is directly proportional to the activity of the CYP450 isoforms. A concentration range between 0.01 to 1000 µg/mL of M. speciosa extracts were tested on the recombinant human CYP450s isoforms. Decreases in luminescence indicate the inhibition of CYP450 enzyme activity by the extracts. Mitragyna speciosa methanolic extract showed higher inhibitory activities with IC50 values of 0.68 ± 0.11 µg/mL for CYP2D6, followed by 199.98 ± 22.45 µg/mL for CYP1A2. On the other hand, Mitragyna speciosa aqueous extract gave a lower IC50 value towards CYP3A4 with an apparent IC50 value of 110.02 ± 17.11 µg/mL. Both extracts did not have any effects on CYP2C19. Ketoconazole, quinidine, alpha-naphthoflavone, troglitazone were used as positive controls for CYP3A4, CYP2D6, CYP1A2 and CYP2C19 respectively. In conclusion, this study suggests that Mitragyna speciosa extracts could potentially cause herb-drug interactions in humans through modulation of CYP3A4, CYP2D6 and CYP1A2 activities.

P200. Inhibition of Human Liver Cytochrome P450 2C8 and 3A4 by Sanguinarine

Xiao-Yi Qi1, Si-Cheng Liang2, Guangbo Ge3, Zhong-Ze Fang3 and Ling Yang2

1Second Affiliated Hospital of Dalian Medical University, Dalian, China, DaLian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 3Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Sanguinarine, a quaternary benzo[c]phenanthridine alkaloid, exhibits extensive pharmacologic actions, such as anti-inflammatory, antimicrobial and anticancer effects [1]. As antibiotics or anticancer drug, sanguinarine is likely to be used in combination with various synthetic drugs in most cases, thus it is necessary to evaluate potential pharmacokinetic drug-drug interactions induced by sanguinarine. An in vitro study revealed that sanguinarine can cause competitive inhibition of human CYP1A2 with K

i of 2 μM [2], but the inhibitory effects on other CYP enzymes remain unreported.

In this study, the inhibition of sanguinarine to CYP3A4 and CYP2C8 was investigated and the results demonstrated that sanguinarine can strongly inhibit the activity of CYP3A4 and CYP2C8 with both reversible inhibition and time-dependent inhibition type. The kinetic parameters of reversible inhibition (K

i) are 2.2 μM and 8.82 μM for CYP3A4 and

145

CYP2C8, respectively. Due to the limited pharmacokinetic data of sanguinarine in humans, it is nigh impossible to evaluate its potential effects to human from in vitro data. Further work will be done in the future.

References

1. Dvorák Z, Simánek V. Metabolism of sanguinarine: the facts and the myths. Curr Drug Metab, 2007; 8: 173-176. 2. Vrba J, Kosina P, Ulrichová J, Modrianský M. Involvement of cytochrome P450 1A in sanguinarine detoxication. Toxicol Lett, 2004; 151: 375-

387.

P201. Inhibitory potential of Chlormadinone acetate (CMA) on five important UDP-Glucuronosyltransferases in human liver

Ting Huang1, Zhong-Ze Fang2, Yan-Yan Zhang3 and Ling Yang3

1National Population and Family Planning Key Laboratory of Contraceptives Drugs & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China,3Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Chlormadinone acetate (CMA), a derivative of 17-à-hydroxyprogesterone, is used as an orally effective progestogen in hormone replacement therapy(HRT). In 1998, the combined monophasic low-dose oral contraceptive ethinyl estradiol (EE) 0.03 mg and chlormadinone acetate (CMA) 2 mg was approved in Germany. Glucuronidation cata-lyzed by UDP-glucuronosyltransferase (UGT) is one of the major steps responsible for the metabolism of many drugs, environmental chemicals and endogenous compounds. Pharmacokinetic behaviours of drugs could be altered by inhibition of these UGT isoforms and the search for drugs that potentially inhibit these UGT isoforms is very significant from a clinical point of view. In the present study, the inhibitory potential of five important UDP-Glucuronosyltransferases in human liver (UGT1A1, 1A3, 1A6, 1A9 and 2B7) by CMA was investigated using 4-MU as nonspecific substrate and recombinant UGT isoforms as enzyme sources. The results showed that CMA exhibited inhibitory effects towards UGT1A3 (IC50 = 8.6 ± 1.4μM) and UGT2B7 (IC50 = 14.2 ± 3.8μM), with other UGT isoforms negligibly influenced. The results obtained from Lineweaver-Burk and Dixon plots showed that CMA noncompetitively inhibited UGT1A3 and UGT2B7. The K

i value was calculated to be 36.9μM and 4.1μM for

UGT1A3 and UGT2B7, respectively. Considering that UGT1A3 and UGT2B7 are involved in the metabolism of many drugs, special attentions should be paid when CMA was administered with the drugs which mainly experienced UGT1A3,2B7-mediated metabolism.

P202. Methoxyalkyl-Guanidines and -Amidines as Inhibitors of Human DDAH-1

Jürke Kotthaus, Ina Lunk, Dennis Schade and Bernd Clement

Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University, Kiel, Germany

Nω-methylated L-arginines represent potent endogenous inhibitors of all three isoforms of NO-synthases (NOSs) and are physiologically degraded by dimethylarginine dimethylaminohydrolase (DDAH) to L-citrulline and (di)methylamine. Thus, inhibition of DDAH activity could be another mechanism to indirectly affect nitric oxide (NO) formation by elevating the Nωmethylated L-arginine (NMMA, ADMA) levels. To date, three different classes of potent inhibitors are known: (1) pentafluorophenyl sulfonates, which are also inhibitors for the arginine deiminase;[1] (2) the most potent inhibitors are based on indolylthiobarbituric acid,[2] but their activity seems to be restricted to bacterial DDAH; and (3) L-arginine analogs whose best representatives are Nω-(2-methoxyethyl)-L-arginine and Nδ-(1-iminobut-3-enyl)-L-ornithine.[3,4] The application of L-arginine analogs is particularly limited by selectivity issues over the other enzymes of the NO-generating system, i.e. NOSs and arginases. Whereas for most compounds a relative selectivity for DDAH over NOSs is well examined, published data regarding selectivity over arginase is very restricted. Within our work we

146

identified several potent DDAH inhibitors and set main focus on selectivity issues. The most promising compound identified during our studies is AbMEG (N(4aminobutyl)-N′-(2-methoxyethyl)guanidine) with a K

i for human DDAH-1

of 18 ± 6 µM, and has only modest effects on NOSs and arginases. Additionally, we performed docking studies to more enlighten the binding mode of this novel compound in order to gain more information on structure-activity relation-ship. However, our attempts to further improve potency and selectivity failed pointing to an exceptional binding mode of AbMEG to the active site of human DDAH-1.

References

[1] P. Vallance et al., Inhibition of dimethylarginine dimethylaminohydrolase (DDAH) and arginine deiminase (ADI) by pentafluorophenyl (PFP) sulfonates, Chem Commun (Camb) 2005, 44, 5563-5

[2] B. Hartzoulakis et al., Discovery of inhibitors of the pentein superfamily protein dimethylarginine dimethylaminohydrolase (DDAH), by virtual screening and hit analysis, Bioorg Med Chem Lett 2007, 17 (14), 3953-6

[3] S. Rossiter et al., Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase, J Med Chem 2005, 48 (14), 4670-8

[4] J. Kotthaus et al., Structure-activity relationship of novel and known inhibitors of human dimethylarginine dimethylaminohydrolase-1: alkenyl-amidines as new leads, Bioorg Med Chem 2008, 16 (24), 10205-9

P203. Pitfalls in the design of metabolism-dependent CYP inhibition (MDI) experiments with a dilution step: Inhibitor depletion by metabolism and/or microsomal binding leads to underestimation of the shifted IC50 value

David B. Buckley, Faraz Kazmi, Phyllis Yerino, Paul Toren, Jeff Holsapple, Brian W. Ogilvie, Brandy Paris and Andrew Parkinson

XenoTech, LLC, Lenexa, KS, USA

We previously demonstrated that, when a dilution step is used to assess MDI potential (i.e. IC50

shift experiments), IC

50 values for direct-inhibition and MDI should be processed based on the final, post-dilution concentration and the

initial, pre-dilution concentration, respectively (Paris et al., 2009). When processed appropriately, the “shifted IC50

values” (those determined following a 30-min pre-incubation of the drug candidate with NADPH-fortified HLM) were notably higher for several known MDIs in experiments conducted with a 10-fold dilution step than those determined by a non-dilution method, suggesting less MDI occurs at the higher concentration of HLM. More than a two-fold difference in shifted IC

50 values was observed between the dilution and non-dilution methods for five of ten inhibi-

tors commonly used positive controls in MDI experiments; namely ticlopidine (CYP2B6), tienillic acid (CYP2C9), paroxetine (CYP2D6), S-fluoxetine (CYP2C19) and azamulin (CYP3A4). Experiments were performed to determine whether these discrepancies in shifted IC

50 values between are attributable to 1) decreased free inhibitor concentration

(fuinc

) and/or 2) extensive metabolism of the inhibitor. In the case of S-fluoxetine, microsomal binding, not extensive metabolism, leads to the discrepancy. Furthermore, correction for microsomal binding alone resulted in shifted IC

50

values (based on fuinc

) that agreed within 6% for dilution (14 →3.1 µM) and non-dilution (5.3 →2.9 µM) methods. Conversely, correction for observed microsomal binding alone did not resolve the descrepanicies in shifted IC

50 values

for ticlopidine, tienillic acid, paroxetine and azamulin. In these cases, extensive metabolism-dependent inhibitor depletion was observed in incubations with high concentrations of HLM (up to 1 mg/mL). In the case of ticlopidine and azamulin, extensive inhibitor depletion was observed even at low concentrations of HLM (≤ 0.1 mg/mL). For these rapidly metabolized inhibitors, the IC

50 shift could be improved not by increasing the concentration of HLM (as occurs

with the dilution method) but by decreasing the concentration of HLM (≤ 0.01 mg/mL) These results establish that the non-dilution method for assessing MDI is more sensitive than the dilution method because the latter can result in substantially less CYP inactivation due to inhibitor depletion and/or a decrease in free inhibitor concentration due to microsomal binding.

Reference

Paris BL, Kazmi F, Buckley DB, Ogilvie BW, Gipson AE and Parkinson A (2009) Pitfalls in the design of CYP inhibition studies incorporating a dilu-tion step to examine time-dependent inhibition (TDI) or metabolism-dependent inhibition (MDI). Drug Metab. Reviews 41: 92-93.

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P204. Abstract Withdrawn.

P205. Rational Design of CYP2C9 Mechanism Based Inhibitors

Michael L. Schrag

ProPharma Services, Superior, CO, USA

It was hypothesized that rational design of a CYP2C9 mechanism based inhibitor could be accomplished by synthe-sizing ethynyl analogs of known substrates such as tolbutamide, sulfamethoxazole, phenytoin, or warfarin. Using this approach, (i) four ethynyl analogs of tolbutamide, (ii) three analogs of sulfamethoxazole, (iii) racemic 4′-ethynyl phenytoin and () racemic 7- and 6-ethynylwarfarin were synthesized. The ethynyl moiety was incorporated in a known metabolic position for each compound. All of the analogs were found to be competitive inhibitors of CYP2C9, and thus had affinity for the active site of the enzyme. Of the ten compounds tested, four were found to result in time dependent inactivation of CYP2C9 – three analogs of tolbutamide and one analog of sulfamethoxazole. When these four inhibitors were tested against a panel of CYPs (2C9, 2C8, 2C19, 2D6, 3A4, 1A2 and 2b6) none were specific for CYP2C9, despite deriving structurally from marker substrates for CYP2C9. Inactivation rates were moderate and ranged from 0.021 to 0.064 min−1. Half-maximal inactivation was achieved in the range of 16 to 274 mM. Protection experiments demonstrated no attenuation of inactivation by glutathione, superoxide dismutase or catalase. Taken as a whole, these results demonstrate that synthesis of ethynyl analogs of known CYP substrates can yield compounds that are mechanism based inhibitors of the target however, specificity for the target CYP may not necessarily be achieved.

P206. Reversible inhibition of four important human liver cytochrome P450 enzymes by diethylstilbestrol

Yan Qing Qu1, Zhong-Ze Fang2 and Ling Yang3

1The Second Affiliated Hospital of Dalian Medical University, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 3Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Diethylstilbestrol (DES), a synthetic estrogen clinically utilized to treat threatened abortion during 1940s and 1970s, has been restricted to treat certain cases of prostatic and breast cancer due to its adverse drug responses such as teratogenic-ity and carcinogenicity. Some reports have demonstrated that the addition of DES to docetaxel could modify tubulin composition and improve the response of prostate cancer to chemotherapy. Given that DES might be co-administered with other drugs such as docetaxel, the present study focused on CYP-based drug-drug interaction (DDI). The results showed that DES could competitively inhibit CYP3A4, CYP2C8, CYP2C9 and CYP2E1. The inhibition constant (K

i) was

calculated to be 4.4 μM, 3.0 μM, 5.0 μM and 8.0 μM for CYP3A4, CYP2C9, CYP2E1 and CYP2C8, respectively. Based on peak serum DES level after drip influsion of 500 mg of fosfestrol (DES diphosphate) in patients, the ratio of [I]/K

i was

calculated to be 4.3, 6.2, 3.7 and 2.3 for CYP3A4, CYP2C9, CYP2E1 and CYP2C8, which suggested that DES was likely to induce in vivo DDI through inhibition of these four major CYP isoforms. These results collectively demonstrated adverse drug responses might exist when DES was co-administered with other drugs.

P207. Time-Dependent Inhibition of CYP2E1 by the Alcohol Cessation Agent Clomethiazole

David M. Stresser1, Eric T Gangl1, Elke S Perloff1, Andrew K Mason1, Andrew P Blanchard1, Sean Ekins2 and Shangara S Dehal1

1BD Biosciences Discovery Labware, Woburn, MA, USA, 2Collaborations In Chemistry, Jenkintown, PA, USA

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Guidance from the USFDA and EMEA recommends assessment of the inhibition of P450 isoforms in vitro to assess risk for drug-drug interactions. Cytochrome P450 2E1 is an alcohol inducible isoform, important in the metabolism of small molecule xenobiotics of low molecular weight (e.g. MW < 300) including ethanol, acetaminophen and certain gaseous anesthetics. In the course of validating our CYP2E1 inhibition assay for both direct and time-de-pendent inhibition, we investigated the inhibition potential of clomethiazole, a compound suggested as a selective chemical inhibitor of CYP2E1 in the FDA′s draft guidance on drug interaction studies. Clomethiazole, ([5-(2-chloroethyl) 4-methylthiazole]), also known as chlormethiazole, Heminevrin®, Distraneurin®, Hemineurin®, is a hypnotic, sedative and anti-convulsant that has been most often used to treat acute alcohol withdrawal among many other indications. The objective of this investigation was to characterize the CYP2E1 inhibition kinetics of clomethiazole. IC

50 values were determined with clomethiazole or diethyldithiocarbamate after 0, 10 and

30 min preincubation in human liver microsomes, with and without NADPH, followed by dilution into a second-ary incubation containing 60 µM of the CYP2E1 probe substrate chlorzoxazone. Follow-on K

I and k

inact values

were determined by preincubation of clomethiazole (0, 0.3, 1, 3, 10, 30, and 100 µM) for 0, 2, 4, 6, 8, and 10 min, followed by a 10-fold dilution and incubation with 300 µM chlorzoxazone. In the direct inhibition assay (0 min preincubation) conducted twice on independent days, clomethiazole exhibited IC

50 values of 54 and 43 µM, which

were consistent with literature values. Clomethiazole also exhibited an IC50

shift of > 11-fold, determined with an IC

50 value of > 10 µM when preincubated for 30 min the absence of NADPH and 0.88 µM when preincubated in

the presence of NADPH. Follow-on KI and k

inact values were determined to be 39 µM and 0.36 min−1, respectively.

The time-dependent inhibition of CYP2E1 by clomethiazole may implicate the need for dose adjustments in the clinical use of clomethiazole and co-administered drugs that are significantly cleared by CYP2E1. A preliminary assessment of the potential mechanism of time-dependent inhibition was conducted. A search for a thiazole ring, a prominent feature of clotrimazole, in 2815 FDA approved drugs yielded 38 that contain this substituent. Notably, ritonavir, a rapid acting time dependent inhibitor of CYP3A4, contains two of these substituents, suggesting the thiazole ring may be important in the mechanism of TDI, under the right physiological conditions. Specificity of the time dependent nature may further depend on the size of the molecule and active site constraints which impact molecule-protein interactions.

P208. Utilization and Optimization of Cryopreserved Human Hepatocytes as a Model to Assess CYP450 Inhibition

LaHoma Easterwood, Lee Robinson, Ben Fontenot, Jenny Smith, Mike Shaw and Jason Wright

Invitrogen/Life Technology, Austin, TX, USA

Pharmacokinetic drug-drug interactions (DDIs) can occur when one drug alters the metabolism of a co-administered drug. To gain a better understanding of a drug′s potential to cause pharmacokinetic-based DDIs via inhibition of drug metabolizing enzymes, a battery of in vitro screens can be employed to determine the extent of CYP inhibition. These assays typically use one or more of the following tools: recombinantly expressed CYPs (rCYPs) or human liver microsomes (HLM). Human primary hepatocytes, which provide an in vitro environment which more closely resembles that of the human liver by providing the full complement of Phase I and Phase II xenobiotic-metabolizing enzymes, have recently been suggested as a model to determine both reversible and time-dependent inhibition of CYPs. For this study we utilized cryopreserved human hepatocytes in suspension as a tool to assess reversible and time-dependent inhibition of CYP enzymes by isoform-specific inhibitors The inhibition potential of these com-pounds for the major drug metabolizing CYP enzymes (CYP1A2, phenacetin O-dealkylation; CYP2B6, bupropion hydroxylation; CYP2C8, paclitaxel 6ahydroxylation; CYP2C9, tolbutamide hydroxylation; CYP2C19, (S)-mephenytoin 4′hydroxylation; CYP2D6, dextromethorphan demethylation; 6hydroxylation; CYP3A4, testosterone 6β-hydroxylation and midazolam 1′-hydroxylation) was assessed by measuring IC

50 and K

i values for direct inhibition as well as K

I

and kinact

values for time-dependent inhibition using known isoform-specific positive control inhibitors. Prior to determining the inhibition potential, the reactions conditions were optimized by assessing the linearity of time and cell density dependence and the K

m and V

max parameters for each of the isoforms were determined. These resulting

kinetic parameters obtained are comparable to those determined for human liver microsomes and demonstrate that cryopreserved hepatocytes can be used for assessing both direct and time-dependent CYP inhibition potential by drug candidates.

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P209. Absorption and Biotransformation of the Model Xeno-Estrogen Bisphenol A by the Skin

Carine Jacques1, Helene Duplan2, Elisabeth Perdu1, Sandrine Bruel1, Anne Riu1 and Daniel Zalko1

1UMR 1089, Xenobiotiques, INRA, Toulouse, France, 2Laboratoire de Pharmacocinétique Cutanée, Pierre Fabre dermo-cosmetique, Toulouse, France

The hypothesis that skin contact could contribute to human exposure to bisphenol A (4,4′-dihydroxydiphenyl dimethyl-methane, CAS # 80-05-7; BPA) has been repeatedly raised and debated at recent international conferences on endocrine disruptors. Among other things, it is based on the fact that BPA residues are known to be present in many manufactured goods and certain types of paper in daily use (thermal paper). We previously published several articles regarding the metabolic fate of BPA and other bisphenols(1,2). Recently, we completed a study focusing on the use of short-term cultures of pig ear skin as an alternative method to study the absorption and fate of xenobiotics by the skin. This model was vali-dated using model compounds such as 7-ethoxycoumarin [7-EC], benzo(a)pyrene [B(a)P] and testosterone, addressing in detail the metabolic capabilities of short-term cultures of pig ear skin. Both oxidative (phase I) and conjugative (phase II) activities were shown to be expressed by this ex vivo system(3,4). We investigated the diffusion and biotransformation of BPA using ring labeled 14C-BPA [50-800 nmol] applied on the surface of pig skin ear short term cultures. Next, we compared the results with experiments carried out using commercial human skin explants. Radioactivity distribution was measured in all skin compartments and in the diffusion cells of static cells diffusion systems. BPA and metabolites were further quantified by radio-HPLC. Results demonstrate extensive absorption of this model xenoestrogen [viable pig ear skin: 65%; viable human explants: 46%; non viable (previously frozen) pig skin: 58%], as well as biotransformation (in viable systems only). Major BPA metabolites produced by the skin were conjugates, which were identified as BPA glucuronide and BPA sulfate, respectively. Experiments with viable skin models unequivocally demonstrate that BPA is readily absorbed and metabolized by the skin. The trans-dermal route is expected to contribute to BPA exposure in human, when direct contact with BPA (free monomer) occurs. This is the first report documenting the fate of BPA in viable skin models.

References

(1) Jaeg, J.P., Perdu, E., Dolo, L., Debrauwer, L., Cravedi, J.P., Zalko, D. (2004). Characterization of new bisphenol A metabolites produced by CD1 mice liver microsomes and S9 fractions. J Agric Food Chem. 52(15):4935-4942.

(2) Zalko D, Prouillac C, Riu A, Perdu E, Dolo L, Jouanin I, Canlet C, Debrauwer L, & Cravedi JP (2006) Biotransformation of the flame retardant tetrabromo-bisphenol A by human and rat sub-cellular liver fractions. Chemosphere 64(2):318-327.

(3) Jacques, C., Jamin, E.L., Perdu, E., Duplan, H., Mavon, A., Zalko, D., Debrauwer, L. In Press (2010). Characterisation of B(a)P metabolites formed in an ex vivo pig skin model using three complementary analytical methods. Anal Bioanal Chem. doi:10.1007/s00216-009-3389-1

(4) Jacques, C., Perdu, E., Dorio, C., Bacqueville, D., Mavon, A., Zalko, D. In press (2010). Percutaneous absorption and metabolism of 14C-ethox-ycoumarin in a pig ear skin model. Toxicology In Vitro. doi:10.1016/j.tiv.2010.04.006

P210. Characterization of Esterase, Glucuronyl and Sulfo transferase Activities in Skin and Reconstructed Human Skin Models

Guillaume Lereaux, Joan Eilstein, Jean-Roch Meunier, Jacques Leclaire and Daniel Duche

Life Sciences Department, L′Oréal Research, Cedex, France

The 7th European amendment to the cosmetic directive bans the use of animal testing to develop new cosmetics in European Union. Cosmetic industry could adjust to that by developing and optimizing reconstructed human skins to substitute them to the animals. The use of these in vitro models to test efficacy and/or safety of new compounds and determine their mechanisms of action and/or toxicity needs to acquire knowledge of their metabolic equipment and its performances. The characterization of metabolic capabilities for skin models developed by L′Oreal (Episkin™ and SkinEthic-RHE™) was initiated and compared with a normal human skin (NHS). It was managed by measuring the expression level of mRNA coding for phases 1 and 2 metabolizing enzymes and their catalytic activities. Main cytochrome P450 (CYP450) families involved in drug metabolism as well as N-acetyl (NAT) and Glutathione (GST) transferases were already estimated. Esterases (ES), glucuronyl (UGT) and sulfo (SULT) transferases were probed to get on this work. Their catalytic activities were quantified both in models and NHS using 4-methylumbelliferyl acetate (4-MUac), 4-methylumbelliferone (4-MU) and p-nitrophenol (PNP) as substrates, respectively. Apparent V

max, Km and

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clearances were determined. Whatever the tissue, results showed a high ES activity, a much lower UGT activity and a very weak SULT activity hardly detectable with phenol substrates while more strongly observed with steroids. To conclude, NHS and models are equipped well with hydrolytic and glucuronyl transferase activities and less for sulpha-tion reactions. This study enriches the existing data on skin metabolism and indicates that reconstructed human skin models express the same functional metabolic equipment than a normal human skin and can be useful tools to design or select skin care molecules.

P211. Characterization of N-Acetyl and Glutathione S-Transferase Activities in Skin and Reconstructed Human Skin Models

Joan Eilstein, Guillaume Lereaux, Edwige Daronnat, Jean-Roch Meunier, Jacques Leclaire and Daniel Duche

Life Sciences Department, L′Oréal Research, Cedex, France

Skin is considered as the body envelope and a physical barrier to its environment. However, it contains numerous metabolizing enzymes which give to it a potential role in terms of metabolism and detoxification. The 7th European amendment to the cosmetic directive bans the use of animal testing to evaluate the efficacy and safety of new cosmet-ics. This policy has forced the cosmetic industry to develop reconstructed human skin models as tools for alternative methods to the animal experimentation. For these reasons, the models must be characterized and compared with a normal human skin (NHS) in terms of metabolic capabilities and detoxifying functions. In this work, we characterized the N-acetyl (NAT) and Glutathione-S (GST) transferase activities. Previous studies showed that NHS and reconstructed human epidermis such as Episkin™ and SkinEthic-RHE™ expressed several NAT and GST isoforms. Their global cata-lytic activities have been quantified in NHS and models using p-aminobenzoïc acid (PABA) and chlorodinitrobenzene (CDNB) as substrates, respectively. Apparent V

max, Km and clearances were measured for each tissue. Results showed

that for both activities, although Vmax

and Km between tissues are different, probably due to the substrate bioavailability, the clearances were equivalent. A high variability between samples was observed for NAT and GST activities of NHS or models made of keratinocytes from single donors. Tissue spoiling or genetic polymorphism could explain this result. To conclude, these findings confirm that NAT and GST activities are expressed and functional in skin and models. Skin model reconstruction with keratinocytes from single donors should be considered to better reproduce phenotype variability of NHS with models.

P212. CYP2A6/2A13 and CYP1A1/1B1 activity is conserved in cultures of differentiated primary human tracheobronchial epithelial cells

Nik Newland, Andrew Baxter, Katherine Hewitt and Emmanuel Minet

Risk Characterization, British American Tobacco, Southampton, United Kingdom

Introduction

The respiratory tract is the primary route of exposure to inhaled toxicants such as environmental pollutants and tobacco smoke. Metabolic-dependent activation of inhaled toxicants is a potential contributor to the onset of lung disease. In vitro models are frequently used for toxicological assessment however; few in vitro models retaining adequate mor-phology and metabolic activity are currently available to investigate lung toxicology. Normal human tracheobronchial epithelial cells (NHTBE) are a potentially interesting respiratory tract in vitro model since they conserve a pseudostrati-fied epithelia morphology. Their metabolic competency however has not been investigated. Objective: Our objective was to characterize the expression and activity of toxicologically relevant CYPs, CYP1A1/1B1 and CYP2A6/2A13, in polarized NHTBE cells cultured at the air-liquid interface.

Methods: CYP2A6/2A13 and TCDD-induced CYP1A1/1B1 expression was investigated by quantitative real-time reverse transcriptase PCR (qRT-PCR) and CYP2A6/2A13, CYP1A1/1B1-dependent activity was assayed by coumarin

151

7-hydroxylation and luciferin chloroethyl ether O-dealkylation, respectively. Enzyme expression and activity was tested in NHTBEs from three donors taken at 7 and 28 day culture and compared to NCI-H292 and A549 cells.

Results: CYP1A1, 1B1, 2A6, and 2A13 mRNAs were detected in NHTBE cells at both 7 and 28 days in culture. CYP1A1 and CYP1B1 were induced by TCDD (range of 400-1500 fold), and the overall level of CYP2A13 was increased at 28 days (range of 5-70 fold). Oxidation and excretion of metabolic probes confirmed that CYP1A1/1B1 and CYP2A6/2A13 are active. CYP2A enzyme activity was higher at 28 days (1.5 -5 pmol/mg prot/min) when compared to 7 days (0.16 to 1.6 pmol/mg prot/min). Inter-individual differences were also recorded between each donor. In comparison, CYP1A1, 1B1, 2A6, and 2A13 activity was limited in H292 and A549 cells.

Conclusion: These results suggest that cultures of NHTBE cells may represent a useful metabolically competent model to study the effects of lung toxicants in vitro.

P213. Cytochrome-P450 Activities in Rat Brain Microsomal Preparations

Nicola Caradonna1, Massimo Valoti2, Giulia Franco2, Stefania Dragoni2, Ugo Zanelli3 and Goran Westerberg4

1Drug Profiling Unit, SienaBiotech SpA, Siena, Italy, 2Neurosciences, University of Siena, Siena, Italy, 3Screening & Technologies, Siena Biotech SpA, Siena, Italy, 4SienaBiotech SpA, Siena, Italy

Although the overall cytochrome P450 (CYP) level in the brain is approximately 0.5-2% of that in liver microsomes [1], it can play an important role in therapeutic and side-effects responses of centrally acting drugs [2]. Furthermore the CYPs present in the various brain regions differ in concentrations and isoform distribution. The objective of the current study was to investigate the metabolism of CYP marker substrates (testosterone, dextromethorphan, fluoxetine and bufuralol) in rat brain microsome preparations [3], comparing brain and liver metabolism of these compounds. UPLC/MS-TOF system allowed high resolution of isomer metabolites like testosterone hydroxylates, while accurate mass (0.01Da win-dow) allowed the detection of different types of transformations with low background noise, relatively high sensitivity and wide dynamic range. Dextromethorphan O- and N-dealkylation, fluoxetine demethylation and bufuralol hydroxyla-tion activities were all observed with brain microsomes, but the formation rate of metabolites was about 3 orders of magnitude lower than the activity observed in liver. Furthermore, testosterone showed a different metabolite pattern respect to those observed in liver: in rat brain microsomes testosterone metabolism mainly produced the 2ß-, 6α-and 16ß-hydroxytestosterone instead of the 2α-, 6ß- and 16α-hydroxytestosterone found with liver microsomes. Moreover, gender differences in testosterone brain metabolism were observed: 2ß- and 6α- hydroxytestosterone formation were more abundant in male than in female brain microsomal preparations, while 2α- and 16α-hydroxytestosterone were found only in incubations with male rat brain microsomes. Our data highlight a qualitatively different CYP-dependent metabolism of xenobiotics in the brain compared to the liver and this could contribute to unpredictable therapeutic and/or toxicological effects of CNS drugs. This work was supported by the European Union. FP6 PRIORITY LSH-2005-2.2.0-8: Small-ligand libraries: improved tools for exploration and prospective anti-tumour therapy. DePPICT Project (Designing Therapeutic Protein-Protein Inhibitors for Brain Cancer Treatments) Contract number: LSHC-CT-2007- 037834 (http://www.deppict.eu/home.jsp)

References

1. Miksys SL et al. (2002) J Psychiatry Neurosci 27:406 2. Meyer RP et al. (2007) Curr Drug Metab 8:297 3. Dragoni S et al. (2003) J Neurochem 86:1174

P214. EFFECTS of Aspirin Overdose ON Balf VEGF of Endotoxemic Rats

Ozlem Aydin1, Neslihan Tekin1, Tayfun Aydin2, Halide E. Temel3, Dilek Burukoglu4, Erinç Aral4 and Fahrettin Akyuz1

152

1Department of Biochemistry, Eskisehir Osmangazi University, Faculty of Medicine,, Eskisehir, Turkey, 2Department of Anesthesiology and Reanimation, Dumlupinar University, Faculty of Medicine, Health Research and Training Hospital, Kutahya, Turkey, 3Department of Biochemistry, Anadolu University, Faculty of Pharmacy, Eskisehir, Turkey, 4Department of Histology and Embryology, Eskisehir Osmangazi University, Faculty of Medicine,, Eskisehir, Turkey

LPS-induced acute lung injury is characterized by pulmonary neutrophil infiltration, pulmonary endothelial cell dam-age, and increased capillary permeability. Salicylate poisoning affects multiple organ systems. Acute aspirin overdose may cause non-cardiogenic pulmonary edema. The normal human lung contains significant amounts of VEGF. VEGF is produced by many types of cells such as fibroblasts, vascular smooth muscle cells, endothelial cells, macrophages. VEGF regulates endothelial cell survival, and vascular permeability. Many studies have suggested that VEGF may contribute to the development of non-cardiogenic pulmonary edema. Other studies have proposed a more protective role on the alveolar epithelium following injury. We hypothesized that aspirin overdose may decrease VEGF levels by increasing epithelial injury and may prolongs recovery period. Forty two adult, male, Spraque Dawley rats were divided into six groups. Intraperitonal saline was administered to control group. Aspirin 300mg/kg and 500 mg/kg was administered to Group 2 and Group 3 by gavage, respectively. Intraperitonal LPS (E. coli, serotype 055-B5) 1 mg/kg was administered to Group 4, 5 and 6, respectively. Aspirin 300 and 500 mg were given to group 5 and 6, 24 hours after intraperitoneal LPS. Blood samples were taken by intracardiac route, bronchoalveolar lavage preformed and lung tissues were taken. VEGF, NO, MMP2 and total protein in BALF and VEGF, MMP2 and salicylate levels were measured in plasma. Arterial blood gases were determined. The wet/dry lung ratio was calculated. MPO activity was measured in lung tissue homogenates. Total protein levels were raised in group 2, 4, 5, and 6. Wet/dry lung weights were not differed among groups. VEGF and NO levels in BALF were increased in group 2 and 3, and decreased in group 5 and 6, respectively. MPO activity was higher in group 2, 4, 5 and 6. Plasma salicylate levels were higher in group 5 and 6 versus group 2 and 3,respectively. We thought that aspirin overdose may increase the severity of LPS-induced acute lung injury and prolongs recovery period by decreasing VEGF and NO levels.

P215. Flavin Monooxygenases (FMOs) and Species Differences in Pulmonary Metabolism

Mark D. Wrona1, Amandine Chefson1, Jean-François Chiasson2, France Landry1, Jean-François Lévesque1, Richard Tschirret-Guth1 and Leanne L. Bedard1

1Preclinical Drug Metabolism and Pharmacokinetics, Merck Frosst Canada & Co., Kirkland, QC, Canada, 2Medicinal Chemistry, Merck Frosst Canada & Co., Kirkland, QC, Canada

The flavin-containing monooxygenase (FMO) protein the oxidation of heteroatoms and is comprised of five known functional isoforms (FMO1 through FMO5). The protein expression of FMOs demonstrates tissue- and species-selective differences, as well as developmental-controlled expression. FMO1 is the predominant isoform expressed in liver in preclinical species. Although FMO1 is present in fetal human liver, it is absent in adult human liver, where FMO3 is the predominant isoform. FMO2, also known as the “lung FMO”, is highly expressed in pulmonary tissue of rodents and rabbits. In humans, however, FMO2 is typically nonfunctional due to a genetic mutation in which the protein is truncated. This mutation occurs with a genotype frequency of ∼ 100% in Caucasian and Asian populations. Both FMOs and CYPs can catalyze N- or S-oxygenation of xenobiotics leading to identical metabolites. The use of CYP-specific inhibitors (such as 1-aminobenzotriazole, ABT) and other techniques can be used to discriminate CYP versus FMO involvement. However, precise diagnostic tools such as FMO specific substrates and/or inhibitors to elucidate specific FMO isoform involvement are currently lacking. The objectives of this study were to: 1) highlight the species differences in in vitro pulmonary metabolism; and 2) elucidate the relative contribution of CYPs versus FMOs, in the hepatic and pulmonary metabolism of an N-methylpiperazine derivative (MRL-A) in preclinical species and humans. in vitro In liver microsomes, both N-demethylation and N-oxidation were observed as major pathways of metabolism in rat, dog, cynomolgus monkey and human upon incubation with NADPH. In rat lung microsomes, N-demethylation was not observed and N-oxidation was found to be the major metabolic pathway. N-oxidation was not inhibited by pretreatment of rat lung microsomes with ABT, indicating that CYP enzymes were not involved and suggesting the involvement of FMO2 instead. In beagle dog and cynomolgus monkey lung microsomes, N-oxidation was also found to be the major metabolic pathway. In contrast, no metabolism was observed in human lung microsomes from either smokers or non-smoker consistent with the potential involvement of FMO2 in the N-oxidation of MRL- in lung microsomes of preclinical

153

species. N-oxidation of MRL-A was found to be catalyzed by human recombinant FMO1 and FMO3, suggesting that FMO3 contributes to its hepatic metabolism. In summary, this study demonstrates that due to the overlapping substrate specificities of FMO isoforms, and differential tissue expression across species, extrapolation of metabolism data for FMO-catalyzed biotransformations from preclinical species to humans must be executed with caution.

P216. Intestinal First-pass Metabolism of TNP

Hua Li1, Jing-ting Deng2 and Xiao-mei Zhuang1

1Drug Metabolism, Beijing Institute of Pharmacology and Toxicology, Beijing, China, 2Drug Metabolism, Beijing Institute of Pharmacology and Toxicology, Beijing, China

TNP, [N-cyclopropylmethyl-7(-[(R)-1-hydroxy-1-methyl-3-(thien-2-yl)-propyl]-6,14- endo-ethanotetrahydronororipa-vine], is a potent and long-acting partial opioid agonist and currently under development as a promising drug candidate for treatment of opium addiction. The animal PK data showed that, TNP was metabolized into several metabolites via multiple biotransformation pathways in rat and undergone a significant first-pass metabolism after oral administra-tion. The concentrations of TNP glucuronide conjugate were much higher than that of the parent in plasma in orally dosed rats and Beagles. Despite the extensive metabolism of TNP in liver, it was assumed that intestine may also play an important role in quick metabolic clearance of TNP, as a number of metabolites were detected in rat feces. In this study the contribution of intestine to the first-pass metabolism of TNP was evaluated using in situ perfused rat intes-tinal preparation and intestine-liver preparation. The in situ perfused rat intestinal preparation and intestine-liver preparation were made as per Pang′s method[1]. TNP (3 mg/kg) was dosed via duodenum of the preparations. The parent compound and its metabolites were quantitatively analyzed by a validated LC-MS/MS method. On the in situ perfused rat intestinal preparation the absorption rate of TNP was 0.95 1·h−1 and the C

max (1250 ng·mL−1) was observed

at 150 min of perfusion. The conjugate and oxidative metabolites were detected. The Cmax

of the conjugate in perfusate was 174 ng·mL−1. When perfused through the in situ intestine-liver preparation, TNP was detected in the perfusate in a constantly low level (200 ng·mL−1), while the concentration of its glucuronide conjugate was higher (250 ng·mL−1). The absorption and metabolism of the parent reached a steady state at 10 min of perfusion, indicating that the absorbed TNP could immediately metabolized by the liver. The AUC of the conjugate in the intestine preparation was counted as 70% of that in the intestine-liver preparation, suggesting that the majority of the conjugate was formed in the intestine. The intestine was the primal organ for phase II metabolism of TNP. The first-pass effect of the drug was intensified by its slow and incomplete absorption in intestine, resulting in the low oral bioavailability.

References

1. KS Pang et al: Disposition of enalapril in the perfused rat intestine-liver preparation:absorption,metabolism and first-pass effect. J Pharmacol Exp Ther. 1985, March,Vol.233.No.3:788-795.

P217. Skin metabolism review

Joan Eilstein, Guillaume Lereaux, Jean-Roch Meunier, Jacques Leclaire and Duché Daniel

Life Sciences Department, L′Oréal Research, Cedex, France

The skin metabolism studies would seem less exciting than those of the liver. Indeed, skin appears to be a tissue of weak catalytic activity with less various produced chemicals and less funny reaction mechanisms. However, this assertion could be due to the lack of specific tools to study skin metabolism such as particular sample preparation protocols or accurate analytical methods. Thus, even if low enzymatic activities are observed, they can become con-sequent when we consider the total skin area. Indeed, the skin is the largest organ of the human body and represents the major protective barrier to the environment and chemicals exposure. Consequently, skin metabolism research would require a real scientific effort and dynamism to characterize skin metabolizing enzymes and their activities. In addition, the 7th European amendment to the cosmetic directive forbids the use of animal testing to evaluate the efficacy and safety of new cosmetics. This policy has forced the cosmetic industry to develop reconstructed human

154

skin models as tools for alternative methods to animal experiments. For these reasons, the models have to be char-acterized and compared with normal human skin in terms of metabolic capabilities. In this talk we will present a review on the L′Oreal research and its strategy in the field of the characterization of skin metabolic equipment and its catalytic capabilities. Thus, characterization for the expression of several enzymes (CYP450, Esterase, NAT, GST, UGT, SULT…) and their catalytic activities (Apparent Km, Vmax and clearance) in reconstructed tissues compared to normal human skin samples will be presented. Also, skin manipulation, experimental process, analytical methods and their problems will be discussed in order to start a real discussion around the skin metabolism and try to com-municate our experience and passion.

P218. The newly discovered molybdenum-containing enzyme mARC is a novel extrahepatic drug metabolizing enzyme

Bernd Clement1, Antje Havemeyer1, Carmen Krischkowski1, Florian Bittner2 and Ralf R. Mendel2

1Institute of Pharmacy, Universitat Kiel, Kiel, Germany, 2Plant Biology, TU Braunschweig, Braunschweig, Germany

The newly discovered molybdenum-containing enzyme mARC is a novel extrahepatic drug metabolizing enzyme The investigation of oxygen-insensitive nitrogen reductive drug metabolism in mitochondria leads us to the discovery of completely unknown mammalian molybdenum in our laboratory. Upon reconstitution with the electron transport pro-teins cytochrome b5 and its reductase this molybdenum enzyme is capable of reducing N-hydroxylated compounds. Thus it was named “mitochondrial amidoxime reducing component” (mARC), because initially the N-reduction of amidoxime structures were studied with this isolated enzyme. Thus this newly discovered reductive enzyme system plays a major role in drug metabolism though its physiological relevance is not still clear. Unusual for drug metabo-lizing enzymes, we found unexpected high extrahepatic reductase activities, for example in kidney and thyroid. The determined activities exceed even the hepatic activities, depending on the tissue investigated. For 40 years, it was believed that only three molybdenum containing enzymes are present in the human body. With the recently isolated protein, it became clear that a fourth molybdenum containing enzyme exists and that the human genome codes for two homologues proteins, mARC1 and mARC2. The N-reductive complex located in the outer mitochondrial mem-brane with its mARC protein represents the first eukaryotic molybdenum enzyme with separate electron transport proteins. In continuation of our drug metabolism studies we found out that the mARC-containing enzyme system is able to reduce several types of N-hydroxylated compounds like amidoximes, N-hydroxylated guanidines, and sul-fonamides. Furthermore it is obviously involved in the detoxification of mutagenic N-hydroxylated DNA bases and hydroxylamines.

P219.NActivated Sterol Regulatory Element-Binding Protein-2 Suppresses Hapatocyte Nuclear Factor-4-Mediated Cyp3a11 Expression in Mouse Liver

Shin-ichi Inoue, Kouichi Yoshinari, Mika Sugawara and Yasushi Yamazoe

Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sedai, Japan

Sterol regulatory element-binding protein-2 (SREBP-2) is a ubiquitously expressed transcription factor. It is activated under the condition of low cellular cholesterol level and plays key roles in the cholesterol homeostasis. In the present study, we have investigated a possible involvement of SREBP-2 in Cyp3a11 expression in mouse liver. Hepatic Cyp3a11 mRNA and protein levels were decreased in mice fed a low-cholesterol diet (LCD), while hepatic nuclear SREBP-2 levels and the mRNA levels of SREBP-2 and SREBP-2-responsive genes were activated. These phenomena were reversed by cholesterol supplementation to LCD. In in vitro reporter assays, the overexpression of SREBP-2 nuclear form sup-pressed Cyp3a11 reporter activity through the region from -1581 to -1570 of Cyp3a11, which contained a DR1 (direct repeat separated by mononucleotide)-type motif, a putative binding site for hepatocyte nuclear factor-4α (HNF-4α). The binding of HNF-4α, but not SREBP-2, to the motif was confirmed in electrophoretic mobility shift assays and DNA-affinity assays. Furthermore, the SREBP-2-dependent suppression of Cyp3a11 expression was disappeared with the mutation or deletion of this motif in reporter assays. In glutathione S-transferase (GST) pull-down assays, SREBP-2

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nuclear form directly interacted with N-terminus of HNF-4α containing DNA binding domain in solution through the region containing DNA binding domain. SREBP-2 was, however, unable to interact with HNF-4α bound to the DR1 motif and had no influence on the HNF-4α binding to the motif in GST pull-down assays and DNA-affinity assays. With in vitro binding assays, SREBP-2 inhibited the interaction between HNF-4α and its representative coactivator, peroxi-some proliferator-activated receptor γ coactivator-1α (PGC-1α) through interacting with PGC-1α via its transactivation domain. Consistently, a mutant SREBP-2 lacking the transactivation domain did not reduce Cyp3a11 reporter activity in reporter assays. PGC-1α overexpression relieved the SREBP-2-mediated reduction of Cyp3a11 reporter activity in reporter assays. Finally, chromatin immunoprecipitation assays demonstrated that PGC-1α and HNF-4α, but not SREBP-2, bound to the DR1 motif in Cyp3a11 promoter and the extent of in vivo binding of PGC-1α, but not HNF-4α, to the motif was significantly reduced by LCD feeding in mouse liver. In conclusion, SREBP-2 is activated to interact with PGC-1α in mouse liver at reduced intake of cholesterol. This results in the reduced recruitment of PGC-1α to HNF-4α on the Cyp3a11 promoter and the subsequent down-regulation of Cyp3a11 expression.

P220. Activity of Aryl hydrocarbon receptor (AhR) in the placental trophoblast and expression of AhR and its translocator ARNT expression in rat and human placentas during pregnancy

Lucie Stejskalova1, Lenka Vecerova2, Laura Messa Perez1, Lenka Hahnova1, Petr Nachtigal2, Radim Vrzal3, Zdenek Dvorak4 and Petr Pavek1

1Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic, 2Department of Biological and Medical Science, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic, 3Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic, 4Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and member of the bHLH/PAS (basic-Helix-Loop-Helix/Per-Arnt-Sim) family that controls the expression of a diverse set of genes like CYP1A1, CYP1A2, CYP1B1, GST1, UGT1A1, UGT1A6, NQO1 and ALDH3A1 involved in xenobiotic biotransformation. In addition to the AhR function in detoxification of endo- and xenobiotics, it also plays multiple roles in fundamental cell biology, development and normal physiology. At the same time, AhR is a mediator of the toxicity of particular xenobiotics such as dioxin, polycyclic aromatic hydrocarbons and halogenated biphenyls that are involved in tumor initiation, promotion, and progression. Importantly, elevated CYP1A1 activity through activated AhR in placentas of smoking women has been associated with pregnancy complications such as premature birth, intrauterine growth retardation, structural abnormalities, fetal death or placenta abruption and risk of low birth weight. The aim of this work was to study placental expression of aryl hydrocarbon receptor and its translocator throughout of pregnancy both at the level of mRNA employing real time-RT-PCR and proteins in rat placenta using Western blotting. At the same time, we studied the protein localization of AhR/Ahr and ARNT/Arnt in both rat and human placentas employing immunohis-tochemistry. In addition, we studied induction of AhR target genes such as CYP1A1, CYP1A2, CYP1B1 and UGT1A1 in human placental trophoblast cultures. Our preliminary results show Ahr protein reached maximum expression during day 15 and Arnt protein at gestation day (gd) 21. We also found diverse localization of Ahr and Arnt in the rat placentas during the pregnancy and describe localization of AhR and ARNT in the first trimester human placenta and in the term placenta. Finally, inducibility of AhR target genes by model ligands in the cultivated trophoblast confirms its function in the human placenta. We conclude that AHR/Ahr and ARNT/Arnt are differentially expressed in the rat and human placenta during pregnancy.

This project was supported by Grant agency of Charles University No.125709/C and by SVV-2010-261-003.

P221. Association of the DNA-PK Component, KU, with IKAROS Isoforms and ITS EFFECTS On DNA Binding Affinity

Zahide Ozer

Hemotology/Oncology, CHLA(Children Hospital Los Angeles)-USA, Baltimore, MD, USA

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Ikaros (Znfn1a1) is a kruppel-type zinc-finger transcription factor that plays a critical role in both lineage commitment and differentiation of lymphoid cells. In this current study we demonstrate binding between Ikaros and the DNA-repair protein KU (70/86) both in vivo (co-immunoprecipitation) and in vitro interaction studies. The magnitude of binding interaction was also determined by surface plasmon resonance(SPR) analysis on a Biocore instrument. These experiments demonstrated decreased to absent affinity of Ku for the smaller dominant negative (IK4-8). Therefore the N-terminal zinc fingers, which are lacking in the negative acting IK isoforms, may play a role in IK-Ku interaction. The role of the N-terminal zinc fingers of Ikaros and Ku (70/86) in their interaction is also suggested by modeling of the protein interaction. We also demonstrate a strong increase in the ds-DNA binding affinity of positively acting Ikaros isoforms upon in-vitro binding to the Ku compo-nents (Ku70 and Ku86) and DNA-PKcs. Since the positively acting Ikaros isoforms differ from the negative isoforms by the presence of functional DNA binding domain, these results suggest that the DNA binding function may also play a role in the interaction between Ku and Ikaros. Furthermore, we observed that interaction between DNA-PK components resulted in phosphorylation of Ikaros on serine by DNA-PK

cs and stimulation of its DNA binding affinity by approximately 10 fold.

These results demonstrate for the first time a potential for direct interaction of Ikaros with the DNA repair machinery and provide evidence that phosphorylation of Ikaros may play a role in regulation of its DNA affinity. It also shows that Ikaros can interact with DNA-PK in mammalian cells and that Ikaros is a very effective DNA-PK substrate in vitro.

P222. Automated imaging, quantification and classification of NF-kB and Nrf2 responses to identify adverse drug reactions in HepG2 cells

Bram Herpers, Zi Di, Lisa E. Fredriksson and Bob van de Water

Toxicology, LACDR, Leiden, Netherlands

The liver is the major site for drug metabolism and as such, the most heavily exposed organ to drug metabolites. Idiosyncratic adverse drug reactions are hypothesized to occur because of loss of liver cell function due to drug metabo-lite stress interfering with periods of inflammation, leading to accelerated cell death. To test this hypothesis, we set up automated live cell imaging methods in HepG2 cells to visualize the onset and dynamics of drug metabolite-induced soft electrophile reactive metabolite formation (i.e. activation of Nrf2 signaling) and to follow cytokine-induced nuclear translocation oscillations of NF-κB under drug-exposure conditions. HepG2 cells stably expressing GFP-tagged Nrf2 and p65 are exposed to a panel of compounds linked to idiosyncratic adverse drug reactions (e.g. diclofenac, troglita-zone, carbamazepine) and challenged with cytokines (e.g. TNFα and interleukin-1β) followed by automated live cell time-lapse imaging on BD Pathway 855 and Nikon TiE2000 imaging systems. The image sequences are analyzed using in house developed algorithms for ImagePro and ImageJ. Using our imaging systems, we show that all hepatotoxicants cause a time dependent reactive metabolite stress-induced Nrf2-GFP stabilization; the time of onset and maximal Nrf2-GFP stabilization differs per compound. In addition, pre-exposure with the various hepatoxicants interferes with the cytokine-induced NF-κB nuclear translocation responses by extending the NF-κB oscillation pattern. These drug-cytokine synergies can accelerate cell death (either through apoptosis and/or necrosis). We apply these imaging systems in the context of dedicated siRNA-mediated knockdown screens to identify the signaling pathways that modulate the occurrence of idiosyncratic adverse drug-cytokine synergies.

P223. Effect of Promoter Region Polymorphisms in N-Acetyltransferase 2 (NAT2) Phenotype

Rika Yuliwulandari1,2, Koji Okamoto1,3, Dharma Permana 2, Katsushi Tokunaga1 1Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, 2Department of Pharmacology, Faculty of Medicine, YARSI University, Jakarta, Indonesia, 3Department of Nephrology and Endocrinology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

N-acetyltransferase 2 (NAT2) has been associated with the risk of drug toxicities and development of various diseases. The phenotype of NAT2 so far is mainly determined by the polymorphisms in the coding region. However, the pos-sibilities of other factors such as the polymorphisms in the non-coding regulatory regions, some confounding factors during the phenotype measurement and possible gene-gene interaction affecting metabolic activity of NAT2 may also influence the function of the gene. This might also underlie the discordance of NAT2 genotype-phenotype reported in some previous studies. Our study in 212 Indonesian subjects has reported 16 common polymorphisms in the promoter

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and coding regions and 13 inferred haplotypes (7 in the promoter and 6 in the coding regions). We hypothesize that the promoter region polymorphisms may also affect NAT2 phenotype. The functional assays were done to further examine the promoter activity by measuring the ratios of urinary caffeine metabolites. A total of 18 subjects with controlled diet exposure who carry those particular haplotypes participated in the study. Urine samples were collected before and 8 hours after a dose of 100 mg Caffeine. The metabolites 5-acetylamino-6-amino-3-methyluracil (AAMU), 1-methylx-anthine (1X), 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylurate (1U) were quantified using previously established HPLC method. The NAT2 activity was calculated based on the molar ratio of (AFMU+AAMU)/(AFMU+AAMU+1U+1X). Based on the coding region haplotype, the mean metabolic ratio of fast acetylator group showed a higher level (0.4) than the one of intermediate group (0.25). Haplotype analysis of promoter region in the same coding region haplotype group showed that promoter haplotypes significantly influenced the NAT2 activity level (p=0.002 when adjusted by NAT2*4/*6A and p=0.04 when adjusted by NAT2*4/*7B). None of the promoter haplotypes showed significant association with NAT2 activity level in unadjusted coding region haplotypes analysis. In the single promoter SNP analysis, rs4646246 (A/G) significantly associated with NAT2 activity level after adjusted by phenotype group based on the coding region haplotype (p=0.02). Further analysis was done to determine which allele affecting low NAT2 activity. As a single marker, G allele that is mainly associated with NAT2*4 was significantly associated with lower NAT2 activity (G allele, n=9, mean=0.20 ± 0.10; A allele, n=25, mean=0.30 ± 0.13; p=0.015). Subjects with G/G homozygote at rs4646246 were supposed to have lower NAT2 activity, but they showed high NAT2 activity level. Therefore, the result of this study indicated that promoter polymorphisms showed significant influence on the NAT2 activity. However, in the combination analysis of promoter and coding regions polymorphisms, the later showed stronger influence on NAT2 phenotype.

P224. Expression of wild type p53 is induced in HepG2 cells by a Sri Lankan herbal decoction comprised of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome)

Sameera R. Samarakoon1, Prasanna B. Galhena2, M. Ira Thabrew3 and Kamani H. Tennekoon3

1Institute of Biochemistry, Molecular Biology & Biotechnology, Colombo 3, Sri Lanka, 2Department of Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka, 3Institute of Biochemistry, Molecular Biology, and Biotechnology, University of Colombo, Colombo 3, Sri Lanka

A decoction of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome) has been reported to prevent chemically induced hepatocarcinogenic changes in rats and to exert a significant cytotoxic effect on human hepatoma cells (HepG2) which possess wild type p53 (p53wt) protein. However, the exact mechanism by which the above decoction mediates its anti-carcinogenic effect is poorly understood. The success of cancer chemotherapy is critically dependent on the ability of the chemotherapeutic agent to induce effective apoptosis in the target cancer cells. Of pivotal importance in this regard is the p53 tumor suppressor protein which induces apoptosis via the transcriptional activa-tion of an array of pro-apoptotic genes and probably also through transcription-independent mechanisms. Therefore the present study was carried out to investigate the effect of the above decoction on p53wt expression of HepG2 cells. HepG2 cells were treated for 12, 24, and 48 h with different concentrations of the decoction (50 µg/ml – 1200 µg/ml). Expression of p53wt was determined by (a) reverse transcriptase PCR (RT-PCR), (b) western blot analysis, and (c) immu-nohistochemistry. Expression of p53wt in HepG2 cells was significantly upregulated by the decoction in a time and dose dependant manner. Detectable up-regulation was observed initially at a dose of 100 µg/ml with 12 h post-incubation. Both mRNA and protein levels were increased. Therefore the upregulation of p53wt expression at the transcriptional and translational level could be one of the mechanisms by which the anti-carcinogenic effect of the decoction comprised of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome) is mediated.

P225. Med25 as a coactivator of HNF4α, reveals vital role in RNA Polymerase II recruitment and defective drug metabolism in human liver

Sailesh Surapureddi, Ritu Rana and Joyce Goldstein

Laboratory of Toxicology and Pharmacology, National Institute for Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA

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Coactivators are recruited by DNA-bound transcription factors to change the chromatin architecture from the inactive to the active state of the transcription process. The Mediator complex occupies a central role in recruiting RNA Polymerase II to the protein coding gene promoters. Generally the association of Med I with the cis acting ele-ments of bound nuclear receptors is thought to be sufficient for the formation of an active transcriptional complex. However, we report here that Med25, associated with but not part of the Mediator core complex, is responsible for the conversion of an inactive to an active transcriptional complex. Further, Med25 associates with the components of Mediator and CBP-p160 coactivator complexes and is recruited to the HNF4a bound target promoter region. Increase and reduction of endogenous Med25 is reflected on the expression of HNF4a bound target genes as well as on the composition of the HNF4a bound transcriptional complex, their recruitment to the target gene promoters and their functional significance. A novel feature of Med25 is that it imparts the desired “selectivity” for the coac-tivators. Med25 affects not all but only a significant sub-set of HNF4a target genes that effectively and exclusively regulate drug metabolism. These results define a role for Med25 and the Mediator complex in the regulation of drug metabolism.

P226. Minor changes in 2,4,6-tryphenyldioxane-1,3 inducer structure cause changes in CAR mediated CYP2B induction

Vladimir Pustylnyak1, Andrey Yarushkin1, Ekaterina Kachaylo2, Nikolay Slynko3 and Lyudmila F. Gulyaeva4

1Molecular carcinogenesis, Institute of MolBiol & Bioph SB RAMS, Novosibirsk, Russia, 2Novosibirsk State University, Novosibirsk, Russia, 3Institute of Cytology and Genetics, Novosibirsk, Russia,4Molecular carcinogenesis, Institute of MolBiol and Bioph SB RAMS, Novosibirsk, Russia

2,4,6-Tryphenyldioxane-1,3 (TPD) is a highly effective species-specific inducer of CYP2Â in rats. Several analogs of TPD were synthesized using a reaction with 1,3-diphenylpropane-1,3-diol and corresponding substituted benzal-dehydes to verify a hypothesis that minor changes in the inducer structure can cause changes in induction abili-ties (R=H, cisTPD and transTPD; R=N(CH3)2, pDMA; R=NO2, pNO2; R=F, pF; R=OCH3, pMetO). Male Wistar rats were treated by TPD or its analogs (single injection of 10 mg/kg body weight in corn oil). In rat liver cisTPD and transTPD, pDMA, pNO2, pF administration significantly increased total CYP content (1.3 – 2.5 fold) and the level of PROD (12 – 20 fold), CYP2B specific activity, whereas pMetO did not have any effects. Western blot and real-time RT-PCR showed that the increase of PROD in liver is related to the high content of CYP2B proteins and paralleled the increase of CYP2B1 (10 – 43 fold) and CYP2B2 (8 – 26 fold) mRNAs. At the same time content of CYP2B proteins and CYP2B1 and CYP2B2 mRNA levels were unchanged in rat liver after pMetO treatment. Induction effects were not observed for content of CYP3A proteins, CYP3A specific activity, and CYP3A gene expression. Thus, our results provide evidence to support the conclusion that the differences of CYP2B induction by TPD analogs occur because of differences transcriptional activation of CYP2B gene. These dramatical changes in TPD analogs ability to activate CYP2B gene expression can be explained by various interactions with CAR. Results of Western-blot and ChIP showed that cisTPD and transTPD, pDMA, pNO2, pF treatment stimulated nuclear accumulation of CAR and evoked CAR receptor PBREM-binding activity in rat liver. Thus, our results demonstrate that analogs TPD as well as TPD modulate CYP2B gene expression and activity by CAR activation.

This work was supported by RFBR grant 09-04-00801-a, Russia.

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P227. The Influence of Extracellular Signal-Regulated Protein Kinase (ERK) on Pregnane X Receptor-Mediated Expression of CYP3A4 and MDR1 Genes in Hepatocellular Carcinoma Cell Lines and in Primary Human Hepatocytes

Michal Bitman1, Radim Vrzal2, Zdenek Dvorak3, Lucie Stejskalova1 and Petr Pavek1

1Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic, 2Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic, 3Department of Cell Biology and Genetics, Faculty of Science, Palacky University, Olomouc, Czech Republic

Extracellular-signal related kinase (ERK, member of mitogen-activated protein kinases, MAPKs) signaling pathway is critical in proliferation, differentiation and carcinogenesis in various cells including hepatocytes. Its cross-talk with the pregnane X receptor, a master xenobiotic regulator, should be very important in terms of CYP3A4 and MDR1 gene regulation during many pathophysiological processes in the liver such as tumorigenesis or cirrhosis. The aim of the study was to elucidate whether modulation of ERK1/2 signaling cascade affects PXR-mediated transactivation of CYP3A4 and MDR1 genes in hepatocarcinoma cell lines and in primary human hepatocytes. CYP3A4 and MDR1 promoter activation was determined using gene reporter luciferase assays in HepG2 and MZ-Hep1 cell lines and mRNA expression level monitored by RT-PCR in primary human hepatocytes and hepatocarcinoma cell lines. As positive and negative controls, pharmacological inhibitors (U0126, PD98058) and activators (epidermal growth factor, EGF; hepatocyte growth factor, HGF) of ERK, small interfering RNA (siRNA) method and constitutively active MEK1 (upstream ERK kinase) expression construct were employed. The rate of proliferation/cytotoxicity was measured using CellTitre AQueous Cell Proliferation Assay. Our preliminary data show that basal and/or rifampicin-inducible expression of CYP3A4 mRNAs in human hepa-tocytes was inhibited by EGF. In contrast, siRNA targeted against ERK1/2 reduced both basal and rifampicin-induced CYP3A4 transactivation in hepatocarcinoma cells. Transactivation af CYP3A4 and MDR1 reporter construct in gene reporter assay and proliferation of hepatocellular cell lines is differently modulated by activation of ERK pathway. We conclude that ERK modulates CYP3A4 and MDR1 expression and inducibility via PXR-mediated regulation in human hepatocytes and hepatocarcinoma cell lines although the mechanism should be elucidated in further experiments.

The project was supported by Grant Agency of Charles University (GA UK) 170/50/85007, Grant Agency of Czech Republic (GA ČR) 303/07/0128 and SVV-2010-261-003.

P228. Cocaine and Amphetamine Regulated Transcript (CART) blood levels increase after Risperidon treatment in psychotic disorder patients

Tulin Yanik1, Canan Kursungoz1, Levent Sutcigil2 and Fuat Ozgen2

1Biological Sciences, MIDDLE EAST TECHNICAL UNIVERSITY, Ankara, Turkey, 2Psychiatry Department, GULHANE MILITARY MEDICAL SCHOOL, Ankara, Turkey

Atypical antiphysicotic drugs are the first line treatment for psychotic disorder, Schizophrenia. They have been shown to be more effective than other antiphysicotic medications, however, they also cause severe side effects such as obesity. Patients under these drugs treatment are tend to leave the treatment because of weight gain.To explain the weight gain mechanisms and find a primary sign to show the weight gain in these patients would be a solution to formulate drugs with less side effects and may increase the consistency of treatment for such disorder that needs long term therapy. To date, there is none drug selection criteria for these patients. In this study, we studied effects of one of those atypical antipsychotic drugs, risperidone (Risperdal®) which has antagonistic effect on the Type 2 dopamine (D2) and Type 2 serotonin (5-HT2) receptors, in combination. Hypothalamic eating control mechanisms are mainly achieved by neurohormones such as proopiomelancortin (POMC), Neuropeptide Y (NPY), Cocaine and amphetamine regulated transcript (CART), synthesized and secreted from the arcuate nucleus of hypothalamus. CART has a number of biological roles which are important for the mammalian physiology that controls feeding behavior, drug reward, stress and anxiety. CART is modulated by leptin which is responsible for decreasing the apetite by increasing POMC and CART expression in the hypothalamus. CART peptides became a focus of research investigations predominantly because of their potent anorexigenic properties and as being a mediator peptide in the interaction between stress and drug abuse. Blood levels of CART have been shown to be a marker for anorexia nervosa and obesity. Therefore,

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we investigated CART plasma levels in psychotic patients. Twenty four male patients who applied to Gülhane Military Medical School (GATA), Ankara, Turkey were diagnosed as having psychotic disorder according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The control group, also, includes similar age, sex, and sociodemogrophical features of 17 subjects. After patients′ diagnosis, their height, weight and body mass index (BMI) were noted, blood samples were collected and hospitalized with a daily antipsychotic treatment (risperidone 4-8 mg/day). During hospitalization period, they were fed with food having 2500 calorie. Except for the meals, they were not allowed t extra food intake. For 4-weeks period, their clinical state was followed in hospital and at the end of the 4th week their weight was again measured, together with blood collection for the biochemical examination. Meanwhile, control group′s height and weight were measured to determine their BMI, and after 4-week, their weight were also noted. Following, we determined CART and leptin levels from the plasma samples by ELISA. We showed that after 4-week risperidone treatment, CART and leptin plasma levels were increased significantly. CART levels were 0.53 ± 0.2 ng/ml in controls and 1.19 ± 0.2 ng/ml (p<0.05) in patients after the treatment. Leptin levels were 1.47 ± 0.27 ng/ml in controls and patients’ were 1,68 ± 0.35 ng/ml (p<0.05). This result is an indicator for weight gain in these patients even though their psycotic state were being treated.

P229. Integrated mass spectrometry based metabolomics for the biomarker discovery on the hepatotoxicity induced by carbon tetrachloride, acetaminophen and methotrexate

Byung Hwa Jung1, Bhowmik Salil Kumar1, Oh Seoung Kwon2 and Bong Chul Chung1

1Integrated Omics Center, Korea Institute of Science and Technology, Seoul, South Korea, 2Doping Control Center, Korea Institute of Science and Technology, Seoul, South Korea

Drug-induced liver toxicity represents an important healthcare issue because it causes significant morbidity and mortal-ity and can be extremely difficult to predict. Using mass spectrometry based metabolomic technique, we try to discover biomarkers of hepatotoxicity induced by carbon tetrachloride (CCl

4), acetaminophen (AAP) and methotrexate (MTX).

Liver injury induced by CCl4 is a commonly used model for hepatotoxicity. The AAP and MTX have been known as

drugs with hepatotoxic potential. On this scheme, we treated rats (n=5 for each treatment) with CCl4 (0.1mL/100g),

AAP (500mg/kg and 1000mg/kg) and with MTX (50 mg/kg) by orally. We have tested serum biochemical parameters including alkaline aminotransferase, aspertate aminotransferase, alanine aminotransferase and found that those param-eters were increased, which demonstrated the liver toxicity was induced by CCl

4, AAP and MTX. Global metabolomic

profiling was performed with UPLC-TOF-MS (ultra performance liquid chromatography-mass spectrometry). To find out hepatotoxic metabolic biomarkers, multivariate data analysis (PLS-DA: partial least square-discriminant analysis, HCA: hierarchical analysis) was used. From those analysis, it was found that some steroids, amino acids, carboxylic acids, nucleotides were metabolically changed between control and drug treated groups. As targeted metabolic profil-ing we analyzed steroids by GC-MS and amino acids by CE-MS. From global and targeted metabolic profiling, glycine, alanine, valine, leucine, DL-ornithine, 3-methylhistidine, ββα-androstanediol, 11-dehydrocorticosterone, α-muricholic acid, cholic acid and lithocholic acid were selected as liver toxicity biomarker. Progesterone was selected as marker for AAP drug adverse effect. Tetrahydrocorticosterone and aspartic acid were selected as markers for MTX drug adverse effect. These metabolites could be used as biomarkers in the prediction of hepatotoxicity and adverse effect of those of those drugs

P230. Investigating of oral constraceptives effects on weight gaining mechanisms in polycystic ovary sendrome (PCOS) patients

Tulin Yanik1, Deniz Sezlev2 and Bulent Okan Yildiz3

1Biological Sciences, Middle East Technical University, Ankara, Turkey, 2Biological Sciences, Middle East Technical University, Ankara, Turkey, 3Internal Medicine, Endocrinology, Hacettepe University, Ankara, Turkey

We investigated one of the oral contraceptive used in the treatment of polycyctic ovary sendrome (PCOS), estra-diol/drospirenone (Yasmin®) adverse effects on weight gaining in PCOS patients. Drospirenone (DRSP) is a pro-gestogen which is a class of steroid hormone and its properties resemble progesterone, in terms of competing

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with mineralocorticoids for their receptor, thus it has antimineralocorticoid effects and shows antiandrogenic activity. Patients use the drug is prone to cardiovascular problems and weight gain with long term usage. Weight gain mechanisms are controlled by the arcuate nucleus of hypothalamus that secrete neurohormones such as pro-opiomelanocortin (POMC) which diminish apetite, and agouti-related peptide (AgRP) which inceases apetite. The secretion of these neurohormones are controlled with molecules such as leptin, insulin and neuropeptide nesfatin-1, being one of the processed molecule of NEFA/nucleobindin2 (NUCB2). AgRP is the antagonist for melanocortin receptors (MC3 & MC4) and the midsegment of nesfatin-1 which has an aminoacid sequence similar-ity with AgRP active site which inhibits food intake in leptin resistant conditions. Moreover activation of nesfatin-1 neurons leads to electrophysiological inhibition of AgRP/NPY neurons and with the injection of nesfatin-1 mid-segment. We used case-control association design to compare the plasma levels of nesfatin-1 and AgRP in the lean PCOS and control groups from Endocrinology Department of Hacettepe University in Ankara, Turkey. The PCOS group was treated with Yasmin for 6 months. Enzyme-linked immunosorbent assay (ELISA) revealed that in the PCOS group, before treatment, plasma nesfatin-1 levels were 2,8 ± 0,2 ng/ml and the control group levels were 2,07 ± 0,08 ng/ml (p<0,05) and after treatment nesfatin-1 levels were 2,6 ± 0,1 ng/ml (p<0,001). AgRP plasma levels were 140,2 ± 5,5 pg/ml and the control group levels were 158,9 ± 8.8 pg/ml (p<0,05) and after treatment AgRP levels were 134,3 ± 8,3 pg/ml (p<0,05). Whereas, there were no significant alteration of the peptides after the treatment. The results indicate that Yasmin primarily does not have an effect on weight gaining mechanisms in PCOS patients.

P231. Prediction of Drug-induced Liver Injury in Humans by Using Chimeric PXB-mice® with Highly Humanized Liver

Shin-Ichiro Nagatsuka1, Darina Hynes1, Shin-Ichi Ninomiya1, Masakazu Kakuni2, Chise Tateno2, Takashi Shimada2 and Yasushi Yamazoe3

1ADME & Tox. Research Institute, Sekisui Medical Co., Ltd., Tokai-mura, Ibaraki, Japan, 2PhoenixBio Co., Ltd., Higashihiroshima, Japan, 3Division of Drug Metabolism and Molecular Toxicology, Graduate school of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Drug-induced liver injury (DILI) takes a significant position in the leading causes of withdrawal or black-box warning of a newly developed drug. In order to find out promising biomarkers for the early prediction of DILI in humans, omics approaches have been imployed using in vivo (experimental animals, mainly rats) or in vitro(human hepatocytes or hepatoma cell lines) systems. However, there are significant species and in vitro/in vivo differences that make the prediction unreliable. We have been using chimeric PXB-mice®, in which more than 70% of hepatic parenchymal cells are replaced by human hepatocytes, for the toxicogenomic analyses of hepatotoxicants. This animal model, which mimics human-type drug metabolism and disposition, has a potential to bridge the gap between rodent-type and human-type livers and to explain the difference of in vivo and in vitro response of human hepatocytes against hepatotoxicant treatments. By using 23 different hepatotoxicants (acetaminophen, amiodarone, diclofenac, d-penicillamine, flutamide, erythromycin, valproate, sulindac, indomethacin, perhexilene, methyldopa, amitriptyline, tamoxifen, acetylsalicylic acid, methotrexate, demeclocycline, hydrazine, hydroxyurea, imipramine, orotic acid, troglitazone, tolcapone and ibufenac) and 10 non-hepatotoxicants, we have analyzed changes in hepatic gene expression in PXB-mice®. These drugs were orally administered to PXB-mice® three-times daily at relatively high doses (ca. 20% of reported LD

50), followed

by hepatic total RNA preparation and gene expression analyses using oligonucleotide microarray chips. For each of hepatotoxicants and non-hepatotoxicants treatments, positive and negative incidents of significant change in expression were analyzed, e.g. when statistically significant induction (or reduction) of a gene expression occurred in 8 out of 10 tested hepatotoxicants, the positive incident was expressed as 80% and negative incident was expressed as 20%. For the prediction of DILI susceptibility due to the drug-treatment, true positive rate was calculated for each gene as the product of positive incident in hepatotoxicant treatment and negative incident in non-hepatotoxicant treatment. Genes which showed the true positive rate of larger or equal to 0.5 were selected as biomarkers for DILI. About 1000 genes were extracted as biomarker candidates for DILI prediction. The method for score calculation for DILI prediction and lot-to-lot difference of chimeric mice on the effectiveness of score method will be discussed.

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P232. Profiling in vivo and in vitro PPARγ-mediated gene expression changes to monitor for adverse thiazolidinedione-related side effects

Jonathan M. Maher, Wayne R. Buck, Keith F. McDaniel, Dale J. Kempf, Timothy R. Middleton, Kevin A. Kurtz, Michael J. Liguori, Tatiana Sharapova, Rita X. Ciurlionis, Eric A. Blomme and Yi Yang

Investigative Toxicology, Abbott Laboratories, Abbott Park, IL, USA

The thiazolidinedione (TZD) class of drugs, which includes rosiglitazone and pioglitazone, is widely used to improve insulin sensitization and lipid homeostasis, but has been associated with increased incidence of adverse side effects. PPARg activation is a key aspect of the efficacy, but on-target side effects can include cardiac hypertrophy, peripheral edema, and increases in adipogenesis. The purpose of the current study was to use gene expression profiling using Affymetrix microarrays to develop gene signatures that correlate with the observed adverse effects in both in vitro human adipocytes from pooled donor samples and from in vivo rat visceral adipose tissue. We hypothesized that gene expression signatures from adipose tissue (in vivo or in vitro) could distinguish PPARg agonists with adverse effects from those with a benign profile. To generate a gene signature in human adipocytes, an analysis of variance (ANOVA) between rosiglitazone administered at the IC50 either alone, or in combination with the potent PPARg antagonist GW5662 was utilized to create a gene signature for PPARg activity. An ANOVA between rosiglitazone and nTZDpa, a TZD compound without adverse PPARg effects in rodents, was utilized and combined with the original set to create a 128 gene set that correlated with PPARg-mediated adverse effects. Similarly in rat visceral adipose tissue, a unique profile of genes was created between TZD compounds known to induce adverse effects to distinguish compounds devoid of these effects. The positive control compounds utilized for these repeat-dose studies caused detectable increases in plasma volume and increases in serum adiponectin levels in a timeframe of approximately one week. From the gene signature, 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) and Fatty Acid Binding Protein 3 (FABP3) had the best correlations with adverse effects in vitro and in vivo, respectively, and may be useful for rapid profiling using a single-gene marker. We propose that one can use gene expression profiling of adipose tissue to accelerate identification of compounds without PPARg-related adverse effects.

P233. Abstract Withdrawn.

P234. The Chemotherapeutic DRUGS at the Metronomic Doses Decrease Vascular ENDOTHELIAL GROWTH FACTOR (VEGF) Secretion of SOLID TUMORS

Hande E. Aktas1, Hakan Akbulut2 and Nalan Akgun2

1Ankara University Institute of Biotechnology, Ankara, Turkey, 2Medical Oncology, Ankara University School of Medicine, Ankara, Turkey

Recently, administration of the lower doses of conventional chemotherapeutic drugs, called metronomic chemotherapy, has been widely used in daily practice. One of the main objectives for this strategy is the anti-angiogenic effects of the drugs at the lower doses. However, the effects of conventional chemotherapy drugs on angiogenesis are not well known. In this study we tested the effects of lower doses of different widely used chemotherapeutic drugs on VEGF secretion tumor cell lines. The VEGF levels secreted by tumor cells, were assayed by sandwich ELISA. A human colon cancer cell line (HT-29), a breast cancer cell line (MCF-7) and a primary gastric cancer cell line established in our lab were used for in vitro tests. IC50 values were estimated by using MTT assay of varying doses of doxorubicine, 5-florouracil, irinotecan, oxaliplatin, docetaxel and paclitaxel. The 1 and 2 log doses of the IC50 of the drugs were tested for the efficacy on VEGF secretion of tuýmor cell lines. The mean VEGF levels following 3 days of incubation were 720 pg/mg.protein for HT-29 cell line, 54 pg for MCF-7 and 234 pg for primary gastric cancer cell line. The test drugs yielded significantly more decrease in VEGF levels at the doses of -2log of IC50 in MCF-7 and primary gastric cancer cell lines than the dose of -1log. However, the same doses did not cause any significant change in VEGF secretion of HT-29 colon cancer cell line. The results of the current study suggest that the anti-angiogenic effects of conventional chemotherapy drugs

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might have a role in the efficacy of the treatment and the metronomic doses of the chemotherapeutic drugs could be different in various tumors.

P235. Xenobiotic Metabolizing Enzyme Polymorphisms in a Turkish Population

Ahmet Oguz Ada1, Volkan Karacaoglan1, Semih C. Kunak2, Emre Soydas1 and Mumtaz Iscan1

1Department of Toxicology, Ankara University, Faculty of Pharmacy, Ankara, Turkey, 2Pharmacology, Giresun University, Medical Faculty, Giresun, Turkey

It is well known that cytochrome P-450 (CYP) enzymes are able to metabolize many xenobiotics including drugs, poly-cyclic aromatic hydrocarbons, pesticides and aromatic amines to reactive metabolites which could be very toxic and/or carcinogenic. Among them, CYP2E1 is responsible for the transformation of xenobiotics to their carcinogenic inter-mediates. The glutathione S-transferases (GST)s are a family of cytosolic enzymes generally involved in the detoxication of activated, electrophilic, xenobiotics. Thus genetic polymorphisms of these enzymes can have great impact on the interindividual susceptibility to toxicities of xenobiotics. In this study, CYP2E1, GSTM1, GSTP1 and GSTT1 gene poly-morphisms were determined in 302 healthy individuals. On the basis of polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) methodology, the frequency of CYP2E1*5B (RsaI), GSTP1 codon 105 and GSTP1 codon 114 polymorphisms were determined. The frequencies of c1/c1 (wild type), c1/c2 (heterozygous variant) and c2/c2 (homozygous variant) CYP2E1 genotypes were 94.7 %, 5.3 % and 0 %, respectively. The GSTP1 genotype frequencies were Ile

105/Ile

105: 56.0 %, Ile

105/Val

105: 33.4% and Val

105/Val

105: 10.6%, and Ala

114/Ala

114: 84.8%, Ala

114/Val

114: 14.2% and

Val114

/Val114

: 1.0%. The genetic polymorphism analysis for the GSTM1 and GSTT1 genes was determined simultaneously in a single assay using a multiplex PCR approach. The frequencies of the GSTM1 and GSTT1 null genotypes were 54.6% and 22.2%, respectively. These results reveal that the frequencies of CYP2E1*5B (RsaI), GSTP1, GSTM1 and GSTT1 gene polymorphisms in a Turkish population are similar to Caucasian populations (Supported by the Research Fund of Ankara University, Grant No: 2006-08-03-002HPD).

P236. A Medium-throughput In Vitro System Incorporating Hepatocyte Sandwich Cultures And High Resolution Mass Spectrometry For Evaluating Metabolism And Biliary Clearance

Mary Grubb and Jonathan Josephs

Biotransformation, Bristol-Myers Squibb Co, Princeton, NJ, USA

A common study conducted with development candidates involves dosing a radiolabeled isotope orally to bile-duct cannulated rats and analyzing excreta for radioactivity. This yields a profile of the percent of the dose in each type of excreta, as well as the total amount of drug excreted. This study can be conducted in discovery to evaluate the contribution of biliary excretion to the clearance of a compound. Based on data indicating that sandwich cultures of hepatocytes differentiate and form biliary canaliculi and tight junctions, a technique was developed to evaluate the fate in vitro of radiolabeled compounds (Y. Liu, E.L. LeCluyse, K.R. Brouwer, R.M. Lightfoot, J. I. Lee, and K.L.R. Brouwer, Use of Ca2+ modulation to evaluate biliary excretion in sandwich cultured rat hepatocytes, J Pharmacol Exp Ther 289:1592-1599, 1999 ). This presentation demonstrates that the selectivity and sensitivity of high resolu-tion mass spectrometry allows for the study of non-radiolabeled compounds in this system. Rat hepatocytes in sandwich cultures (B-Clear, Qualyst) were pre-incubated with either standard HBSS or Ca2+ - free HBSS. Buspirone, rosuvastatin and losartan (3 uM) were added and incubation was continued for up to 30 min. At time points, media samples were collected. At 30 min, cell lysates were also harvested. Samples were injected into a UHPLC interfaced with an Orbitrap mass spectrometer. Metabolite identification in lysate and media samples was conducted by data-dependent LC-MS-MS. Quantitation of parent and metabolites in cell lysates and media was achieved using LC-MS and integration of peak areas in high resolution/exact mass ion extracted chromatograms of the parent drug and metabolites. Intrinsic biliary clearance was calculated from parent and metabolite quantitation. As expected, there was no measured biliary excretion of buspirone, as determined by the difference in cell lysate concentration between cultures pre-incubated with HBSS (tight junctions present) or Ca2+ - free HBSS (no tight junctions). Media and cell lysates contained metabolites of buspirone, including those formed by hydroxlyation and glucuronidation. In

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contrast, the biliary excretion of rosuvastatin was 30% in sandwich cultures, and there were no metabolites observed in media or cell lysates, consistent with published data. There were a few metabolites for losartan, including the aldehyde oxidation product and the glucuronic acid conjugate of the parent alcohol. Both losartan and its carboxylic acid metabolite were excreted in bile, as evidenced by a difference observed for cultures pre-incubated in HBSS or Ca2+ - free HBSS. Thus, intrinsic biliary clearance and metabolite profiles were obtained at physiological concentra-tions without the use of radiolabeled compounds, and using much lower quantities of compound than in vivo studies conducted with bile-duct cannulated animals.

P237. Characterization of NTCP, OATP and OCT Function in Human Cryopreserved Hepatocytes in Suspension

Tom De Bruyn1, Zhi-Wei Ye1, Jasminder Sahi2, Patrick Augustijns1 and Pieter Annaert1

1Lab Pharmacotechnology & Biopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium, B-3000, 2Life Technologies, Durham, NC, USA

The limited availability of freshly-isolated human hepatocytes has led to a rapid development of cryopreservation protocols of surplus hepatocytes. As a result, experiments can be planned and all human donor material can be consumed as efficiently as possible. The utility of cryopreserved human hepatocytes to study drug metabolism has clearly been demonstrated, however data regarding transporter activity levels using multiple substrates in a larger number of batches of cryopreserved human hepatocytes are currently not available. The aim of this study was to conduct a systematic evaluation of activities of NTCP (Sodium Taurocholate Co-transporting Polypeptide), OATP (Organic Anion Transporting Polypeptide) and OCT (Organic Cation Transporter) in cryopreserved human hepatocytes from different donors. The uptake of 5 different substrates was assessed among 14 different indi-vidual and 4 pooled batches of cryopreserved human hepatocytes (CellzDirect, Inc.) and known inhibitors were used to evaluate the contribution of active processes to overall substrate uptake. Hepatocytes were thawed with Cryopreserved Hepatocyte Recovery Medium and resuspended in Krebs-Henseleit buffer. All uptake values were determined after 1 min at a substrate concentration of 1µM. The function of NTCP was evaluated by measuring the uptake of taurocholate (TC). The average uptake clearance of TC was 27.5 µl/min/million cells and decreased with 88.9% when extracellular sodium was replaced by choline. To assess the function of OATP, the uptake of estrone-3-sulphate (ES), digoxin (DGX) and estradiol-17β-d-glucuronide (E17G) was determined. Uptake clear-ance amounted to 99.1 µl/min/million cells, 9.5 µl/min/million cells and 5.2 µl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25µM) decreased the uptake clearance of ES and E17G to 48% and 70% of the control value, respectively, while no significant decrease in DGX uptake clearance could be observed. To study the activity of hOCT, 1-methyl-4-phenylpyridinium (MPP+) was used and average uptake clearance amounted to 19.8 µl/min/million cells. Co-incubation with the OCT inhibitors prazosin (3µM) and corticosterone (1µM) showed a decrease to 71% and 85% of the uptake clearance in control conditions, respectively. Experiments in pooled hepatocytes generally showed uptake clearance values which were comparable with the average of all individual batches. Our data provide clear evidence that cryopreserved human hepatocytes are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of various batches of cryopreserved human hepatocytes for transporter activity supports rational selection and optimal use of the hepatocytes depending on the application.

P238. Comparison of the effects of the Clofibrate and its siliconized analogue in freshly isolated rat hepatocytes

Mohammad A. Eghbal1, M. Ziaee2, AR Garjani1, H. Hamzeiy1, N. Saeedi Kozehkonani2, M. Bolourtchian3, M. Ghaffarzadeh3 and M. Ahmadi3

1(1) School of pharmacy, (2) Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, 51664-14766, 2(1) School of pharmacy, (2) Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, 3Chemistry and chemical engineering Research center of Iran, Tehran, Iran

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Peroxisome proliferator-activated receptors (PPARs) play key roles in the regulation of energy homeostasis and inflammation. Fibrates, agonists of PPAR?, are currently used in clinical practice widespread. A siliconized analogue of clofibrate (CLF) was synthesized, whereby the chlorine atom in the phenoxy ring was replaced by silicon and ethyl 2-(4-(trimethylsilyl)phenoxy)-2-methylpropanoate (silafibrate, SIF ) was prepared. The aim of this study was to inves-tigate the effects of SIF and compare it with CLF on isolated rat hepatocyte. Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method, and Cell viability was observed by 0.4% trypan blue. The protective effects of CLF and SIL in freshly isollated hepatocytes against oxidative stress were compared by making use of trypan blue exclusion method, measurement of ROS formation using Dichlorofluorecin and measurement of mitochondrial membrane potential using Rhodamine 123.. RESULTS: SIF was more protective than CLF against oxidative stress–induced cell damage in freshly isolated hepatocytes.

P239. Development and Validation of a Novel Post-Thaw Recovery Media for Human Cryopreseved Hepatocytes

Jie Wang, Rongjun Zuo, Haiyan Xia, Sweta N. Parikh, Mercyanne Andes, Charles L. Crespi and Christopher J.

Patten BD Biosciences, Bedford, MA, USA

Human hepatocytes provide the most physiologically relevant model for studying hepatic metabolism since they contain the full complement of enzymes and transporter proteins. With progress in cryopreservation, cryopreserved hepatocytes have become an “off-the-shelf” reagent with many advantages including consistency and flexibility. A high post-thaw viability is required for successful studies, so it is critical to have both an effective cryohepatocyte recovery media and high-quality cryopreserved hepatocytes. We have developed and validated a new post-thaw recovery media for human cryopreserved hepatocytes. The recovery media maintains hepatocyte post-thaw functions while improving cell health and recovery. The newly developed recovery media was optimized with BD Human Inducible CryoHepatocytes for viability and recovery. It showed an on average 5% post-thaw viability increase compared to traditional Percoll™ methods with more than 20 lots tested. Cell recovery increased on average 1.6 million cells/ vial compared to Percoll. 24- Hour cell confluency and morphology are slightly better with the new recovery media. 8-Hour midazolam metabo-lism and CYP3A4 basal and fold-induction remained consistent. One tube of the recovery media was able to recover up to 5 vials of cryopreserved hepatocytes. The results indicate that the previous method of recovering cryopreserved hepatocytes, like Percoll recovery method, was not optimal. The newly developed post-thaw recovery media is shown to consistently provide improved cell health and recovery while maintaining hepatocyte function. The new recovery media has other advantages such as requiring only one centrifugation and the ability to recover multiple vials without sacrifice in viability and recovery.

P240. Evaluation of Cryopreserved, Differentiated HepaRG Cells for the Simultaneous Determination of CYP450 Induction and Metabolism

Robert Annand1, Mary Jacewicz2, Christophe Chesné3, David Steen4 and Katherine Tsaioun2

1Cell Biology, Apredica, Watertown, MA, USA, 2Apredica, Watertown, MA, USA, 3R & D, Biopredic international, Rennes, France, 4Biopredic international, Rennes, France

Understanding metabolism is an important part of the drug discovery process. Characterization of Cytochrome P450 (Cyp) induction by xenobiotics is vital for characterizing the potential for drug-drug interactions. Traditional studies of metabolism and induction using primary hepatocytes are hampered by the irregular availability and scarcity of donors. HepaRG is a cell line that is highly differentiated and displays many hepa-tocyte-like functions, including Cyp expression and induction. HepaRG has been used to measure metabolism and induction, but requires a lead time for preparation of the cells prior to the experiment. As part of our effort to speed the characterization of Cyp metabolism and induction, we have investigated the use of cryopreserved HepaRG cells to study metabolism and Cyp induction in the same well. The cryopreserved cells consist of

166

differentiated HepaRG cells which are cryopreserved in suspension. The cells were thawed and plated, and then treated as in the same manner as plated differentiated HepaRG cells. Carbamazepine, a tricyclic anti-depressant, is known to be both a potent inducer of, as well as a substrate for, Cyp3A4. Carbamazepine was incubated with HepaRG cells, and aliquots at various time points were analyzed by LC/MS/MS to measure its metabolism. Subsequent to metabolism determination, the cells were washed and incubated for 2-3 days with carbamazepine for Cyp induction. Cyp activity was determined by measuring testosterone 6b-hydroxylase activity of induced cells compared to a vehicle control using an LC/MS/MS-based assay. Carbamazepine was shown to both be metabolized by HepaRG cells, as well as induce Cyp3A4 in the same cells. Studies with other known Cyp inducers and substrates confirmed the utility of using cryopreserved HepaRG cells. By using the combined metabolism-induction assay, we are able to speed drug discovery by rapidly identifying compounds that have potential for metabolism or Cyp induction in a single, rapid assay using cells that are readily avail-able with reproducible characteristics. Routine use of this assay will allow the identification of surprises early in the drug discovery process, thereby reducing the amount of time and money invested in compounds with liabilities. The convenience of using cryopreserved cells to allow for increased throughput for measuring drug interaction properties of new compounds is demonstrated here, allowing the experiment to be conducted at a time that is convenient for the lab.

P241. Human Hepatocyte Cultures Allow Repeated Application of Drugs Resulting in Repeatable Effects On Hepatocellular Metabolism

Anett Ullrich1, Maciej Patrzyk2, Dagmar Braun3, Beatrice Grossjohann4 and Dieter Runge1

1PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany, 2Clinic for Surgery, University of Greifswald, Greifswald, Germany, 3Cheplapharm Arzneimittel GmbH, Mesekenhagen, Germany, 4Riemser Arzneimittel AG, Greifswald, Germany

Introduction Human hepatocytes are the in vitro system of choice to study drug-induced processes in man. Here, we present HEPAC2: A standardized and validated culture system in which functional human hepatocytes can be maintained serum-free for several weeks. Anabolic and catabolic hepatocellular functions and cellular vitality are monitored daily. Albumin and urea are produced on a relatively constant level for up to 2-3 weeks, while the cells remain viable. Based on this, a standard protocol was established that allows repeated exposure of hepato-cytes to test substances for studying drug metabolism. Methods: Human hepatocytes were isolated from healthy tissue of liver resections due to cancer. Hepatocytes are cultured in HHMM (Human Hepatocyte Maintenance Medium) with daily medium changes. After adaptation to the in vitro conditions during the first 3 days, the cells are exposed to test pieces for 24 hrs beginning on day 4 of culture. Subsequently, the culture medium is replaced by medium without the test substance and the same exposure scenario is repeated in intervals of 4 days. Results: As a first model substance we used acetaminophen (APAP) to assay the feasibility of this system. High doses of APAP (2815 mg/l) diminished urea production by 25 % and albumin secretion by 70 %. In addition, APAP let to a complete loss of glycogen and a switch from rough to smooth endoplasmic reticulum. These effects were reversible. After removal of APAP, secretion of urea and albumin returned to control levels, the glycogen stores were refilled and a high content of rough ER was found again. Human hepatocytes are able to compensate functional as well as ultrastructural changes due to APAP within 48-72 h after removal of the compound. Within one cell culture this exposure scenario could be repeated 4-5 times without loss of reproducibility. As a second model substance we used Riemun, an immuno-modulator derived from human blood. Riemun had no effect on the albumin secretion, but led in a concentration dependent manner to an increase of urea release. After removal of Riemun, secretion of urea returned to control levels. Again, within one cell culture this exposure scenario could be repeated 4-5 times without loss of reproducibility. Next, APAP and Riemun were applied alternately. This rotatory application of APAP and Riemun did not alter the effects that each substance had on its own on albumin and urea metabolism. In conclusion, these data demonstrate the robustness and the suitability of our long-term culture system to serve as a tool for repetitive screening of drug-mediated changes on hepatocellular functions and it can become an alternative to animal testing. One human hepatocyte culture may be used multiple times for toxicity testing of xenobiotics. In case of nontoxic effects of the compound, the same cells can metabolise a second compound after a recovery period of 3-4 days.

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P242. Modulation of Antioxidant and Xenobiotic-Metabolising Enzymes in Primary Rat Hepatocytes by Glucosinolates

Margherita La Marca1, Pascale Beffy2, Pier Giovanni Gervasi2, Renato Iori3 and Vincenzo Longo1

1Institute of Agricultural Biotechnology, CNR, Pisa, Italy, Pisa, Italy, 2Institute of Clinical Physiology, CNR, Pisa, Italy, Pisa, 3Research Center for Industrial Crops, Agricultural Research Council (CRA-CIN), Bologna, Italy

Glucosinolates (GLs) have recently attracted intense research because of their protective attributes against cardiovas-cular disease and some kind of cancer. They are present in sixteen families of dicotyledonous angiosperms including a large number of edible species. GLs are beta-thioglucoside-N-hydroxysulfates with a variable side chain (R) and a sulfur-linked beta-D-glucopyranose moiety. Experimental data indicates that certain vegetables may offer an indirect protection against oxidative stress by activating endogenous cellular defense systems at genetic and protein levels. GLs have shown a low bioactivity as such, but once converted by myrosinase enzyme (also present in intestinal flora), they originate isothiocyanates (ITCs) [1], which induce antioxidant enzymes through the activation of nuclear factor E2-related protein (Nrf2) receptor. In the present study, by using primary rat hepatocytes, we investigated the effects of three glucosinolates (glucosinalbin, sinigrin, and gluconapin) on the antioxidant and drug-metabolising enzymes at transcriptional, catalytic and immunoblotting levels. The isolation of hepatocytes was made by De Smet method [2] using two layers of rat-tail collagene (type 1). Cells were treated with the myrosinase-hydrolysed GLs at the doses up to 40microM and analysed by PCR for the expression of NAD(P)H: quinone oxidoreductase-1 and heme oxygenase-1 genes. On the other hand, in the hepatocyte microsomes and 10000 x g supernatants, prepared in the standard way, it was investigated the enzymatic activity of NAD(P)H: quinone oxidoreductase-1, glutathione-S-transferase, catalase, heme oxygenase-1, GSSG-reductase and ethoxycumarin-O-deetylase (a marker of several CYPs). Additionally, the acti-vation of Nrf2 was verified in cytosol and nuclei by western blotting analysis. All the analyzed GLs were able to activate, in a dose dependent manner, the transcription of NAD(P)H: quinone oxidoreductase-1 and heme oxygenase-1 genes and the corresponding activities. In addition, it was also observed a significant induction of glutathione-S-transferase, catalase, GSSG-reductase antioxidant activities. On the other hand, the CYP-dependent ethoxycumarin-O-deetylase activity was decreased by sinigrin and gluconapin at the highest dose suggesting a their cytotoxicity. In contrast, the treatment with glucosinalbin produced a dose dependent induction of this CYP activity, possibly due to the presence of an aromatic moiety in its structure. By western blotting analysis it was ascertained that most of cytosolic Nrf2 was translocated into the nuclei after the GLs treatments indicating that the induction of above mentioned antioxidant enzymatic activities was regulated by this transcriptional factor. In conclusion, it was found that these GLs might have a protective potential against oxidative stress similar to that shown by the classic sulforaphane. In vivo experiments are needed to ascertain this possibility.

References

[1]Fahey J. W., Zhang Y., Talalay P. (1997) Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc. Natl. Acad. Sci. USA. 94: 10367-10372.

[2] De Smet K., Beken S., Vanhaecke T., Pauwels M., Vercruysse A., Rogiers V. (1998) Isolation of rat hepatocytes. Methods Mol. Biol. 107: 295-301.

P243. Plated human cryo-hepatocytes for metabolic stability assay

Haiyan Xia, Kirsten Cooper, Charles L. Crespi, Chris Patten and Rongjun Zuo

BD Biosciences, Bedford, MA, USA

Cryo-hepatocytes cultured in suspension are widely used for metabolic stability assay, however the short life time (generally less than 4 hours) makes it inappropriate for slowly metabolized compounds. Replatable human cryo-hepatocytes in plated culture, on the other hand, maintain hepatic functions longer than suspension cul-ture, making it a better tool for evaluating metabolic stability of such compounds. This study aims to optimize a metabolic stability assay using plated human cryo-hepatocytes and assess the correlation between the metabolism activities in these 2 culture formats. Metabolism activities of CYP3A4, 1A2, 2C9, 2D6 were measured with more

168

than 40 lots of human cryo-hepatocytes in plated culture. Cryo-hepatocytes were seeded with 200uL seeding medium (containing 10% FBS) on a 48-well plate and incubated for 4 hours at 37°C/5% CO2 incubator before the seeding medium was aspirated and an incubation medium was added. After another 12- to 16-hour incubation, the incubation medium was removed and fresh medium containing probe substrate added. Stop solution was added at different time points (0.5hr, 1hr, 2hr, 3hr, 4hr, 6hr, 8hr) and cells were scrapped from plates for HPLC and LC/MS analysis. The plated assay was optimized by assessing effect of BSA supplementation on activity and the effect of cell sonication on parent compound recovery. Standard suspension metabolic stability assay was conducted similarly on each lot to assess a correlation between the activities in these two different cultures. The results showed that no impact on metabolism activity was observed by adding BSA into incubation medium nor did sonicating cells make difference in parent compound recovery. CYP3A4 activities showed a correlation coefficient of 0.7638 across all lots tested between metabolic stability assays in the two culture formats, an improved correla-tion coefficient of 0.9934 was seen with CYP3A4 activity above 800 pmol/min/106cells. To conclude, metabolic stability assay using plated human cryo-hepatocytes was optimized and compared to that using suspension culture. A good correlation was seen in CYP3A4 activities between these two culture formats when the cryo-hepatocytes have high metabolism activities.

P244. Primary Human Hepatocytes Cultured in a 3D Bioreactor Maintain Cytochrome P450 Activity up to 3 Weeks

Malin Darnell1, Thomas Schreiter2, Therese Söderdahl3, Daniel Knobeloch4, Andreas K.N. Nüssler5, Birgitta Dillner6, Anna-Lena Berg6, Jörg C. Gerlach7, Katrin Zeilinger2 and Tommy B. Andersson1

1AstraZeneca R&D Mölndal, Clinical Pharmacology and DMPK, Mölndal, Sweden, 2Div. of Experimental Surgery, BCRT, Charité Universitätsmedizin Berlin, Berlin, Germany, 3AstraZeneca R&D Lund, Global Safety Assessment, Lund, Sweden, 4Department of Surgery, Charité Universitätsmedizin Berlin, Berlin, Germany, 5Department of Traumatology, TU Munich, MRI, 6AstraZeneca R&D Södertälje, Global Safety Assessment, Södertälje, Sweden, 7McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA, USA

Drug metabolizing enzymes and drug transporters in the liver play a critical role in the clearance and drug-drug interactions of many drugs. Isolated hepatocytes used today represent a good model of liver metabolism because they are able to perform the full range of known in vivo biotransformation, synthetic and detoxification func-tions. However, hepatocytes rapidly lose their liver specific functions, when maintained under standard in vitro culture conditions. It has been shown that liver cells cultured in a perfused three-dimensional (3D) bioreactor retain in vivo like properties and form tissue like structures, enabling liver specific functionality to be extended over several weeks. Miniaturized bioreactor prototypes, based on a multi-compartment bioreactor technology for clinical bioartificial liver support, have been developed for preclinical drug testing. The device is based on the use of interwoven hollow fibre capillary membranes for independent medium and gas perfusion of the cells located in the extra-capillary space (cell compartment). Freshly isolated human hepatocytes were inoculated into two different bioreactor prototypes with a cell compartment volume of 2ml or 0.5ml. A cytochrome P450 (CYP) substrate cocktail including phenacetin, bupropion, midazolam and diclofenac was added to the circulating medium to evaluate the maintenance of the critical drug metabolizing enzymes CYP1A1/2, CYP2C9, CYP2B6 and CYP3A4 activities. All enzymes under investigation showed activity and the CYP activities were maintained over 12 days in both the 2 ml and the 0.5 ml bioreactor. In addition, CYP1A1/2, CYP2C9 and CYP3A4 showed activity up to 23 days in the 2 ml bioreactor. Moreover, the presence of markers for hepatocytes, biliary cells and nonparenchymal cells as well as hepatic transporter proteins in the cell aggregates was demonstrated by immunohistochemical staining. In conclusion, this study shows that liver cells cultured in a 3D bioreactor build up in vivo like structures and maintain liver specific functions for several weeks, including CYP activity. The bioreactor system allows repeated studies in the same experimental setup and could provide a useful instrument for in vitro drug metabolism studies.

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P245. Real Time PCR Analysis of Selected Oxidant/Antioxidant and Apoptotic Genes in D-Galactosamine-Lipopolysaccharide Hepatotoxicity in Rat

Natasa Lekic, Dalibor Cerny and Hassan Farghali

Pharmacology, Charles University in Prague, First Faculty of Medicine, Prague, Czech Republic

Hypothesis: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepa-totoxicity. The aim of this work is to find early signals of xenobiotic-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic gene expressions. As well, to address the relationship between conventional liver dysfunction markers and the measured genes in D-Galactosamine (D-GalN)/ Lipopolysaccharide (LPS) hepatotoxicity model that resembles clinical hepatitis.

Methods: Intraperitoneal administration of D-GalN 400 mg/kg and LPS 50 μg/kg in male Wistar rats induced hepato-toxicity. Following 24h, hepatocellular damage was evaluated by standard biochemical methods including ALT, AST, billirubin, thiobarbituric acid reacting substance (TBARS), catalase and conjugated dienes (CD) level measurements. The gene expressions of the antioxidant/oxidant enzymes - inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase 1 (SOD1), glutathione peroxidase (GPx1) and apoptotic genes- caspase 3 (CASP3), BH3 interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax) mRNAs were analyzed by RT-PCR. Activity of caspase 3 was measured using an assay kit.

Results: LPS/D-GalN combination produced fulminant hepatic injury as shown by increases in several hepatotoxicity biochemical markers (ALT, AST) and antioxidant status markers (catalase activity). However, there were no measur-able changes in GSH and TBARS. Heme oxygenase-1 and nitric oxide synthase-2 gene expressions were significantly increased, along with the levels of their products, billirubin and nitrite. The analysis of gene expression of antioxidant enzymes, glutathione peroxidase 1 and superoxide dismutase 1, remained unchanged and decreased respectively. Early activation of apoptosis was confirmed by the significant increase in the gene expression of apoptotic genes Bid, Bax and caspase 3, while the measured caspase 3 activity remained unchanged.

Conclusions: Overall, the results of this study have revealed the early activation of oxidative stress and apoptosis as seen by the relevant changes in apoptotic gene expressions, which also enabled us to address an intricate relationship between oxidative stress and apoptotic parameters. As well, the real time PCR analysis of certain genes combined with the conventional biochemical markers is a potentially useful tool in understanding various steps of xenobiotic induced fulminant hepatic injury with the potential of intervention. Supported by grants GAÈR 305/09/0004 and GAÈR 305/07/0001.

P246. Resveratrol Decreases Ethanol-Induced Oxidative STRESS IN Rat Liver

Yesim A. Gocmen1 and Saadet Gumuslu2

1Biochemistry Department, Bozok University, Medical Faculty, Yozgat, Turkey, 2Biochemistry Department, Akdeniz University, Medical Faculty, Antalya, Turkey

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status. Ethanol was administered to male Wistar rats for 20 days by ip injection. During this time, trans-resveratrol (20 mg/kg per day) was intraperitoneally (i.p.) administered and rats were sacrificed for the collection of liver and blood. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) were studied. Particularly the activities of hepatic GPx, GR and CAT were found reduced by ethanol treatment while SOD and GST activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic GSSG levels and ameliorated SOD, GPx, GR and and GST activities in the liver. Trans-resveratrol reduced the extent of GSSG production changes. In conclusion, we can propose that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by ethanol administration, which might be the basis of the hepatoprotective effect of trans-resveratrol.

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P247. The effect of multiple cryopreservation cycles on drug-metabolizing enzymes in human hepatocytes

Kevin Lyon, Chad Pope, Melissa Ostermann, Maciej Czerwinski and Andrew Parkinson

XenoTech, LLC, Lenexa, KS, USA

Cryopreserved hepatocytes provide a convenient in vitro test system to study both the phase 1 and phase 2 metabolism of new chemical entities; however freeze-thaw cycles can damage the cells. Cryoinjury is associated with rapid dehydration of cells (osmotic effects) and formation of intracellular ice during the freezing process, which in turn leads to a disrup-tion of cellular membranes, changes in protein conformation and nucleic acid damage (single and double DNA strand breaks). This study characterized the effects of multiple cryopreservation cycles on the activities of drug-metabolizing enzymes in single donor human hepatocytes or pooled hepatocytes from multiple donors. Hepatocytes isolated from four donors were cryopreserved once, twice or three times according to a stepwise protocol. Pooled hepatocytes (n=5) were prepared according to two protocols: (1) for the multiple-freeze pool, cryopreserved hepatocytes were thawed, combined and then cryopreserved for a second time; (2) for the single freeze pool (CryostaX™), cryopreserved pellets were combined without thawing or refreezing the cells. All cells were stored in the vapor phase of liquid nitrogen and were thawed under the same conditions. Viable hepatocytes were separated from non-viable cells by Percoll gradient centrifugation. The following enzyme activities were measured in situ: CYP3A4 (testosterone 6b-hydroxylation), CYP1A2 (phenacetin O-dealkylation), CYP2B6 (bupropion hydroxylation), FMO (benzydamine N-oxidation), UGT (4-methyl-umbelliferone and 7-hydroxycoumarin glucuronidation), and SULT (7-hydroxycoumarin sulfonation). Compared with individual lots of hepatocytes that were cryopreserved once, hepatocytes that were cryopreserved twice lost 33% of SULT, 18% of CYP3A4, 7-13% of UGT, and 6% of FMO activity. Hepatocytes that were cryopreserved three times lost even more enzyme activity (at least twice as much as the cells cryopreserved twice). Compared with pooled hepatocytes that were cryopreserved once (CryostaX™), pooled hepatocytes that were cryopreserved twice lost 33% of SULT, 30% of UGT, 24% of CYP1A2, 14% of FMO, 13% of CYP3A4/5, and 10% of CYP2B6 activity. In summary, multiple cryopreservation cycles have a deleterious effect on the activity of drug-metabolizing enzymes in human hepatocytes. In general, the loss of UGT and SULT activity was more extensive than that of CYP and FMO activity.

P248. Aldose reductase natural inhibitors from ethyl acetate extracts from southern Turkey

Melih Onay1, Nursen Çoruh2 and Belgin İşgör3

1Biochemistry, Middle East Technical University, Ankara, Turkey, 2Chemistry, Middle East Technical University, Ankara, Turkey, 3Materials Engineering, Atılım University, Ankara, Turkey

Aldose reductase is a key enzyme involved in diabetic patogenesis. Its presence can cause accumulation of sugar alcohol in eyes following the formation of cataract such as neuropathy and retinopathy in diabetes mellitus. Investigations have showed that cataract formation is inhibited by medicinal plants including flavonoids derivatives compounds. Glycyrrhiza glabra (GG) presents in Fabaceae family. GG is used in medicinal chemistry for the therapy of ulcer. Lavandula stoechas (LS) belongs to in family Lamiaceae and used as antispasmodic, antiseptic. Melissa officinalis (MO) exists in Lamiaceae family and MO is generally utilized in the nervous disorders. Ocimum basilicum (OB) appear in the Lamiaceae family and it is benefited in treatment of kidney malfunction. Tilia tomentosa (TT) is belongs to family Tiliaceae. TT is assigned for the treatment of flu and migraine. In the present study, ethyl acetate extracts of these plants often used as medicinal features and consumed hot drinks in southeastern Turkey have been studied for bovine lens aldose reductase inhibi-tion activities and have been investigated for antioxidant capacities with DPPH radical scavenging activity and total phenol contents. In comparing aldose reductase inhibitions, Inhibitory effects of G. glabra, L. stoechas, M. officinallis, O. basilicum, and T. tomentosa extracts were examined and 50% inhibitory concentrations (IC 50 ) were calculated as 41.55 ± 1.4, 50.04 ± 1.5, 50.32 ± 2.46, 82.96 ± 1.85 and 75.96 ± 3.51 µg/mL respectively. According to this, G. glabra is found more effective than other four plants as aldose reductase inhibitor. Also, fifty percent effective concentrations (EC 50) with DPPH radical scavenging activity were calculated in (µg/mL) and found as 80.63 ± 0.91, 413.55 ± 3.46for G. glabra and L. stoechas respectively. In addition,the highest amount of tatal phenolic content (TPC) was displayed by G. glabra as 44.54 ± 0.754 µg GAE/mg of extract. These results have exhibited that ethyl acetate extracts of G. glabra and L. stoechas can be pioneer for improvement of diabetic pathogenesis.

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P249. Dietary Aromatase Inhibitors in Human Non–Small Cell Lung Cancer

Alaattin Sen1, Hakan Akca2 and Gurbet Celik3

1Department of Biology, Faculty of Arts & Sciences, Pamukkale University, Kinikli, Turkey,2Medical School,, Pamukkale University, Denizli, Turkey, 3Biology, Pamukkale University, Denizli, Turkey

Recent studies have implicated that estrogen and estrogen receptors play essential role in some lung cancers progres-sion. It has been demonstrated that this steroidal growth-stimulatory effect may be promoted by tumor expression and activity of aromatase (CYP19), an estrogen synthase, in some lung cancers. Therefore, in this study we have investigated the effect of extracts obtained from well-known vegetable diets consumed commonly in Mediterranean and Aegean regions of Turkey on the expression of aromatase enzyme in human non–small cell lung cancer (NSCLC) cells- PC14 and PC3 cell lines. For this purpose, the expression of aromatase activity, protein and mRNA levels were determined after incubating the cell lines with dietary extracts at varying concentrations for 72 hours and are compared with control levels. Aromatase activity was determined flourometrically by using dibenzylfluorescein as a substrate. The expression of aromatase at protein and mRNA levels was detected by Western blotting using anti-hCYP19 and RT-PCR using suitable primers, respectively. Extracts obtained from Allium porrum, Mentha piperita, Lepidium sativu, Laurus nobilis, and Tamus communis reduced aromatase activity 52%, 44%, 33%, 32% and 58% respectively; without significant changes in protein and mRNA levels in PC14 and/or PC3 cell lines. The results obtained in this study have shown that Allium por-rum, Mentha piperita, Lepidium sativu, Laurus nobilis, and Tamus communis do contain effective aromatase inhibitors and consumption of these foods would be protective in preventing the risk of cancers and/or may present alternative options for patients afflicted with lung cancers. Further advanced and sophisticated scientific studies should be car-ried out to identify the individual compounds responsible for these effects and to elucidate the molecular mechanism behind these actions.

P250. Dimethyl-Diphenyl-Bicarboxylate (DDB) as Antiapoptotic Agent Targeting Death Ligand and Caspases Signaling Pathway in a Model of Hepatic Ischemia Reperfusion Injury

Alshaymaa Amin El-Bahy1, Lobna A. Kassem2, Ola A. Heikal1 and Laila G. Mahran1

Pharmacology and toxicology, The German university in Cairo, Cairo, Egypt 1Pharmacology & Toxicology Department & 2Physiology Department Faculty of Pharmacy & Biotechnology, GUC, Cairo, Egypt

Background and aim: Dimethyl-Diphenyl-Bicarboxylate (DDB) is a hepatoprotectant used in treatment of chronic viral hepatitis and other liver diseases; it′s a synthesized derivative of Schizandrin C, active component of tra-ditional Chinese herb Fructus Schizandrae. However, the mechanism of DDB against liver injury needs further elucidation. Our objective is to explore whether DDB exerts hepatoprotective effect against hepatic ischemia reperfusion injury (I/R) and to identify its potential effect on apoptotic cell death targeting the extrinsic apoptotic pathway.

Methods: Rats were divided into four groups: Group I (sham-operated), Group II (I/R group) upon which I/R was per-formed, Group III (Pretreated groups): where rats were treated with DDB at 3 dose levels (100mg/kg, 300mg/kg, 500mg/kg) for 10 days, then subjected to I/R and Group IV (treated group): rats were treated with DDB at the same dose levels but without being subjected to I/R. The following parameters were measured: serum ALT & AST, tissue levels of GSH, MDA & TNF-α, activity of signaling Caspases 8 & 9 and effector Caspase 3 as well as expression of nuclear factor-κB. End point of apoptosis was detected by DNA fragmentation.

Results: DDB administration before I/R, significantly attenuated the elevated levels of ALT, AST, MDA and TNF-a. It also significantly increased the GSH content. Hepatic apoptosis was inhibited as evidenced by significant decrease in all caspases activities & expression of nuclear factor-κB as well as by DNA fragmentation. DDB administration to normal rats, inhibited lipid peroxidation, enhanced the antioxidant activity, decreased the TNF-a and downregulated the expression of nuclear factor-κB. These effects are dose-dependent. Conclusion: DDB exerts potent antioxidant effect in both normal and injured livers. DDB inhibited hepatic I/R induced apoptosis through inhibiting signaling Caspases 8, 9 and 3, inhibiting TNF-a production which regulates the extrinsic apoptotic pathway and down regulates NF-κB expression in liver tissue.

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P251. Effects of Avaricon® on High Fat Diet-induced Hepatosteatosis, Oxidative Stress and Lipid Profile in Rats

Jelena Calasan1, Vesna Mijatovic1, Biljana Bozin2, Isidora Samojlik1 and Olga Horvat1

1Department of pharmacology, toxicology and clinical pharmacology, Faculty of Medicine, Novi Sad, Serbia and Montenegro, 2Department of pharmacy, Faculty of Medicine, Novi Sad, Serbia and Montenegro

Troxerutin, a dominant component of drug Avaricon®, is a flavonoid naturally present in tea, coffee, cereal grains and a variety of fruits and vegetables and is known to have broad pharmacological activities, and therefore to reduce risk of various diseases.This study was carried out to investigate the protective effect of troxerutin, against diet-induced obesity and its consequences (hepatosteatosis, oxidative stress and lipid abnormalities). The examination was performed on 36 female Wistar rats (183-265 g) divided randomly into 6 groups and treated for 6 weeks as follows: group 1 (ND) was given normal diet (laboratory rodent chows), group 2 (HFDoil) high fat diet - laboratory rodent chows prepared with sunflower oil, group 3 (HFDfat) - laboratory rodent chows prepared with animal fat; other three groups were given the same diet as previous, but they were perorally pretreated with Avaricon® (3.16 mg/100gBM/day). Body weights were measured daily, while weights of organs were measured after animals sacrificing. The activity of liver enzymes (glutathione per-oxidase - GSH-Px, peroxidase - Px, catalase - CAT, xanthine oxidase - XOD) and lipid peroxidation - LP were measured in the liver homogenate. Serum biochemical parameters (triglyceride - TAG, total cholesterol, LDL, VLDL and HDL cholesterol, aspartate-aminotransferase - AST, alanine-aminotransferase - ALT, gamma-glutamyltransferase - GGT) were also determined. The results showed that the body weight and weight of the liver in the HFD+Avaricon® groups did not significantly differ as compared to the HFD group. The consumption of Avaricon® reduced oxidative stress enhancing the levels of CAT and XOD in the hepatic tissue of HFDoil rats. Significantly increased GSH-Px activity between control ND and Avaricon® treated group and a significantly increased Px activity between all control and Avaricon® treated groups were observed. Avaricon® showed protective effect in group with ND as well as in HFDoil group, where statisti-cally significant (p<0.05) decrease of the liver LP activity was determined. Moreover, serum LDL-cholesterol levels in the HFDoil+Avaricon® group were significantly decreased as compared to those in the HFDoil group. Contrary to the expectations, serum TAG and VLDL levels in the HFDfat+Avaricon® group were significantly higher than those in the HFDfat group. There was a significant difference in serum GGT between HFDoil and HFDoil+Avaricon® group. Other biochemical parameters were not changed. These results demonstrate that intake of Avaricon® can be beneficial for the suppression of HFD-induced dyslipidaemia (especially caused by consumption of sunflower oil), hepatosteatosis and oxidative stress in rats.

P252. Evaluation of selected herbal remedies sold in Libyan market for treatment of diabetes

Mukhtar R. Haman

Pharmacognosy, Faculty of Pharmacy, AlFateh University, Tripoli, Libya

Interest in traditional system of medicine, in a particular, herbal medicine, has increased substantially in both devel-oped and developing countries over the last two decades. Some reported adverse events following the use of certain herbal medicines have been associated with a variety of explanations. Safety and quality measures are needed to overcome these problems and to ensure steady, affordable and sustainable supply of medicinal plant materials of good quality. Three samples (Laxative, Ant diabetes and Aphrodiac) been purchased from the market (herbal shop) [Attar].

Sample A: Claimed use: Laxative. Herbal ingredients: 1- Carum carvi. 2- Foeniculum vulgare. 3- Mentha piperita. 4- Cassia obovate.

The marketed preparation was contaminated by fungi and Hg. –Organoleptic evaluation revealed that it has characterestic odour of ingredients and green colour. –

Sample B: Claimed use: for diabetes. Herbal ingredients: 1- Rhubarb. 2- Laurel paper. 3- Lupine. 4- Garden cress. 5- Halayeb dates. 6- Zarirah cane. 7- Colocynth seed. 8- Sar seed. 9-Shorngan. 10- Looba morra. 11- Samouh.

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Heavy metals were within the limit, but there was fungal and bacterial ontaminations. –Organoleptic evaluation revealed the characteristic odour of foenugreek which is not mentioned as one of herbal –ingredients of the sample. Chromatography (TLC.) revealed the presence of glibenclamide (inferior adultration). –

Sample C: Claimed use: Aphrodiac. Herbal ingredients: 1-Capsicum annum. 2- Cinnamomum aromaticum. 3- Zingeber officinalis. 4- Panax ginseng. 5-Ferula hermonis.

Safety study indicates that Arsenic is above the limit, and Mercury also & there was a fungal & bacterial –contamination. Organoleptic evaluation revealed that the colour was green although the parts used are roots, barks & fruits. –The sample has the charecteristic odour of fennel. –

P253. Ginger consitutents modulate function and expression of p-gp and bcrp

Sabrina Nickel, Anne Mahringer, Gert Fricker and Valeska Reichel

Department of Pharmaceutical Technology and Biopharmacy, Heidelberg University, Heidelberg, Germany

Constituents of many foods like turmeric or grapefruit are known to cause undesireable effects on the bioavailability of many drugs like Anticancerdrugs or Antibiotics by modulating the function or expression of ABC-transporter pro-teins [1,2]. Ginger is often used as a ntural product against nausea and enjoys enormous popularity as food ingredient [3]. The potential of ginger ingredients 6-Gingerol, 8-Gingerol, 10-Gingerol, 6-Shogaol, 8-Shogaol to interact with the ABC-transport proteins p-glycoprotein (ABCB1) and BCRP (ABCG2) was investigated. The potential of ginger ingredi-ents to interact with the ABC-transport proteins p-glycoprotein (ABCB1) and BCRP (ABCG2) was studied in porcine brain capillary endothelial cells (PBCECs) and transfected MDCK-II cells, overexpressing human BCRP. Therefore, fluorescence based assays were applied, namely Calcein-assay for p-glycoprotein and pheophorbide export for BCRP. A potential induction of the p-glycoprotein was studied by long-term incubation of the PBCECs, subsequent Microarray and Western-Blotting. Regulatory effects of the ginger constituents on the expression of BCRP was tested by Western-Blotting of the export pump in MDCK-cells. The data suggest that ginger constituents interfere with ABC-transport proteins p-glycoprotein and BCRP, and therefore ginger products may exhibit a certain risk, when given concomitantly with drugs which are substrates of the studied ABC transport proteins.

P254. Living knowledge of the healing plants: ethno-phytotherapy in the Local people, Santal and Orao ethnic groups from the Rangpur district of Bangladesh

Md. Ariful Haque Mollik

Epidemiology, Biostatistics, Community Nutrition and Noncommunicable Diseases, Peoples Integrated Alliance, Bogra, Bangladesh

Every-year, the people of Rangpur district is subjected to infectious diseases like cholera, dysentery, diarrhea, mea-sles, diphtheria, pertussis, tetanus, poliomyelitis; and parasitic diseases such as malaria, filariasis, and helminthiasis were responsible for widespread illness and numerous deaths. On the other hand; noncommunicable diseases such as diabetes mellitus, cardiovascular diseases, mental illness, gastrointestinal disorders, cancer, rheumatoid arthri-tis, respiratory diseases, and urogenital diseases were increasing in frequency in the 1980s. Local people and two important ethnic groups are live here called Santal and Orao. In absence of effective modern medications, the Local people rely mostly on treatment with plants administered by the folk medicinal practitioners of Rangpur district. In the same way, the Santal and Orao ethnic groups have their own medicinal practitioners, who usually administered plant formulations for treatment of the above-mentioned diseases. The objective of this ethnopharmacological survey was to document the plants that the folk medicinal practitioners, Santal and Orao ethnic group′s medicinal practi-tioners use for treatment of the above-mentioned diseases. Ethnopharmacological data were gathered interviewing by the folk medicinal practitioners, Santal and Orao ethnic group′s medicinal practitioners using a semi-structured questionnaire. Details pertaining to the preparation and administration of plant drugs are provided. Plant samples

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were collected and identified at the Bangladesh National Herbarium. The plant names (with family name given in parenthesis) obtained in this ethnopharmacological survey included Andrographis paniculata (Acanthaceae), Smilax zeylanica (Smilacaceae), Clerodendrum indicum (Verbenaceae), Piper cubeba (Piperaceae), Ficus racemosa (Moraceae), Plumbago indica (Plumbaginaceae), Vitex negundo (Lamiaceae), Justicia adhatoda (Acanthaceae), Leucas aspera (Lamiaceae), Morus alba (Moraceae), Mimosa pudica (Leguminosae), Hedyotis corymbosa (Rubiaceae), Terminalia belerica (Combretaceae), Bombax ceiba (Malvaceae), Piper nigrum (Piperaceae), Ricinus communis (Euphorbiaceae), Abrus precatorius (Leguminosae), Curculigo orchioides (Amaryllidaceae), Costus speciosus (Costaceae), Aloe vera (Asphodelaceae), Kalanchoe pinnata (Crassulaceae), Aphanamixis polystachya (Meliaceae), Terminalia arjuna (Combretaceae), Achyranthes aspera (Amaranthaceae), Terminalia chebula (Combretaceae), Rauwolfia serpentina (Apocynaceae), Zingiber officinale (Zingiberaceae), Swertia chirata (Gentianaceae), Morinda citrifolia (Rubiaceae), Cajanus cajan (Leguminosae), Withania somnifera (Solanaceae), Cassia occidentalis (Leguminosae), Cissus quadran-gularis (Vitaceae), Wedelia chinensis (Asteraceae), Bacopa monnieri (Plantaginaceae), Syzygium cumini (Myrtaceae), Heliotropium indicum (Boraginaceae), Cinnamomum camphora (Lauraceae), Scoparia dulcis (Scrophulariaceae), Cynodon dactylon (Gramineae), Curcuma longa (Zingiberaceae), Aegle marmelos (Rutaceae), Amaranthus spinosus (Amaranthaceae), Oxalis lobata (Oxalidaceae), Aristolochia indica (Aristolochiaceae), Datura metel (Solanaceae), Azadirachta indica (Meliaceae), Ocimum tenuiflorum (Lamiaceae), Asparagus racemosus (Asparagaceae), Coccinia cordifolia (Cucurbitaceae), Phyllanthus emblica (Phyllanthaceae), and Paederia foetida (Rubiaceae). The present investi-gation also indicated that most of the preparations made for curing of the above-mentioned diseases were derived from single plant sources. Since the Local people, Santal and Orao ethnic groups of Rangpur district in Bangladesh mostly does not have access to primary medical facilities; these plants can form the basis of treatment for the above-mentioned diseases without resorting to costly urban visits or allopathic medicinal practitioners. Information on indigenous use of plants has led to discovery of many medicines in use today. Scientific studies conducted on the above-plants may lead to discovery of more effective drugs than in use at present.

P255. Natural resveratrol analogs arachidin-1, arachidin-3 and piceatannol bind human and mice cannabinoid receptors CB1 and CB2

Paul L. Prather1, Lisa K. Cortez1, Luis Nopo-Olazabal2, Vipin Nair2, Fabricio Medina-Bolivar2 and Anna Radominska-Pandya3

1Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA, 2Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA, 3Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

Resveratrol (tRes) and other polyphenols produced in plants show biological properties by acting on multiple molecular targets, resulting in protective effects against cardiovascular disease, ageing, and cancer. Due to similar antioxidant and anti-inflammatory actions of tRes and cannabinoids, we theorized that tRes and structurally related analogs might bind to cannabinoid receptors. This hypothesis was tested by employing competition receptor binding experiments. Here we report that tRes, two natural prenylated analogs, trans-Arachidin-1 and -3 (tA1, tA3) extracted from peanut hairy root cultures, and trans-Piceatannol (tPice), a hydroxylated analog of tRes, selectively bind human CB1 and mouse CB2 can-nabinoid receptors. tRes and all analogs bind to CB1Rs expressed in mouse brain with an affinity (K

i) in the low 5-20 µM

range and a rank order of tA1 > tPice > tRes = tA3. Interestingly, while tA1 and tA3 also bind to CB2Rs with similar affinity, the affinity of tRes and tPice for CB2Rs is 6 to 10-fold lower than that observed for CB1Rs. In addition, all compounds except for tA3 exhibit approximately 2-10-fold selective binding to CB1Rs relative to CB2Rs. Based on these observations, it appears that the addition of the isoprenyl group, found only in tA1 and tA3, significantly enhances the binding affinity of these analogs for CB2Rs (∼6-10-fold). Furthermore, combination of the isoprenyl group and an additional hydroxyl group in tA1 apparently also modestly enhances the binding affinity of this analog for CB1Rs (∼2-fold). Preliminary experiments with the glucuronide metabolites of tRes found in humans, tRes-3- and -4′-O-glucuronide, show that these compounds do not bind CB2Rs in a concentration up to 100 µM. In summary, these studies clearly establish that tRes and several structurally related analogs bind to both receptors with a low µM affinity. Comparable µM levels appear to be required to produce most of the in vitro protective effects observed for tRes. Based on this data, we hypothesize that CB1Rs and CB2R play an important role in the molecular mechanism of action for tRes, but not for its predominant glucuronide metabolite. Most importantly, observations that minor modifications to the basic tRes structure result in marked alterations in the affinity of various analogs for CBRs, indicate that tRes likely represents a useful scaffold for

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future design of highly selective and efficacious CB1R and CB2R ligands for use, as therapeutics to treat obesity and/or drug dependence. (NIH-GM075893 to AR-P; NSF-EPSCoR EPS-0701890 to FM-B)

P256. Phenethyl Isothiocyanate and Sulforaphane Mediated Apoptotic Cell Death in Human Prostate Cancer Cells Is Initiated by Mitochondria Generated Reactive Oxygen Species

Shivendra Vikram Singh, Dong Xiao, Anna A Powolny, Su-Hyeong Kim and Eun-Ryeong Hahm

Dept of Pharmacology, Univ of Pittsburgh Cancer Inst, Pittsburgh, PA, USA

Cruciferous vegetable-derived isothiocyanates [e.g., phenethyl isothiocyanate (PEITC) and sulforaphane (SFN)] not only afford significant protection against chemically-induced cancer in experimental rodents, but also inhibit growth of human cancer cells in association with induction of apoptosis. However, the mechanism by which PEITC and SFN cause cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we demonstrate that the PEITC- and SFN-induced cell death is initiated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiratory chain (MRC). Exposure of prostate cancer cells to pharmacologic con-centrations of PEITC resulted in ROS production, which was accompanied by inhibition of MRC complex III activity. SFN-mediated ROS production correlated with inhibition of complex I, II and III activities. The Rho-0 variants of LNCaP and PC-3 cells lacking normal oxidative phosphorylation were significantly more resistant to PEITC- and SFN-mediated ROS generation, apoptotic DNA fragmentation, caspase-3 activation, and/or collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, Bax and Bak knockdown conferred significant protection against PEITC-induced apoptosis. In conclusion, the present study provides concrete experimental evidence to implicate ROS in initiation of PEITC- and SFN-induced apoptosis, at least in human prostate cancer cells. Clinical implication of these findings is that cancer chemopreventive efficacy of PEITC and SFN may be compromised in the presence of antioxidants. This investigation was supported in part by the National Cancer Institute grants CA101753-06 and CA115498-05.

P257. Plasma Pharmacokinetics and Urinary Excretion of Ginsenosides after Oral Administration of Panax notoginseng Extract to Human Subjects

Feifei Du1, Fang Xu2 and Chuan Li3

1Dept of DMP, Shanghai Institute of Materia Medica, Shanghai, China, 2Dept of DMPK, Shanghai Institute of Materia Medica, Shanghai, China, 3Dept of DMPK, Shanghai Instiyute of Materia Medica, Shanghai, China

Background and aim: Ginsenosides are believed responsible for most of therapeutic effects of Panax notoginseng (Sanqi), a Chinese cardiovascular herb. Our earlier studies in rats indicated biliary excretion being the predominant route for eliminating most Sanqi ginsenosides and their deglycosylated metabolites, resulting in low systemic exposure to the saponins. However, biliary excretion of drugs could vary between rats and humans. For this reason, we examined plasma pharmacokinetics and renal excretion of ginsenosides from orally administered Sanqi extract in humans.

Methods: Serial plasma and urinary samples were collected from five healthy male human volunteers who received a single p.o. dose of Sanqi extract at 35 mL/subject (17.5 g of Sanqi yielding 35 mL of extract). The samples were analyzed for the ginsenosides and metabolites using liquid-chromatographic mass spectrometry.

Results: Ginsenosides Ra3, Rb

1, Rd, F

2, 20-gluco-Rf, Re, Rg

1, and notoginsenoside R

1 were detected in plasma follow-

ing ingestion of Sanchi extract, which were also found in urine, except for ginsenosides F2. Compound-K was a major

metabolite measured in plasma and urine. The levels of major plasma ginsenosides Ra3, Rb

1, and Rd declined more

slowly than that of ginsenoside Rg1, and significant differences also occurred in their systemic exposure levels. Renal

excretion of ginsenoside Rg1 and notoginsenoside R

1 appeared to be associated with active tubular secretion.

Conclusion: The PK profiles of the Sanqi ginsenosides in humans were comparable to those found in rats. Slight interspecies differences could have been due to humans being relatively poor biliary excretors compared with rats.

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References

Liu H-F., Yang J-L., Du F-F., Gao X-M., Ma X-T., Huang Y-H., Xu F., Niu W., Wang F-Q., Mao Y., et al. (2009) Absorption and disposition of ginseno-sides after oral administration of Panax notoginseng extract to rats. Drug Metab. Dispos. 37:2290-C2298.

P258. Prooxidant and Antioxidant Effects of Three Dietary Flavonoids and Their Mixtures

Burcu Eren and Hande Gurer-Orhan

Toxicology, Ege University, Faculty of Pharmacy, Izmir, Turkey

Dietary flavonoids have gained a reputation for being antioxidants and beneficial on breast cancer, prostate cancer, menopausal symptoms, osteoporosis and cardiovascular disease. On the other hand several studies have demonstrated that flavonoids exert biphasic effects; they can also act as prooxidant and procarcinogen. In the present study, in vitro antioxidant and/or prooxidant capacity of three dietary flavonoids; genistein, daidzein and quercetin was evaluated by the examination of their ability to produce/inhibit reactive oxygen species (ROS) formation by using a fluorescent probe, 2′,7′-dichlorofluorescein. Individually genistein and daidzein both induced formation of ROS in red blood cells in a concentration dependent manner in the presence and absence of “transient metals and H2O2” while they inhibited ROS formation in a microsome model where their metabolites were formed. The combinational effect of two flavonoids had a similar effect pattern. However, both effects in both models were observed at high concentrations of flavonoids that were not physiologically relevant. Quercetin showed significant antioxidant effects even at its lowest concentrations (1µM) in both RBCs and microsomes and in every condition tested (in the presence and absence of “transition metals and H2O2”). Another major finding of the present study is that the prooxidant effect of genistein and daidzein were suppressed by quercetin in a mixture of three at physiologically relevant concentrations. A total antioxidant effect was observed with the mixture of three both in RBCs and microsomes. Similar prooxidant effects of genistein and daidzein which were prevented by quercetin in a mixture of three was also observed in an oxidative hemolysis model initiated by a water-soluble free radical initiator 2,2 -azo(2-asmidinopropane)dihydrochloride (AAPH). Our study indicated that flavonoids can act as a prooxidant as well as an antioxidant depending on their concentrations and the incubation conditions. The present study also points to the importance of considering the mixture effect while evaluating possible risks of dietary flavonoids.

P259. Can silymarin prevent cardio and hepatotoxicity induced with doxorubicin?

Nebojsa Stilinovic, Roberto Sederino, Aleksandar Raskovic, Sasa Vukmirovic and Milica Paut

Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine Novi Sad, Novi Sad, Serbia and Montenegro

The main reason of the cardio and hepatotoxicity of doxorubicin is the production of free oxygen radicals, lipid peroxida-tion and DNA damage of these tissues. The aim of the investigation was to examine effects of the interaction of silymarin and doxorubicin on the electrophysiological changes on the myocard after continous infusion of lidocaine, on the liverhomogenate level and activity of parameters, which indicate oxidative stress, followed by histological evaluation of the heart and the liver. The first control group was treated with physiological, sodium-chloride solution, the second one was threated with olive oil. The experimental groups where treated with silymarin once per day, doxorubicin every second day and with the combination of silymarin and doxorubicin. After the treatment, the animals were anaesthetized with urethane and prepared jugular vein was connected to the infusion pump with lidocaine in the course of recording ECG until the ECG analyses encompassed toxic effect. Then animals were sacrificed by cardiopunction and the heart was taken for histological examination. After abdominal section rat livers were taken for biochemical and for histological examination. The results showed that the treatment with doxorubicin causes statistically significant increase of lidocaine dose necessary to produce toxic effect (26,33:2,65, p<0.01) in comparison to the control groups, the group treated with silymarin and the group treated with the combination of doxorubicin and silymarin. Observing the values of the parameters, which indicate the intensity of lipid peroxidation, we could see that the treatment with silymarin leads to a significant decrease of activity of prooxidative parameter (lipid peroxidation, 2,25:0,28 p<0,005) and a decrease of activity of catalase (5,98:0,40, p<0,05) and glutathion peroxidase (5,30:0,45, p<0,005). The levels of reduced glutathione

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were almost unchanged in all groups. The results of the histological examination show no pathological signs in any group. Based on the results it can be concluded that silymarin prevents electrophysiological changes on the myocard, induced by doxorubicin, after continuous infusion of lidocaine. Prooxidative effects of doxorubicin are diminished by the treatment with silymarin. The treatmant with doxorubicine (1,66 mg/kg) leads not to pathohistological changes in the heart and liver tissues.

P260. Deoxyschizandrin improved the absorption of P-glycosprotein (P-gp) Substrate Drugs in Human Intestinal Caco-2 Cells

Ying Hu, Hui Xin Liu, Na Li and Ling Yang

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Schisandra fruits are extensively used as traditional medicine in China, Japan and Korea. Deoxyschizandrin(DSZ), the major active components of S. chinensis, have been reported to show anti-inflammatory, anti-cancer, and anti-hepatocarcinogenesis activity. Recently, schizandrin B was reported to have an inhibitory effect on P-glycoprotein (P-gp), a drug efflux pump. The report suggested that lignans from S. chinensis can influence intestinal absorption of drugs that are P-gp substrates. In present study, we investigate the permeability of DSZ and its effect on P-gp mediated transport of rhodamine 123 (R123) and digoxin in Caco-2 cell monolayer. Firstly, we employed Sulforhodamine B (SRB) cytotoxicity experiment to investigated cytotoxicity of DSZ to Caco-2 cell. Based on the cytotoxicity assay results, DSZ did not decrease the viability of Caco-2 cells significantly when its concentration up to 200μM, indicating that DSZ exhibit high safety. In the study on the permeability of DSZ, DSZ showed moderate permeability and its passive permeability (PappA-B) is 5.6 × 10−6 cm/s, greater than 2 × 10−6 cm/s. Subsequently, bidirectional transports of R123 and digoxin in Caco-2 were determined with DSZ. In the bidirectional transport study, DSZ increased R123 and digoxin absorptive transport and reduced their excretive transport in a time dependent manner. At the presence of 50 μM DSZ, the transport rations (B-A/A-B) for R123 and digoxin were 1.2 and 1.1 compared the control ratios of 4.8 and 4.5. In conclusion, DSZ significantly increased the transport of P-gp substrate drugs (R123 and digoxin) by inhibiting the P-gp-mediated efflux. The results suggested that DSZ could potentially increase the absorption of drugs that can act as a P-gp substrate. Therefore, due to its high safety and good permeability property, intake of DSZ as a health supplement or herbal medicine may increase the intestinal absorption of bioavailability of co-administered drugs that are P-gp substrates. Simultaneously, DSZ could be a good candidate for absorption enhancers of P-gp substrate drugs.

P261. Differential Effects of Cyclamen Extract on P450 Gene Expression

Sevki Arslan1, Ozden Ozgun2 and Alaattin Sen3

1Department of Biology, Pamukkale University, Faculty of Arts & Sciences, Denizli, Turkey, 2Biology, Pamukkale University, Denizli, Turkey, 3Department of Biology, Faculty of Arts & Sciences, Pamukkale University, Kinikli, Turkey

Cyclamen trochopterantum is one of the endemic species of cyclamens that grows naturally in the south-western part of Turkey. It is used as an ethno-medicine for treatment of hemorrhoids, infertility and eczema and expelling digestive tract worms. It is well established that extracts prepared from different cyclamen species show interesting spermicidal, anti-microbial, anti-inflammatory and anti-nociceptive activities. Although their wide usage and diverse biological activities, there is no available information about the effect of cyclamens on carcinogens and other xenobiotics metabo-lism and possible drug interaction potential. In this respect; the aim of this study is to determine the effect of ethanol extracts of cyclamen′s tubers on hepatic CYP450 gene expression. For this purpose, Male Wistar rats were treated with four different dose of cyclamen tuberose extract for 10 consecutive days. The mRNA levels of hepatic CYP1A2, CYP2E1, CYP2C6, and CYP3A1 were determined by RT-PCR. Relative CYP2E1 mRNA level was increased up to 8.8-fold in cycla-men treated rats compared to control animals (p<0.05). Similarly, CYP1A2 and CYP2C6 mRNA levels were increased significantly in between 1.50-to10.65-fold as a result of cyclamen (p<0.05). On the other hand, cyclamen treatment was not changed relative CYP3A1 mRNA level with respect to control rats. In the light of the present study, it is expected that people using this plant as a herbal remedy may have increased risk of carcinogenesis due to co-exposure of chemicals such as nitrosamines, aromatic amines and benzo(a)pyrene may be increased because of induction of CYP1A1/2 and

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CYP2E1 enzymes. Moreover, alteration of drug clearance and clinical drug toxicity due to induction of CYP2C6 may be observed in the people used this plant as a medicine. In conclusion, the induction of cytochrome P450 enzymes by cyclamen may cause not only potential and documented impairment of drug clearance but also carcinogenesis, tissue necrosis and toxicity.

P262. Effect of Naringenin on Metabolic Activity of Cytochrome P450 1a2 and 3a in BCG - Immune Liver Injury in Mice

Guo-Liang Zhang, Tao-Yuan Li, Xin Wang, Shu-Mei Wang, Dan Li and Xuan Zhao

Department of Pharmacology, Basic Medical School, Beijing University, Beijing, China

Background and Objective: Naringenin is a natural flavonoid and widely exist in citrus fruits such as grapefruit. It has been known that naringenin was one of inhibitors for cytochrome P450 (CYP450) metabolic enzymes in vitro and in vivo. Moreover, it was also reported that reactive oxidation species were produced during the CYP450 catalyzed substrate oxidation, which consequently may account for hepatocyte and tissue injury. However, whether and how naringenin involved in the metabolic activity of CYP450 in liver damage was still unknown. In the present study the effect of naringenin on the formation of hepatic oxidative stress elements and cytochrome P450 metabolic activity were investigated in BCG-induced immune liver injury in mice.

Methods: The immune liver injury was induced by intravenous injection of Bacillus Calmette Guérin(BCG 125mg/kg,2 weeks) in BALB/c mice. Naringenin (Nar 50,100,150mg/kg) was administered by gavage during the succedent one week. After 2 weeks of BCG injection, hepatic microsomes were prepared by ultrahigh speed centrifugation and total P450 content was determined by spectrophotography. Serum alanine aminotransferase(ALT) level, liver microsome superoxide dismutase(SOD) were evaluated by colorimetric method. The metabolic activities of cyp3a and cyp1a2 were assessed by the oxidation of probe substrate nifedipine and phenacetin in vitro using high performance liquid chromatography (HPLC) method.

Results: BCG pretreatment increase relative liver weight (P<0.01) and relative speen weight (P<0.01), serum ALT levels (P<0.01), but liver microsome SOD activities were declined. Under the BCG-immune stimuli condition, total P450 content was significantly decreased(P<0.01), and the metabolic activity of cyp3a (P<0.05) and cyp1a2 (P<0.05) were also significantly decreased. Oral administration of naringenin (50,100,150mg/kg) decreased the elevated level of ALT and the high dose (150mg/kg) of naringenin increased the suppressed activity of SOD in vivo. Moreover, naringenin reversed the decrease of cyp3a and cyp1a2 metabolic activity induced by BCG-stimulus. However, administration of naringenin alone suppressed the metabolic activity of cyp3a and cyp1a2 in mice microsome in vitro.

Conclusion: The present results showd that naringenin improved BCG-induced immune liver injury, which might be due to antioxidation by enhancing SOD activity in mice in vivo. Moreover, naringenin inhibited metabolic activities of cyp3a and cyp1a2 in mice hepatic microsomes in vitro. These results suggested that the affects of naringenin on CYP450 oxidative metabolism might participate in the hepatoprotection, but the underlying mechanism needs to be further investigated.

P263. Local administration of medicinal plants in treating neurogastroenetrology & motility disorders: results from a cross-sectional study in Khulna district of Bangladesh

Md. Ariful Haque Mollik

Epidemiology, Biostatistics, Community Nutrition and Noncommunicable Diseases, Peoples Integrated Alliance, Bogra, Bangladesh

Neurogastroenetrology & motility disorders encompass a broad spectrum of problems that includes gastroesophageal reflux disease, non-cardiac chest pain, functional dyspepsia, irritable bowel syndrome, constipation, and obesity. These ailments are spread world-wide and particularly in the developed countries. Allopathic medicine has no known rem-edy for cure of neurogastroenetrology & motility disorders but merely serves to control secondary symptoms. On the other hand, traditional health practitioners claim to treat neurogastroenetrology & motility disorders with medicinal plants resulting in complete cure. The traditional health practitioners rely on decoctions made from medicinal plants

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or plant parts to treat neurogastroenetrology & motility disorders, which decoctions can vary widely between tradi-tional health practitioners in the various districts of the country. The objective of this present study was to conduct a systematic identification of medicinal plants used to treat neurogastroenetrology & motility disorders of people in Khulna district, Bangladesh which area because of its proximity to the Sunderbans forest region contains a diversity of medicinal plants. Semi-structured questionnaires were administered to twenty-four traditional health practitioners to evaluate the traditional health practitioners′ perceptions and practice relating to causation and treatment of one or more neurogastroenetrology & motility disorders related infections. Medicinal plant specimens were photographed and brought to Bangladesh National Herbarium for identification. The collected information indicates that the follow-ing medicinal plants are used to treat neurogastroenetrology & motility disorders: Terminalia arjuna, Lepidagathis hyalina, Abrus precatorius, Nigella sativa, Aconitum napellus, Olea europaea, Plantago ovata, Heritiera fomes, Calamus rotang, Smilax zeylanica, Amaranthus gangeticus, Calotropis procera, Zingiber officinale, Lawsonia inermis, Aloe vera, Piper cubeba, Ficus religiosa, Centella asiatica, Datura metel, Aegle marmelos, Vitex negundo, Ricinus communis, Cocos nucifera, Kalanchoe pinnata, Ocimum sanctum, Withania somnifera, Azadirachta indica, Curcuma longa, Dillenia indica, Bacopa monnieri, Swertia chirata, Piper nigrum, and Ceriops tagal. Information on traditional medicinal uses of mangrove medicinal plants is scant in the scientific literature. From that view point, it is interesting to know that two true mangrove medicinal plants like Ceriops tagal and Heritiera fomes are used by the traditional health practitioners of Khulna district to treat difficult to cure diseases like neurogastroenetrology & motility disorders. Since allopathic medicine does not have a cure for neurogastroenetrology & motility disorders, the above-mentioned medicinal plants are potentially interesting for discovery of novel compounds, which can be effective in controlling or curing neurogas-troenetrology & motility disorders.

P264. Mechanistic study on the metabolism inhibition of Oseltamivir by herbal components

Xiaoan Wang, Siukwan Wo and Zhong Zuo

School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, N/A

Objectives: As the pro-drug of a neuraminidase inhibitor Oseltamivir Carboxylate (OC), Oseltamivir (O) is currently recommended for the prophylaxis and treatment of avian influenza together with Chinese Medicine Formulae (CMF) in Hong Kong. Our previous animal studies observed the pharmacokinetic herb-drug interactions between O and Yin Qiao San/Sang Ju Yin (CMF1) with a significant decrease in OC formation in vivo. The current study aims to provide the mechanistic explanations for the inhibition of metabolism of O by coadministered herbal components from CMF1.

Methods: Metabolism of O in rat GI mucosa, rat plasma, rat liver microsome (RLM), and recombinant human Carboxylesterase1 (hCE1) were investigated by in vitro incubation systems. Nine components from CMF1 were selected as marker compounds and tested for their effects on the metabolism of O. Dose response curves on enzyme inhibition were obtained and compared among different components. Mechanism of enzyme inhibition of the most potent marker from CMF1 on the metabolism of O in hCE1 was further studied using different plots such as Direct Plot, Dixon Plot, Lineweaver-Burk Plot and Eadie-Hofstee Plot.

Results: It was found that metabolism of O to OC in rat plasma and rat liver microsome was much more prominent than that in rat GI mucosa. Such metabolism was found to be inhibited by CMF1/CMF1 components in a dose-dependent manner. Among the studied components, Arctiin, MC, Forsythoside A and Forsythin have demonstrated relatively strong inhibition on the metabolism of O to OC in rat plasma with MC exhibited the strongest inhibition activity. Mechanistic enzyme inhibition study of MC indicated a pattern of competitive inhibition.

Conclusion: Our results demonstrated that metabolism of O could be significantly inhibited in the presence of CMF1/CMF1 components. Among all the studied CMF1 components, MC demonstrated the strongest inhibition with a com-petitive inhibition pattern. (Acknowledgement: CUHK direct grant 2041449)

P265. Mechanistic understanding of the effects of 6 schisandrins on the absorption and first-pass intestinal and hepatic metabolism of Tacrolimus

Hui-chang Bi, Xiao-ling Qin, Xiao Chen and Min Huang

* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China

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Tacrolimus (FK506) is a well-known potent immunosuppressant agent for the prevention and/or treatment of graft rejection in solid organ transplantation patients. We recently reported that the blood concentrations of Tacrolimus in rats were markedly increased following co-administration of Wuzhi Tablet (WZ), a preparation of ethanolic extract of Wuweizi (Schisandra sphenanthera). The major active schisandrins of WZ include Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A, Schisandrol B and Schisantherin A. Till now, the interaction between FK506 and WZ and the underlying mechanisms are not clearly clarified. Therefore, the purpose of this study was to investigate the effects of these schisandrins on the absorption and first-pass intestinal and hepatic metabolism of FK506. The effects of the schisandrins on the transport of FK506 were examined using Caco-2 cell monolayer, and the effects of the schisandrins on the metabolism of FK506 were investigated in rat and human liver microsomes. Pharmacokinetic behavior of FK506 following oral or intravenous dosages was studied and compared with or without the co-administration of the schisandrins. When FK506 was orally administered to rats with the schisandrins, whole blood concentrations of FK506 were all increased. Schisandrol B showed the strongest effect, which increased 6.75 folds of the AUC0-24h of FK506, and the next was Schisantherin A and Schisandrin A, which increased 1.48 and 1.37 folds of the AUC0-24h, respectively. When FK506 was administered the rats intra-venously with the schisandrins, whole blood concentrations of FK506 in rats were also increased and elimination was delayed, but the extent of effect was much slighter than that of oral groups. In the presence of Schisandrol B, the oral bioavailability (Foral) of FK506 was increased from 5.40% to 28.32%, and the (FabsxFG) of FK506 was 332.6%greater than that of FK506 alone group. However, the FH of the two groups were only 20.6% greater than that of FK506 alone group, indicating that the reduction of intestinal first-pass effect contributes most to the increase in oral bioavailability of FK506 when coadministered with Schisandrol B. Results from transport experiment using Caco-2 cell monolayer showed that the transport ratio of FK506 pretreated with 100 uM Schisandin A (1.0) or Schisandin C (1.5) or Schisandrol B (1.3) were lower than that of without pretreatment (1.9), suggesting Schisandin A, Schisandin C or Schisandrol B could inhibit P-gp-mediated efflux of FK506. Results from metabolism study using liver microsomes showed that the inhibitory effect of these 6 schisandrins on FK506 liver metabolism were dose dependent. 100 uM of Ketoconazole and Schisandrol B almost completely inhibited FK506 metabolism in rat and human liver microsomes, indicating Schisandrol B can strongly inhibit the CYP 3A-mediated metabolism of FK506. In conclusion, the exposure of FK506 in rats was increased when coadministered with Schisandrins, and Schisandrol B showed the strongest effect. Schisandrins inhibited P-gp-mediated efflux and CYP3A-mediated metabolism of FK506, and the reduction of intestinal first-pass effect by Schisandrins was the major cause of the increased FK506 oral bioavailability. (The work was supported by the Science and Technology Ministry of China 2009ZX09304-003.)

P266. Protective effect of Globularia alypum against doxorubicin induced Cardiotoxicity

Abidli Nacira1, Karaali Wahib1, Benrebai Mouad 1

1Laboratory of biology and environment, Faculty of Science of Nature and Life, University Mentouri - Constantine, Chaabet Ersas Campus, Constantine, Algeria

Doxorubicin (DOX) is an efficient chemotherapeutic agent used against several types of tumors; however, its use is limited due to severe cardiotoxicity. Since it is accepted that DOX induced myocardiopathy is the consequence of Oxidative stress through the mediation of free radicals, antioxidant agents have been used to attenuate its side effects. The aim of this work was to investigate the effect of the aerial parts butanolic extract of Globularia alypum (GABE), witch is known for their medicinal properties in traditional medicine such as the treatment of cardiovascular and renal diseases, on the acute cardiac toxicity induced by doxorubicin in rats. The analytical chemical study (The UV absorbance spectrum in the methanolic solution and also the analytical chromatography (PC, TLC) carried out on the GABE showed the presence of flavonoids belongs to a group of flavonols and Flavones. In Vitro the GABE exhibited a significant antioxidant activity, based on the scavenging activity of the stable free radical DPPH° where the amount of the extract needed for 50% inhibition (IC50) was 0.021 mg/ml. The GABE exhibited significant protection against DOXO induced cardiac toxicity. The pretreatment of rats with the GABE orally at a dose of 100mg/kg for a month resulted in a decrease in the plasma enzymatic cardiac injury( CPK, LDH, and AST); oxidative stress in the heart tissue was estimated by measuring the glutathione (GSH) levels and malondialdehyde (MDA) in the homogenate. The pretreatment of rats with the GABE orally at a dose of 100mg/kg for a month resulted in a decrease in the cardiac cytosolic MDA and maintenance of cardiac cytosolic GSH level as compared to DOXO treated animals at a dose of

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20 mg/kg intraperitoneall. These findings suggest that the butanolic extract of this plant has protective effects against Dox induced cardiotoxicity and may be a useful agent in the combination therapy with doxorubicin to limit free-radical-mediated cardiac injury.

P267. A New HTS Approach to Study the Interactions of Multiple Compounds with Individual Human UDP-glucuronosyltransferase; UGT1A6 as a Model

Anne M. Soikkeli1, Mika Kurkela2, Marjo Yliperttula3, Jouni Hirvonen1 and Moshe Finel4

1Division of Pharmaceutical Technology, University of Helsinki, Helsinki, Finland, 2Centre for Drug Research, University of Helsinki, Helsinki, Finland, 3Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Helsinki, Finland, 4Centre for Drug Research, University of Helsinki, Helsinki, Finland

There is currently no good method to screen many compounds and chemical entities for their interactions with the human UDP-glucuronosyltransferases (UGTs). Such method is highly needed in order to detect potential inhibitors, drug-drug and food-drug interactions, in both the intestine and the liver. The increasing awareness and the rising importance of drug-drug and food-drug interactions prompted us to concentrate on the developing of new approaches to identify such potential interactions at early stages, rather than attempting to develop a method to identify new agly-cone substrates for the different UGTs, another difficult challenge. Hence, the aim of the current study was to develop a high throughput screening (HTS) assay for one of the human UGTs that is expressed in both the liver and intestine, and we have selected UGT1A6. Our strategy was to find suitable fluorescent probe substrates for UGT1A6 and, using automated assay system, to examine the effect of test compounds on the fluorescence of the probe substrate. For the experiments describe in this poster we have used recombinant human UGT1A6 that was produced in our laboratory (Kurkela et al., 2003)1 and to achieve high sensitivity we were looking for probe substrate the fluorescent intensity of which is increased rather than decreased by the conjugation with glucuronic acid. To this end, we have developed an HTS assay protocol to optimize the analyses with respect to different reaction parameters such as composition of the reaction mixture, incubation length, and treatment of the fluorescent signal. Good results were obtained when we used black 96-well plates and measured fluorescence at 2 time points, 0 and 30 min after reaction initiation by the addition of the co-substrate UDP-glucuronic acid (UDPGA). The fluorescence was determined using a Varioskan Flash® plate reader and the area under the fluorescence signal (AUS) was highly linear, R2>0.99, in the 0.1-5 µM concentration range of the glucuronidated probe substrate. The reaction sensitivity was optimal in phosphate buffer, pH of 7.4, and the reaction mixture also contained MgCl2 and organic solvent, 5 % DMSO in the final reaction mixture. Several known substrates of UGT1A6 were used as competitors to validate the HTS method. In addition, the effect of these competitors on the fluorescence signal was independently assayed by HPLC. The quality of the HTS method was demonstrated by the derived Z’ factor values, 0.5 and above, indicating a highly successful HTS assay. The homogeneous assay protocol was also compatible with the automation by TECAN Genesis workstation. Subsequently, the automated assay was employed to test the interactions of a set of non-steroidal anti inflammatory drugs (NSAID), as well as salicylate deriva-tives. In conclusion, a sensitive fluorescence-based HTS assay was successfully developed in 96-well format to study the interactions of new chemical entities with UGT1A6.

References

(1) Kurkela M, Garcia-Horsman J.A., Luukkanen L., Mörsky S., Taskinen J., Bauman M., Kostiainen R., Hirvonen J., Finel M. Expression and char-acterization of recombinant human UDP-glucuronosyltransferases (UGT) J Biol Chem 2003 278(6): 3536-3544

P268. A Universal Spectroscopic Technique for High-Throughput In Situ Measurement of Cytochrome P450 Catalysis

Matthew J. Traylor1, Eric S. Arnon1, Jonathan S. Dordick2 and Douglas S. Clark1

1Chemical Engineering, University of California, Berkeley, Berkeley, CA, USA, 2Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA

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The rate of metabolism of a drug is a key parameter used to evaluate potential candidates during drug development. In vitro experiments with P450 enzymes are used to estimate the metabolic stability, or rate of degradation, of a drug of interest. The standard methodology uses high-throughput liquid chromatography tandem mass spectrometry (LCMS/MS). This technique requires expensive equipment and has limited throughput due to the inherently sequential nature of the separation and analysis. These problems limit the application of this technique to later stages of drug development where there are fewer compounds to evaluate. We have developed an alternative assay to calculate metabolic stabil-ity based on fluorescence intensity measurements. This assay, known as the Metabolic Enzyme Stability Assay Plate (MESA-Plate), requires less expensive equipment and has higher throughput due to the parallel nature of fluorescence measurements. The increased throughput of this method will allow higher quality data to be obtained earlier in the development process. A simple time and cost analysis shows that the MESA-Plate technology is an order of magnitude faster and cheaper than the current LCMS/MS industry standard. An oxygen and electron balance on the P450 reac-tion mechanism yields a general expression for the rate of substrate depletion as a function of O

2, NADPH, and H

2O

2.

Techniques were developed to measure each of these molecules in a time-resolved high-throughput fashion using fluorescence spectroscopy. These measurements were conducted simultaneously with human P450 enzymes acting on model drug compounds. Kinetic constants calculated with these data closely matched literature values. The rate of substrate depletion was also monitored via gas chromatography to further verify the accuracy of the MESA-Plate assay and validate this technique for the measurement of metabolic stability. The MESA-Plate assay is also expected to be a unique platform for reaction optimization and directed evolution of any P450 system, and can be a valuable tool for the biotechnology and pharmaceutical industries.

P269. HTS for selective muscarinic cholinergic receptors antagonists

Valerii D. Tonkopii

Pharmacology, Institute of Limnology Russian Academy of Science, St. Petersburg, Russia

In the experiments on the rats we have developed methodological approach to the evaluation of the selectivity of muscarinic cholinergic receptors (M-ChR) antagonists action in the whole organism conditions. According the results obtained during investigation the protective effect of M cholinolytics during acute poisonings of organophosphates (DDVP, DFP etc.) depends on M1 subtype ChR occupation. The efficiency of antagonists in inhibition of tremor reaction caused by M-ChR agonist arecoline administration associates with interaction of M2 subtype of ChR. It was established by the method of linear regression, that there was a high degree of correlation (r=0.99) for different M cholinolytics between the ratios of ED50 of M antagonists in the tests with arecoline and organophosphates and the ratios of dis-sociation constants of antagonists complexes with M-ChR from the homogenates of rat′s cerebral cortex and heart containing M1 and M2 ChR subtypes respectively. Thus, the ratio of ED50 arecoline/ ED50 DDVP is serve as a meas-ure of the selectivity of drugs action. Pharmacological analysis of Daphnia magna cholinergic system with the use of anticholinesterase compounds, and M-ChR agonists and antagonists was carried out. On the experiments to Daphnia magna the effects of some non selective, mainly M1 and M2 ChR antagonists on the toxicity of DDVP and arecoline were studied. There was a strong correlation between the ED50 of antagonists in the tests with arecoline and DDVP in the experiments on rats and the EC50 of antagonists in experiments on Daphnia magna. For the first time in the experi-ments on Daphnia magna it was shown that a ratio of the average effective concentrations (EC50) of M antagonists in the tests with arecoline and organophosphates also may be used as a measure of the selectivity of M-ChR antagonists action. The principal similarity in action of muscarinic antagonists to Daphnia magna and rats allow to recommend the Daphnia for screening of selective muscarinic receptor antagonists.

P270. In-vitro Distribution Dialysis Technique for Multiple Tissue Protein Binding

Mark G. Qian1, Cindy Xia1, Susan Chen1, Frank Lee1 and Tai-Nang Huang2

1Dmpk, Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, 2Dept of DMPK, Linden Bioscience, Lexington, MA, USA

In vitro distribution dialysis using a multi-compartmental device was proposed more than two decades ago in an effort to simulate the steady state across multiple compartments for measuring tissue binding, and eventually to predict the drug

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tissue distribution in-vivo. However, the original device was clumsy with a long, tedious, and disruptive assay process. To circumvent the defects and at the same time to meet the ever-increasing demand for tissue distribution assessment especially in developing oncology and neurology therapeutics, a convenient new multiplex dialysis system designed for in-vitro tissue protein binding has been used to mimic tissue distribution at steady state. This device is suitable for assessing species dependency in tissue binding and for simultaneous competing binding study among different organ tissues for the unbound drug released from a plasma pool. This presentation will provide results that demonstrate the capability of the simple and cost-effective technique for differentiating drug binding characteristics of different tissue homogenates against plasma proteins. The free drug fraction information available from the studies can be used to better explain/correlate drug exposures to its action. The results generated using this technique are shown to be comparable to those from the in-vivo studies.

P271. Software algorithms for high resolution LC-MS analysis in early drug discovery: quantitative and qualitative screening in the same run

Karen Salomon1, Xiaojie Ding1, Jim Shofstahl1, Thomas McClure1, Rose Herbold1 and Hans Pfaff2

1ThermoFisher Scientific, San Jose, CA, USA, 2ThermoFisher Scientific, Bremen, Germany

New software algorithms have been developed to automatically process high-resolution accurate-mass time course data for quantitative and qualitative information on a parent drug and its metabolites The availability of full scan ultra high resolution LC-MS instruments allows for rapid analyses of drug discovery samples without needing specific MS methods for target compounds. Software algorithms have been specifically developed to process full scan ultra high resolution data in order to identify compounds of interest at mass to charge values which have not necessarily been pre-defined. The overall data processing methodology includes advanced background subtraction and peak picking, as well as a parameter-less peak detection routine that is used for automatic peak integration. A sophisticated elemental composi-tion determination is used to confirm the metabolite chemical formula. The new algorithms are linked to allow for the automated detection and reporting of drug metabolites in both in vitro and in vivo time course studies. In addition to rapid screening for expected metabolites, unexpected metabolites are also uncovered in the same analysis. Software was developed for using the specialized algorithms described above and was used to analyze high resolution full scan time course data from several drug incubations. Each time course sample was processed to separate chromatographic peaks from background noise in an automated fashion, and then compared to a reference sample. Components that were unique to the time course sample were annotated and then searched against an expected metabolism modifica-tion database and any matches were recorded. In addition any large peaks not linked to the metabolite database were identified as putative unexpected metabolites and labeled as such. Confirmation of putative metabolite assignment was carried out with elemental composition determinations based on accurate mass data and the isotopic envelope detected. An area calculation for each putative metabolite was performed using a parameter-less peak picking algorithm. A relative area ratio was then calculated using the area of the parent drug at time zero. A plot was generated using the relative areas of the putative metabolites at different time points to display the appearance profiles of the metabolites found. On the same graph, there is also a plot of the relative parent drug disappearance, thereby providing comprehen-sive information on the metabolic stability of the target drug compound as well as the identity of metabolites formed over time. Examples from several drug incubations will be shown to demonstrate the utility of the software.

P272. Study of the effect of granulation technique on physicochemical properties of piroxicam liq-uisolid compacts

MH Zarrintan1, Y. Javadzadeh2 and K. Salarmand3

1Department of Phamaceutics, Tabriz University of Medical Sciences, Tabriz, Iran, 2Department of Pharmaceutics, Tabriz University of Medical Sciences, Tabriz, Iran, 3Tabriz University of Medical Sciences, Tabriz, Iran

Piroxicam is a poorly soluble, highly permeable drug and the rates of its oral absorption are often controlled by the dissolution rate in gastrointestinal tract. The rates of absorption and dissolution are usually equal. It can be supposed

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that if dissolution of the drug can be increased, the rate and even the extent of drug absorption will be increased. Different methods have been used to improve the dissolution of poorly soluble drugs including the technique of liq-uisolid compact. From the previous works it has been clear that although with this technique the dissolution of drugs gets improved, still lower flowability and compressibility of liquisolid powders bring limitation to this approach. It seems that wet granulation of liquisolid powders of piroxicam with binders may solve the above-mentioned problems. With this in mind, in the present study the effect of binders as alcoholic gelatin solution, hydroxypropyl methylcellulose (3% w/v) and hydroalcoholic solution was studied on the flowability and compressibility of piroxicam liquisolid powder made of avicel, propylene glycol, silica and sodium starch glycolate as carrier, non-volatile solvent, coating material and disintegrant respectively. Flowability of prepared liquisolid granules compared to liquisolid powders was analyzed using flow rate and repose angle data. Friability, tensile strength and dissolution profiles of liquisolid compacts were also investigated. The results showed that the liquisolid granules of piroxicam had better flow properties compared to liquisolid piroxicam powder. This was more significant for the liquisolid granule compacts made of hydroalcoholic solu-tion as binder. It seemed that almost the uniform shape and size of liquisolid piroxicam granules made of hydroalcoholic solution was the reason for better flowability of this compacts. Other properties measured including friability, tensile strength and dissolution profiles of the prepared liquisolid compacts showed improvement in liquisolid compacts made of granules with the examined binders. In can be concluded that using wet granulation technique with a suitable binder as hydroalcoholic solution to prepare liquisolid compacts of piroxicam, can bring improved physicochemical properties compared to powder liquisolid piroxicam compacts and show better flowability and compressibility.

P273. The End of MRM: Ultrafast SPE-TOF Analysis Streamlines Workflow and Increases Throughput of ADME Assays

Michelle V. Romm, Nikunj Parikh, Vaughn P. Miller, William A. LaMarr and Can C. Ozbal

BIOCIUS Life Sciences, Woburn, MA, USA

The throughput of in vitro ADME analysis is gaining an almost equal footing with data quality as a determinant of laboratory workflow. These assays have traditionally utilized tandem MS, which is limited by its requisite MRM method development that requires several minutes per sample of processing time. The use of accurate mass offered by high resolution (time of flight: TOF) mass spectrometers, which eliminates the need for MRM optimization, in conjunction with a high throughput solid phase extraction (SPE) system was investigated to assess its ability to enable a faster and more efficient assay analysis workflow. We compared the assay results for a panel of in vitro ADME assays (CYP inhibition, metabolic stability, PAMPA, and plasma protein binding) using ultrafast SPE-MS/MS and SPE-TOF systems for analysis. A chemically diverse set of 50 compounds was used for each assay and all analyses utilized the same generic SPE conditions. All SPE-TOF analyses were performed using the same generic MS conditions with a RapidFire 360 interfaced to an Agilent 6530 Q-TOF run in ESI-TOF mode. The experimental results obtained for all four of the ADME assays by each analysis system were comparable (R2 >0.9). Using a generic SPE condition, the ultrafast SPE-MS systems consistently produced sample analysis cycle times of 7 seconds/sample with no changes to standard laboratory workflow and no significant sample carryover. Using a generic MS method, the TOF analysis gave comparable results to conventional triple quadruple analysis. Assay results with the SPE-TOF system were comparable to the SPE-MS/MS system for a variety of ADME assays indicating that MRM method development could be eliminated for these assays providing a significant increase in efficiency of laboratory workflow. The SPE-TOF system enabled faster and more efficient assay data analysis across a panel of ADME assays without compromising the quality of results.

P274. Automatically Finding Drug Discovery ‘Rules of Thumb′

Edmund J. Champness1, Matthew D. Segall1 and Tatsunori Hashimoto2

1Optibrium Ltd., Cambridge, United Kingdom, 2Statistics Department, Harvard University, Cambridge, MA, USA

Rules-of-thumb for evaluating potential drug molecules, such as Lipinski′s Rule of Five, are commonly used because they are easy to understand and translate into practice. These rules have traditionally been constructed by observation

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or by following simple statistical analysis. However, application of these techniques to QSAR models or early screening data often ignores the underlying statistical structure. Conversely, when machine learning algorithms are used to classify ‘drug-like′ molecules, they often result in black-box classifiers that cannot be modified to suit a particular target drug profile. We propose a novel hybrid approach to constructing rules-of-thumb from existing data to match a given target product profile for any therapeutic objective. These rules are easily interpretable and can be rapidly modified to reflect expert opinions before application.

P275. Biosimulation of acetaminophen-induced liver injury

Harvey J. Clewell1, Brett Howell1, Yuching Yang1, Alison Harrel1, Scott Siler2, Richard Ho2, Rukmini Kumar2, Melvin Andersen1 and 30Paul Watson1

1The Hamner Institute for Health Sciences, Research Triangle Park, NC, USA, 2Entelos, Inc., Foster City, CA, USA

The long-term objective of this joint Hamner/Entelos/USFDA research effort is to develop a dynamic, mechanistic computer model of liver homeostasis and drug-induced liver injury (DILI). Initial model development focused on acetaminophen (APAP); future efforts will include idiosyncratic liver toxicants such as isoniazid and valproate. The APAP model embodies a quantitative description of the key processes involved in APAP-induced liver injury: APAP metabolism, reactive metabolite formation, glutathione depletion and resynthesis, ROS formation, energy homeostasis, mitochondrial dysfunction, necrosis, apoptosis, and tissue regeneration. Model parameters are estimated using data from published literature and other sources, including acetaminophen toxicity studies per-formed in healthy adults and mining of the AERS database. The first stage of the platform includes a whole-body physiologically based pharmacokinetic (PBPK) model for APAP and its metabolites in the mouse, rat, and human. The PBPK model is linked to a pharmacodynamic (PD) model describing the reaction of APAP-derived N-acetyl-p-benzoquinone imine (NAPQI) with glutathione and the resulting depletion and resynthesis of glutathione. The formation of NAPQI-adducts with cellular proteins and increased oxidative stress resulting from glutathione deple-tion are linked to cellular damage and response. The PBPK/PD model of APAP metabolism and glutathione depletion is being used to investigate the extent to which differences in metabolism and glutathione homeostasis explain interspecies differences in susceptibility to APAP toxicity. As compounds with other modalities of DILI are added to the platform, the resulting model will serve as a flexible biosimulation platform to enhance drug development by quickly identifying drug candidates that are likely to be associated with idiosyncratic hepatic toxicity, and to guide personalized medicine by determining patient characteristics associated with a greater likelihood of adverse liver responses. The DILI simulation model will also serve as a valuable knowledge management tool for organizing the available data on the elements of metabolism, cellular injury, and tissue response that are the determinants of susceptibility to DILI.

P276. Classification models for CYP450 2C8 inhibitors and non-inhibitors

Pei-Pei Dong, Chunzhi Ai, Yan-Yan Zhang and Ling Yang

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Cytochrome P450 2C8 (CYP2C8) is one of the principal drug metabolizing P450 monooxygenases expressed in human liver. It is involved in the metabolism of some therapeutically important drugs such as paclitaxel, repaglinide, rosigli-tazone, and cerivastatin. The inhibition of this enzyme in many cases leads to undesired accumulation of the adminis-tered therapeutic agent. The purpose of this study is to develop in silico models that can effectively distinguish human CYP2C8 inhibitors from non-inhibitors. We present a data set of more than 200 structurally diverse drug compounds classified for their interaction (inhibitor, non-inhibitor) with CYP2C8. Structures of each drug were characterized by a lot of 2D descriptors. Feature selection was performed using Monte Carlo variable selection (MCVS) method to select key descriptors for modle building. Artificial neural network (ANN) and support vector machaine (SVM) methods were introduced for the classification of inhibitor and non-inhibitor of CYP2C8. The SVM model was better than ANN with

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the accuracy of 67.2% and sensitivity of 68.6%. This model is further validated by the test set where both accuracy and sensitivity were 65.8% and 66.4%, respectively. The 2D descriptors sufficiently represented the molecular features of CYP2C8 inhibitors. Our model can be used for the prediction of either CYP2C8 inhibitors or non-inhibitors in the early stages of the drug discovery process.

P277. Comparison of In Silico Tools for the Prediction of Sites of Metabolism by Cytochrome P450

Hoa Le, Young Shin, Cornelis Hop and Cyrus Khojasteh

Genentech, South San Francisco, CA, USA

The majority of marketed drugs are eliminated from the body via metabolism by cytochrome P450 enzymes. Therefore, early identification of the metabolically labile sites affected by this enzyme system helps the medicinal chemists to better design compounds with improved metabolic stability. The typical approach is to identify the metabolites formed from in vitro studies using LC-MS/MS. The interpretation of MS/MS spectra is one of the bottlenecks that the throughput. With the emphasis on higher efficiency, there is growing interest in complementing the in vitro assay with the use of predictive in silico tools as part of the drug discovery stage. The goal of this study is to compare how well MetaSite, StarDrop, and SMARTCyp predict the sites of metabolism for 30 in-house compounds and 30 probe substrates (12 CYP3A4 substrates, 9 CYP2C9 substrates, and 9 CYP2D6 substrates-not sure if these compounds were part of the training set, perhaps they were!). Bear in mind that, none of the software packages predict the rates of metabolism nor predict selectivity of different enzymes in metabolizing the substrates. Also, except for MetaSite that broadly predicts major CYP isoforms, StarDrop only predicts CYP3A4, CYP2D6 and CYP2C9 sites of metabolism, and SMARTCyp predicts only CYP3A4. To measure the degree of predictivity of each software package, we assigned 3 points if the first major metabolite reported is predicted correctly, 2 points for the second choice, 1 point for the third choice and 0 point were given for the fourth choice and beyond. The total points assigned for each enzyme experimentally were compared as a percentage of the total points assigned theoretically for a first choice prediction for all substrates for each enzyme. For probe substrates, all three software packages behaved relatively well with StarDrop performing slightly better: StarDrop (CYP3A4 = 83%, CYP2C9 = 89% and CYP2D6 = 93%), MetaSite (86%, 59%, 70%), and SMARTCyp (CYP3A4 = 72%). We are currently assessing the prediction accuracy of metabolic soft spots from in-house new chemical entities with in-house experimental observations.

P278. Evaluation Of ACD/ADME Suite 5.0 Regioselectivity Predictions

Justas Dapkunas, Remigijus Didziapetris, Andrius Sazonovas and Pranas Japertas

ACD/Labs, Inc., Vilnius, Lithuania, LT

The module predicting regioselectivity of human liver microsomal metabolism was recently released as a part of ACD/ADME Suite 5.0 software. These predictions are derived from an ensemble of models. Each one of them covers one of the five major reactions occurring in human liver microsomes (N- and O-dealkylation, aromatic and aliphatic hydroxylation, as well as S-oxidation). Together they provide an estimate of the possibility to be metabolized for each atom in the molecule. All aforementioned models have been built using GALAS (Global, Adjusted Locally According to Similarity) modeling methodology allowing the evaluation of prediction reliability in the form of calculated Reliability Index (RI). The total size of the training set used was 873 compounds. In this study we evaluate the ACD/ADME Suite regioselectivity predictions using an external validation set with >40 com-pounds for which metabolism data have been published recently. The predictions were divided into four groups according to their quality (“Excellent”, “Good”, “Satisfactory”, and “Unsatisfactory”). At least one metabolism site was found for more than 80% of compounds, and more than 60% of them had “Excellent” or “Good” predictions. Overall, >50% of experimental metabolism sites were predicted by ACD/ADME Suite, on the other hand >80% of predicted sites were experimental metabolism sites. High RI values successfully identified correct predictions. The presented results show reasonable agreement between ACD/ADME Suite drug metabolism regioselectivity predictions and experimental data.

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P279. Guiding Focused Design of Potent Leads with Improved Metabolic Stability

Matthew D. Segall

Optibrium Ltd., Cambridge, United Kingdom

A number of methods have been developed for the prediction of regioselectivity of metabolism by the major drug metabolising isoforms of Cytochrome P450 [1,2,3]. However, while valuable, predicting the relative proportion of metab-olite formation at different sites on a molecule is only a partial solution to designing more stable molecules. Valuable additional information comes from predicting a measure of the lability, or vulnerability, of each site to metabolism. Such a measurement is the site lability, as calculated by StarDrop™, which is a measure of the efficiency of the product formation step, by comparison of the rate of product formation with that of decoupling to form water and inactivation of the oxidative enzyme species. This is an important factor in determining the overall rate of metabolism and, when combined with other descriptors relating to substrate affinity or enzyme oxidation rate, can provide good predictive models of in vitro metabolic rate, at least within ‘local′ regions of chemistry. These models can, in turn, be used to guide design of compounds with improved stability. We will demonstrate this with a case study, targeting a fast follower for a compound that had problems with poor oral bioavailability and short and variable half-life in man. These issues were caused by rapid metabolism by Cytochrome P450 CYP3A4, which corresponded with predictions of highly labile sites and a high logP. The final compound had to be suitable for oral dosing and the target was in the CNS. In the initial phase of the project, the goals were to identify lead compounds which improved the in vitro half-life with respect to CYP3A4 metabolism by a factor of 2 while maintaining receptor IC

50 of less than 200nM. To achieve this, a virtual library of 13,000

novel compounds was enumerated, based on the structure of the initial compound. From this, a set of 100 compounds with a range of chemical diversity and physicochemical properties was chosen for synthesis and testing for potency and in vitro stability with respect to CYP3A4. From these data, local models were built for both CYP3A4 stability and potency. All 13,000 virtual molecules were then assessed with the local models and only 40 were predicted to be both stable and potent. All of these were also predicted to be soluble, absorbable and to cross the blood-brain barrier. The 40 compounds were then synthesised and tested, resulting in 4 high quality lead series, each containing multiple compounds that were both potent and more than twice as stable as the competitor with respect to CYP3A4 metabolism.

References

[1] V.S. Gopaul et. al. (2009) Drug Metab. Rev. 41(s3) p. 187-196 (abstract 242) [2] P. Rydberg et. al. (2010) ACS Med. Chem. Lett. DOI: 10.1021/ml100016x [3] M. Hennemann et. al. (2009) ChemMedChem 4(3) p. 657-69

P280. In Silico Prediction of Biotransformation Pathways: Decarboxylation of Aliphatic and Aromatic Compounds

Ernest Murray, Mohammed A. Ali and Anthony Long

Knowledge Base, Lhasa Limited, Leeds, United Kingdom

The metabolic fate of xenobiotic substances in mammals may be predicted using in silico expert systems. To be of value as a tool which informs the chemical basis for the prediction, an expert system should, at least, contain knowledge of the mechanistic pathways involved, the presence of reactive intermediates and the likelihood of all such processes inferred from the literature. This work highlights the refinement and development of biotransformations relating to the decarboxylation of aliphatic and aromatic compounds and beta-oxidation of carboxylic acids. Recent work on a knowledge-based expert system focused on the decarboxylation of mammalian xenobiotic substrates. Two existing biotransformations in the knowledge base were investigated: 1) Decarboxylation of aliphatic and aromatic carboxylic acids and 2) beta-Oxidation of carboxylic acids. A recent review of the literature indicated that the decarboxylation of aliphatic and aromatic carboxylic acids could be split into the three compound classes based on structure-metabolism relationships: alpha-amino, aromatic and beta-keto carboxylic acids. The majority of these biotransformations were found to be catalysed by decarboxylase or aromatic L-amino acid decarboxylase. However, aryl acetic acids were identi-fied as a separate class of compound which undergo oxidative decarboxylation mediated by cytochrome P450. In the

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case of statins, beta-oxidation of the carboxylic acid side chain occurs, in preference to decarboxylation, by a multistep mechanism that mirrors the two carbon degradation of naturally occurring fatty acids in lipid metabolism. Compounds which are also metabolised by the beta-oxidation pathway include alpha,beta-unsaturated and alpha-branched car-boxylic acids. A comparison between the new and a previous version of our biotransformation dictionary was car-ried out using the following test compounds: N-nitrosoproline, S-3-hydroxybutyrate, 6-fluoro-L-DOPA, ketoprofen, diclofenac, cerivastatin, iloprost, ozagrel and valproic acid. Two biotransformations were updated and two new entries were registered in the biotransformation dictionary: decarboxylation of alpha-amino, aromatic and beta-keto carboxylic acids, beta-oxidation of carboxylic acids, beta-oxidation of alpha,beta-unsaturated carboxylic acids and beta-oxidation of alpha-branched carboxylic acids. In all cases, these test compounds activated one or more of the new or modified biotransformations in the updated dictionary. Structures of the predicted intermediates and metabolites were consistent with those reported in the literature. This investigation has demonstrated that in response to new data, the refinement of existing biotransformations and the addition of new entries into the knowledge base can increase the predictive performance and selectivity for the prediction of decarboxylation of aliphatic and aromatic carboxylic acids, enhancing the overall performance of such in silico expert systems.

P281. Modeling Toxicity Endpoints Using Artificial Neural Network Ensembles

Adam C. Lee, Robert Fraczkiewicz and W.S. Woltosz

Simulations Plus, Inc., Lancaster, CA, USA

As a large number of clinical candidates fail to make it to market due to toxic effects, significant efforts have been made in predicting toxicity endpoints. Modeling biological endpoints is a daunting challenge due to the involvement of multiple known and unknown mechanisms. Here we describe our efforts using artificial neural network ensembles (ANNEs) to generate models for toxicity endpoints including acute rat toxicity (LD50), bioconcentration factor, reproductive toxicity (classification), fathead minnow (LC50), and Tetrahymena pyriformis (IGC50). We also provide comparisons to other QSAR methodologies used by the Environmental Protection Agency′s Toxicity Estimation Software Tool (T.E.S.T.): multiple linear regression, hierarchal clustering, nearest neighbor, and other similarity-based methods. In almost all comparisons made using the same training and test sets, the ANNEs showed superior performance in terms of root mean squared error for regression models and false rate for the classification model. Furthermore, statistics showed significant improvement when models were created using Kohonen maps independent of the Y endpoints to establish new similarly sized training and test sets from the cumulative data with respect to each T.E.S.T. model. Using these and other models to generate new ADMET risk standards can facilitate better decisions in drug discovery.

P282. Pattern-recognition-based Prediction of Metabolic Regioselectivity Mediated by Human Cytochromes P450 3A4

Chunzhi Ai, Yuxi Mao and Ling Yang

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

P450 3A4, the most important human CYP enzyme, is responsible for the metabolism of more than 50%-60% of clinically used drugs. Early knowing the site of metabolism will help the medicinal chemist to decide how best to block the metabolism of candidates in drug discovery. Traditionally, the regioselectivity has always been evaluated by estimating the dehydrogenation energy, and/or the binding poses of substrates in the active site of the enzyme. Here, we present a novel strategy for predicting the metabolic regioselectivity mediated by human cytochrome P450 3A4. We have defined a novel type of fragmental-based descriptors that was used together with the E-state molecular descriptors to establish the model for predicting the site of metabolism. With pattern recognition method, 127 sub-strates of P450 3A4 collected from literatures were employed to develop the model. The cross-validated prediction result is 79% and the accuracy for predicting one right site is higher than 88%, suggesting the efficiency of the newly defined fragmental-based descriptors. The predictability of this model is as good as the recently published QSAR model proposed by Sheridan et al1.

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References

1. Sheridan, R. P.; Korzekwa, K. R.; Torres, R. A.; Walker, M. J., Empirical regioselectivity models for human cytochromes p450 3A4, 2D6, and 2C9. Journal of Medicinal Chemistry 2007, 50, (14), 3173-3184.

P283. Prediction of Drug-Drug Interactions of Two New Antibiotics Using a Mechanistic Static Model

Arnaud M. Bruyere1, Cristina Lopez2, Olivier Barberan2 and Françoise Brée3

1Xénoblis, Saint Grégoire, France, 2Aureus Pharma, Paris, France, 3Xénoblis, Saint Grégoire, France

Drug-Drug interactions (DDIs) are a major concern for drug discovery and development teams today, especially for antibiotics that are often co-administered with other drugs. Since DDIs have led to several drugs being removed from the market due to adverse effects, it is essential not only to understand potential DDIs as early as possible but also as accurately as possible. Aureus′ DDI Predict 2009® provides researchers with an automated facility to predict metabolic drug-drug interactions, based on in vitro–in vivo extrapolation (IVIVE), a mechanistic static model (MSM) approach and an inter-experiment variability. The two main mechanisms of inhibition, reversible and/or irreversible, characterized by time, and inhibitor concentration-dependent loss of enzyme activity, can be used in DDI Predict 2009®. In this study, we determined Michaelis-Menten constants of two new antibiotics (one beta-lactamase inhibitor and one macrolide) and their inhibition constants on CYPs using human liver microsomes. In vitro data demonstrated that both of them were Mechanism Based Inhibitors of CYP3A4. DDI involving MBIs requires not only estimation of in vitro parameters (inactivation rate constant (kinact) and MBIs potency (KI)) but also parameters related to the degradation rate constant of the enzyme (kdeg) and inactivator concentration at the enzyme active site ([I]), respectively, to predict AUCi/AUC ratio. Other parameters related to substrate metabolism in the liver (fm) and also metabolism in the gut (bioavailability across the gut (Fg)) are critical for DDI predictions. Thanks to Aureus′ DDI Predict 2009®, new chemical entity could be considered as a victim of inhibitors as well as a potential inhibitor. Predictions for both antibiotics showed high potential interactions when they were considered as victims. As a perpetrator, only the macrolide antibiotic showed significant potential DDIs, mainly with calcium channel blocker and anti-HIV classes. These two antibiotics were then compared in term of inhibition properties to two other reference antibiotics, clavulanic acid (beta-lactamase inhibitor) and erythromycin (macrolide).

P284. Prediction of regioselectivity and order of the occurrence for CYP-mediated reaction

Yasushi Yamazoe, Kazumi Ito and Kouichi Yoshinari

Div. of Drug Metab. & Mol. Toxicol., Tohoku University, Graduate School of Pharmaceutical Sciences, Sendai, Japan

We have developed an in-silico predicting system for CYP-mediated reaction using isoform-specific secondary-di-mensional templates. These enable us to tell the order of occurrence among possible plural sites as well as the regiose-lectivity. The system and data for human CYP2E1 is shown in the present study. Although CYP2E1 prefers small sized molecules for the substrates, the enzyme mediates oxidations of large sized molecules such as a]pyrene. Overlays of typical substrates on a honeycomb-type template, to assemble their sites of oxidation into an identical position, suggest a range of regions frequently occupied. The regions, termed core template, has a a]pyrene-like shape. In this model, substrate atoms, except for hydrogen atom, are expanded and overlaid on corners of honeycomb structures of the a]pyrene-template. A chemical, whose expanded structure is not accommodated within the template, is judged not to be a substrate of CYP2E1. Using published metabolism data with more than 100 substrates of CYP2E1, the core template was further refined to verify the boundary and to define the relative contribution of template positions. These experiments suggest in general that CYP2E1 interacts with planar shapes of substrates for the oxido/reductions. The positions on the template were classified into four different groups (0 to 3 score) depending on the relative usage. Steric allowance for bulky residues, also differ depending on the position of the template. Therefore, we set independent scores (-5 to 3) for chemical residues to incorporate three-dimensional information. The scores were adjusted to link to the relative abundance of the in vitro reactions. Total sums of scores with weighted template occupancy and three-dimensional

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information were calculated for all the orientations of possible conformers of test substrates, and found to predict the relative abundance (order) as well as the regioselectivity of human CYP2E1 reactions with high fidelities.

P285. Real-Time and Label-Free Monitoring of Bioenergetics and Cell Impedance/Adhesion for Long and Short Term Cell-Based Functional Assays in Vitro

Josef Unger, Sabine Drechsler, Axel Kob, Marcus Wego, Ralf Ehret and Stefanie Ortinau Bionas,

Rostock, Germany

Advanced in vitro methods may help to identify pharmacodynamics properties of substances and the prediction of compound toxicity in early phases of development. Two major aspects are mostly relevant for pharmaceutical studies. The first one is the investigation of short-term effects involving e.g. membrane transporter mechanisms or receptor signaling. The second one deals with long-term effects mediated by toxins and xenobiotics. Therefore the functional analysis of living cells in a physiologically controlled environment which provides information about pharmacologi-cal and toxicological as well as metabolic properties may serve as an alternative method for animal experiments. The Bionas® analyzing systems detect physiological parameters of cell lines and primary cells in label-free and non-invasive assays. The multi-parametric sensor chip continuously measures the oxygen consumption and the extracellular acidifi-cation which both determine the acute rates of the cellular energy metabolism. In addition, cell impedance/adhesion measurement detects alterations in the cellular adhesion and morphology of cells [1, 2]. A perfusion system guarantees highly defined cell environmental conditions and allows parameter recordings in long and short-term studies. By gener-ating profiles of bioenergetics (glycolysis and mitochondrial respiration) and cellular impedance, the systems monitor the action of substances and their cytotoxic effects including potential regeneration of cells in the area of toxicology, oncology and drug discovery, respectively. Therefore specific data from the drug/cell interaction can be determined [3]. These data may either be generated by long-term studies over hours to days or via short-term detection of cellular responses. In the field of drug development, membrane transporter-mediated mechanism and receptor interaction studies are the major focus. In summary, the Bionas® analyzing systems are the only available devices capable of analyzing extracellular acidification, respiration and cell impedance/adhesion in short and long term studies. The combination of these cellular parameters with the functionality of a temporal resolution of compound effects makes it a valuable tool for cell-based functional studies.

References

[1] Ehret, R., Baumann, W., Brischwein, M., Schwinde, A., Stegbauer, K., Wolf, B.: Monitoring of cellular behaviour by impedance measurements on interdigitated electrode structures. Biosensors & Bioelectronics, 12 (1) (1997) 29-41

[2] Ehret, R., Baumann, W., Brischwein, M., Schwinde, A., Wolf, B.: On-line control of cellular adhesion with impedance measurements using interdigitate electrode structures. Medical & Biological Engineering & Computing, 36 (1998) 365-370

[3] Thedinga, E., Kob, A., Holst, H., Keuer, A., Drechsler, S., Niendorf, R., Baumann, W., Freund, I., Lehmann, M., Ehret, R.: Online monitoring of cell metabolism for studying pharmacodynamics effects. Toxicology and Applied Pharmacology, 220 (2007) 33-44

P286. Isolation of Zebrafish (Danio rerio) Liver Microsomes: Protocol and Considerations

Hui Ting Chng1, Han Kiat Ho1, Siew Hong Lam2 and Eric Chun Yong Chan1

1Pharmacy, National University of Singapore, Singapore, Singapore, 2Biological Sciences, National University of Singapore, Singapore, Singapore

Xenobiotic metabolism studies utilizing liver microsomes in pharmaceutical research typically utilize microsomes isolated from rat, human, dog, mouse or monkey. In recent years, zebrafish (Danio rerio) has been increasingly explored in pharmaceutical research as an alternative model for toxicological screening (Eimon PM & Rubinstein AL The use of in vivo zebrafish assays in drug toxicity screening. Expert Opin Drug Metab Toxicol. 5, 393-401 (2009). As metabolism is closely related to drug-induced toxicities, there is an unexplored need to understand xenobiotic metabolism in zebrafish and validate its use as a toxicological model. As such, zebrafish liver microsomes (ZLM) would be needed for in vitro drug metabolism assays. While the isolation of ZLM appeared to be straightforward,

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our preliminary approach, adopted from the isolation and characterization of rat liver microsomes (RLM) (Hill JR in Current Protocols in Pharmacology 7.8.1-7.8.11 (Wiley Interscience, 2003); Guengerich FP, Martin MV, Sohl CD & Cheng Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nat Protoc. 4, 1245-1251 (2009)) demonstrated that the isolated ZLM did not possess CYP450 but instead contained 3.702 nmol/mg protein of metabolically-inactive CYP420. On the other hand, the isolated RLM was found to contain 0.552 nmol/mg protein of CYP450. Fish liver microsomes with metabolically-active CYP450 had been successfully isolated from tropical, marine and freshwater fishes (Leitão MA et al. The liver monooxygenase system of Brazilian freshwater fish. Comp Biochem Physiol C Toxicol Pharmacol. 126, 29-38 (2000); Stegeman JJ, Binder RL & Orren A. Hepatic and extrahe-patic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor. Biochem Pharmacol. 28, 3431-3439 (1979); Vodicnik MJ, Elcombe CR & Lech JJ. The effect of various types of inducing agents on hepatic microsomal monooxygenase activity in rainbow trout. Toxicol Appl Pharmacol. 59, 364-374 (1981)). While the fishes explored in these studies are medium to big-sized fishes with distinct and intact livers, each small-sized zebrafish has a liver comprising a soft tissue that is in close proximity to the gall bladder and gut (Figure 1). As such, isolation of zebrafish liver is prone to contamination by bile salt and blood during dissection. Based on our observed challenges in isolating ZLM, we hypothesize that bile salt and haemoglobin contamination occurs during zebrafish liver isolation leading to denaturation of CYP450 and an intense spectra signal at 420 nm, respectively. In this paper, we performed a systematic investigation to elucidate the roles of bile salt and haemoglobin contamination in compromising the isolation of CYP450. For the former, experiments were performed with and without bile secretion to compare the absence and presence of CYP450 in ZLM. For the latter, experiments were performed to investigate the rinsing of dissected liver and the subsequent centrifugation steps in mitigating the effect of haemoglobin contamination. Finally, the functional activity of the isolated ZLM was tested using metabolism assay. The optimized protocol and considerations to obtain metabolically-active ZLM are discussed in this paper.

P287. Oligomerization of the UGT2B7 wild-type and its mutants: homo- and heterodimerization analysis by fluorescence resonance energy transfer(FRET)

Lingmin Yuan, Lushan Yu and Su Zeng

Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang University, Hangzhou, China

Human UDP-glucuronosyltransferases (UGTs) are major phase II metabolism enzymes that detoxify a multitude of structurally diverse, endogenous, and exogenous substrates through the covalent addition of a glucuronic acid moiety. Mammalian UGTs are located on the endoplasmic reticulum membrane and most of their mass is in the luminal side. That UGTs form homo- or hetero-oligomers was revealed by a variety of techniques[1,2,3,4,5]. When a UGT1A1 mutant, (UGT1A1*5, Q311X) in CNII patients is expressed simultaneously with the wild-type enzyme, the activity is reduced to approximately 6% of that of the wild type[6].This shows a hetero-oligomer UGT1A1-UGT1A1*5 shows dominant nega-tive inheritance. Thus, UGT2B7 wild-type and mutants, UGT2B7*71S, UGT2B7*2 and UGT2B7*5 can form homo- and hetero-oligomers, which may influence the activity and substrate selectivity of UGT2B7 wild-type.

Liver

Gall bladderIntestines

Figure 2 Female zebrafish with the dissected intestines (pale pink tis-sue), liver (darker pink tissue) and gall bladder (green coloured sac)

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Methods: Construct pFastBac1-UGT2B7(*N)-CFP and pFastBac1-UGT2B7(*N)-YFP plasmids, and then express or Co-express by using bac-to-bac insect expression system. the oligomerization is detected by using FRET.

Results: FRET efficiency of UGT2B7*1-UGT2B7*1 is (15.0 ± 1.0)%, FRET efficiency of UGT2B7*71S-UGT2B7*71S is (22.4 ± 2.0)%, FRET efficiency of UGT2B7*2-UGT2B7*2 is (12.9 ± 2.8)%, FRET efficiency of UGT2B7*5-UGT2B7*5 is (9.5 ± 0.8)%, FRET efficiency of UGT2B7*1-UGT2B7*71S is (11.1 ± 0.8)%, FRET efficiency of UGT2B7*1-UGT2B7*2 is (12.9 ± 2.8)% and FRET efficiency of UGT2B7*1-UGT2B7*5 is (11.0 ± 0.4)%, FRET efficiency of UGT2B7*71S-UGT2B7*2 is (6.7 ± 0.8)%, FRET efficiency of UGT2B7*71S-UGT2B7*5 is (17.2 ± 2.7)%, FRET efficiency of UGT2B7*2-UGT2B7*5 is (26.5 ± 2.5)%.

Conclusion: UGT2B7 wild-type and mutants, UGT2B7*71S, UGT2B7*2 and UGT2B7*5 can form homo- and hetero-oligomers, and these models could be used to detect the variance of activity and substrate selectivity by wild-type and mutants homo- and hetero-oligomers.

References

1. Ghosh SS, Sappal BS, Kalpana GV, Lee SW, Chowdhury JR, Chowdhury NR. 2001. Homodimerization of human bilirubin-uridine-diphosphoglu-curonate glucuronosyltransferase-1 (UGT1A1) and its functional implications. J Biol Chem 276:42108–42115.

2. Ishii Y, Miyoshi A, Watanabe R, Tsuruda K, Tsuda M, Yamaguchi-Nagamatsu Y, Yoshisue K, Tanaka M, Maji D, Ohgiya S, Oguri K. 2001. Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 and 2B22 in COS-7 cells enhances UDP-glucuronosyltransferase 2B21-catalyzed morphine-6-glucuronide formation. Mol Pharmacol 60:1040–1048.

3. Kurkela M, Hirvonen J, Kostiainen R, Finel M. 2004. The interactions between the N-terminal and C-terminal domains of the human UDP-glucuronosyltransferases are partly isoform-specific and may involve both monomers. Biochem Pharmacol 68:2443–2450.

4. Kurkela M, Garcia-Horsman JA, Luukkanen L, Morsky S, Taskinen J, Baumann M, Kostiainen R, Hirvonen J, Finel M. 2003. Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than the other UGTs and was purified as an active dimeric enzyme. J Biol Chem 278:3536–3544.

5. Operana TN, Tukey RH. 2007. Oligomerization of the UDP-glucuronosyltransferase 1A proteins. Homo- and heterodimerization analysis by fluorescence resonance energy transfer (FRET) and coimmunoprecipitation. J Biol Chem 282:4821–4829.

6.Koiwai, O., Aono, S., Adachi, Y., Kamisako, T., Yasui, Y., Nishizawa, M., et al. (1996). Crigler-Najjar syndrome type II is inherited both as a domi-nant and as a recessive trait. Hum Mol Genet 5:645–647.

P288. Development of A Novel Culture Plate for 3-Dimentional Cell Culture Aimed for Drug Discovery Screening

Yoko Ejiri1, Masuhiro Nishimura2, Masaya Hosoda1, Motohiro Fukuda1 and Shinsaku Naito2

1Tsukuba Research Center, Kuraray Co., Ltd., Tsukuba, Japan, 2Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan

Recently, some new cell culture technologies, including special scaffolds (eg: gel, fiber) to form three-dimensional (3-D) spherical aggregates, have been developed. In case of hepatocyte, these aggregates have been observed to maintain higher level of hepatocytes specific functions including cytochrome P450 activity and albumin secre-tion. In addition, in the cell-based assays for drug discovery, high throughput screening (HTS) is demanded. Consequently, we studied 3-D culture methods using micro structural scaffolds that are divided by micro-sized wall on the surface, which provide simple handling to be applicable for HTS. Our attempt was to build spheroids with diameter ranging from 50 to 200μm because the size is regarded as suitable for nutrition supply into the center of the spheroid. We prepared cell culture plates, made of polystyrene, with micro-space arrayed regularly on bottom surface (Figure ).

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The shape of each micro-space is a square with 200μm width and 50μm or 100μm depth (Micro-Space Culture Plate). Primary hepatocytes were cultured on 24-well Micro-Space Culture Plate for 5-6 days by the same method as the monolayer culture on flat plates (Initial cell density: 0.2-0.4 million in 500μl). Medium change was done after 4 hr from culture initiation and everyday thereafter. During initial 4 hr period, cells attached to the surface in each micro-space, which is similar to the case on flat plate. The cells began to aggregate and formed spheroid like structure in the center of some micro-spaces at day 3 of culture. This structure was presented in almost all micro-spaces at day 5. We analyzed hepatocyte specific functions at day 3 and 5. mRNA expression of major cytochrome P450 enzymes, CYP1A2, 3A4 and 2C9, were higher than those of monolayer culture by 3-5 fold, 3-5 fold and 2-3 fold, respectively. Also, mRNA expression of phase II enzymes, UGTs, was highly maintained compared to those with conventional culture method. Expression of UGT1A1 was higher than that of monolayer culture by 2-5 fold. These observations indicated that Micro-Space Culture Plate could be employed for 3-D cell culture by the same handling as used for flat plate. Furthermore, In case of hepatocytes, fundamental functions could be maintained during the culture period so that the plate is expected to be useful for an HTS screening tool to evaluate drug metabolism and toxicity.

P289.NInduced Expressions and Activities of Drug-Metabolizing Enzymes in the Human Hepatocellular Carcinoma Cell Line Cultured On the Micro-Space Cell Culture Plates

Akane Yoshida1, Kaoru Kobayashi1, Yoko Ejiri2, Sachiko Takagi1, Hanaka Mimura1, Masaya Hosoda2 and Kan Chiba1

1Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 2Tsukuba Research Center, Kuraray Co., Ltd., Tsukuba, Japan

Human hepatocellular carcinoma cell lines cultured in monolayer have been limited to use for drug metabolism study because of negligible activities of drug-metabolizing enzymes such as cytochrome P450s (CYPs). Several studies showed that the expression levels of CYPs in human hepatocellular carcinoma cells were increased by three-dimensional cul-ture. However, the three-dimensional culture systems are generally hard to handle. To reduce technical complexity 24-well plates arrayed with uniform micro-sized compartments on the bottom (micro-space cell culture plates) have been developed as a novel system. The purpose of the present study was to evaluate whether human hepatocellular carcinoma FLC4 cells cultured on the micro-space cell culture plates are useful for drug metabolism study. In this study, we compared expression levels and activities of drug-metabolizing enzymes in FLC4 cells cultured on the micro-space cell culture plates with those in monolayer FLC4 cells. When FLC4 cells were cultured on the micro-space cell culture plates, the cells formed spheroids. Results of DNA microarray analyses showed that mRNA expression levels of various drug-metabolizing enzymes including CYPs, UDP-glucuronosyltransferases (UGTs), sulfotransferases and glutathione S-transferases in spheroid cells were higher than those in monolayer cells. Since these genes were expressed in human liver tissues at the high levels, it was thought that the expression profiles of drug-metabolizing enzymes in spheroid cells were similar to those in human liver tissues. Results of real-time PCR analyses showed that expression levels of CYP1A2, CYP2C9, CYP3A4 and UGT1A1 mRNAs in the spheroid cells were remarkably higher than those in monolayer cells. When FLC4 cells were treated with diclofenac (CYP2C9 substrate) or triazolam (CYP3A substrate), significant formations of their metabolites were detected in the medium of spheroid cells. On the other hand, the formations of metabolites were negligible in the medium of monolayer cells. In addition, expression levels of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) mRNAs in spheroid cells were higher than those in monolayer cells. In only spheroid cells, expression levels of CYP3A4 and CYP2B6 mRNAs were also induced by rifampicin (PXR ligand) and CITCO (CAR ligand), respectively. These results suggest that the micro-space cell culture plates are useful tools for drug metabolism study using the human hepatocellular carcinoma cell line.

P290. Assessment of a human in vitro system as an alternative for S-9 fraction to predict in vivo metabolic profiles for drugs

Amin Kamel, Mithat Gunduz, Kevin Colizza, Franco Lombardo and Shawn Harriman

Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, Cambridge, MA, USA

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Drug biotransformation plays a pivotal role at all stages of discovery and development of new chemical entities (NCEs). At early discovery stage, metabolite identification can aid drug design efforts to identify metabolic soft spots and thus lead medicinal chemists to the design of more stable analogues. In the drug development stage, in vivo metabolic profiles of a drugs in preclinical species and humans has recently gained more significance as FDA guidance recommends that human circulating metabolites exceeding 10% of the parent should be present in equal or greater quantities in at least one of the preclinical species used in toxicity assessment. Pooled human liver microsomes, liver S-9 fraction, and hepatocytes are the most common in vitro models used to study drug metabolism and each model has its advantages and disadvantages. The goal of the present study is to establish, if possible, an in vitro system that is simple, cost-effective and possibly more proficient in predicting in vivo metabolic profiles for drugs as compared to S-9 fraction which is relatively deficient in P450 content/activity. The second objective is to assess the ability of this system to generate in vivo metabolic profiles compared to human liver microsomes, liver S-9 fraction, and hepatocytes. Human liver microsomes combined with cytosol and supplemented with relevant cofactors was used as an alternative in vitro system to predict human excretory and circulating metabolite profiles for a small set of commercially available drugs. Sample analysis was carried out on a Thermo-Finnigan LTQ-Orbitrap® with accurate mass measurement and MSn capabilities. Metabolic profiles using this alternative in vitro system were then compared to those obtained with human liver microsomes, liver S-9 fraction, and hepatocytes. Preliminary results suggest that this alternative in vitro system not only reliably produces primary in vivo human metabolites but also pro-duces secondary metabolites and thus has the potential to improve the accuracy of the predictability of the in vivo human metabolites including circulating metabolites. For example, ziprasidone secondary metabolites benzisothiazole piperazine sulfoxide (BITP-SO) and benzisothiazole piperazine sulfone (BITP-SO

2) were not

detected in incubations with human hepatocytes, S-9 fraction or liver microsomes. However, these two sec-ondary metabolites were detected using the microsmes/cytosol system supplemented with relevant cofactors. Efforts are ongoing to apply this approach to a larger set of commercially available drugs in order to establish, if possible, similar trends and thus add sufficient confidence in using this in vitro model as an alternative to predict in vivo metabolic profiles for drugs.

P291. Characterization of the Tight Junction Integrity in Different Renal Cell Cultures

Christian Lechner1, Gert Fricker2, Andrea Thiele3, Andreas Reichel1 and Ursula Moenning1

1Research Pharmacokinetics, Bayer Schering Pharma AG, Berlin, Germany, 2Department of Pharmaceutical Technology and Biopharmacy, Heidelberg University, Heidelberg, Germany, 3Imaging Research, Bayer Schering Pharma AG, Berlin, Germany

The kidney plays an important role in the elimination of exogenous and endogenous substances from the blood. Elimination occurs via passive filtration and active secretion into the urine. A cell-based in vitro model which enables an easy analysis of renal secretion would be of particular value for the characterization of new drug candidates. Since the formation of tight junctions is an important prerequisite of a renal cell culture model, we have characterized the tight junction integrity in cultivated monolayers of the renal cell lines MDCKII (dog), NRK-52E (rat), IHKE-1 and Caki-1 (human), as well as in primary rat proximal tubular cell cultures (PRPTCs). The expression pattern of the tight junction proteins occludin, claudin 2, claudin 7, claudin 10, claudin 11, and ZO-1 was investigated by immunohistochemistry and western blotting, the tightness of cell monolayers has been investigated in functional studies by determination of the transepithelial electrical resistance (TEER) and bidirectional permeation of appropriate marker compounds. It could be shown that the evaluated tight junction proteins are expressed in a cell line specific manner. Whereas the expression of occludin and ZO-1 could be dem-onstrated in all cell lines investigated, the expression of claudins was very heterogenous. MDCKII cells showed a strong expression of claudin 2 and claudin 7, the NRK-52E, IHKE-1 Caki-1 and freshly isolated primary cells lacked the expression of at least one of the claudins. These findings were consistent with the observed tightness of the cell monolayer reflected by the TEER values and the permeation behaviour of paracellular markers. Only MDCKII cells, expressing most of the investigated tight junction proteins at high extent, exhibited a tight barrier characteristic. The other cells tested, which express only a subset of the claudins, were found to be rather leaky. These cells are consequently less applicable for the establishment of a vectorial transport assay for the evaluation of renal secretion.

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P292. Microsomal Protein Binding in IVIVE

Alison Meechan, Lisa McBride, Jon Welch, Fiona Milligan and Stephen Madden

In vitro Sciences, Charles River, Edinburgh, United Kingdom

Hepatic microsomal protein binding is an essential parameter in extrapolating in vitro data to predict in vivo outcomes. In particular, estimations of metabolic clearance and the extent of drug/drug interactions are reliant on an accurate estimate of the free drug concentration in in vitro incubations. In some instances plasma protein binding has been used as a surrogate for microsomal protein binding. In this study we have utilised an automated rapid equilibrium dialysis (RED) system to determine the binding characteristics of 7 small molecule compounds under investigation in our laboratories. Plasma and microsomal protein binding were determined experimentally and microsomal protein binding was estimated using the pKa and logP values of the compounds under investigation. Sample analysis was by HPLC-MS/MS. The compounds selected had pKa and logP values in the range 0.31 to 9.67 and 0.43 to 3.84, respectively. The experimentally determined free fraction in human plasma ranged from 0.2 to 95%. Experimentally determined free fraction in human hepatic microsomes ranged from 1 to 83%, whilst the predicted microsomal free fraction ranged from 40.2 to 98.9%. The level of microsomal and plasma protein binding was found to be approximately equal for only 2 of the compounds (one with high and one with a low level of binding). For the other 5 compounds the microsomal binding was substantially lower in all cases. In conclusion, therefore, the use of plasma protein binding as a surrogate for microsomal binding may lead to significant errors in extrapolating from in vitro to in vivo data, and for some compounds predictive models may also have similar issues.

P293. Evaluation of skin discs for in vitro metabolism studies

Alison Meechan and Daniela Gentile

In vitro Sciences, Charles River, Edinburgh, United Kingdom

The skin is an important organ providing protection to the body from the foreign chemicals that it may become exposed to, either intentionally or by accident. As well as acting as a physical barrier to systemic exposure the skin also possess significant metabolic capability and can activate prodrugs, detoxify noxious chemicals and bioactivate otherwise innocuous compounds. Therapeutically, it has been demonstrated that dermal administration can be more effective for providing systemic exposure to a wide of range compounds rather than can be achieved using the traditional oral route. Although the presence of drug metabolising enzymes (expression and activities) has been demonstrated in a number of human cellular and subcellular dermal models there is a paucity of detailed characterisation data available for the skin disc model which is increasingly employed in skin metabolism stud-ies. It is particularly important to determine the most appropriate compounds to utilise as positive controls (to demonstrate enzyme functionality) when performing such studies. By far, the enzyme activity most widely assessed in skin metabolism is ester hydrolysis. Increasingly the reduction of MTT by mitochondrial reductase enzymes is an assay being utilised as a generic marker of the viability of such models. The validity of the skin disc model will be assessed by detailing the activities of selected Phase I and Phase II enzymes in freshly excised and frozen skin samples from a number of human donors and by demonstrating bioactivation of known toxicants (via assessment of irreversible protein binding).

P294. Vinorelbine′s effects on the cell cycle progression in HeLa cell line

Ozlem Gokce, Akin Yilmaz, Venhar Gurbuz, Ece Konac and Abdullah Ekmekci

Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Ankara, Turkey

Microtubules, having roles in many biological functions including cell motility, cell division, intracellular transport, cellular architecture, are essential structures for cells (1). Their dynamic property involved in cell division makes out of microtubules major targets for anticancer drugs. One of the commonly used drug groups known to suppress micro-tubule dynamics are named vinca alkaloids. By destabilizing the microtubules, these molecules inhibit the metaphase

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anaphase transition, abolish mitosis and induce apoptosis (2). Vinorelbine, a vinca alkaloid, is used for the treatment of many cancers (3). In this study, we aimed to investigate apoptotic and necrotic effects of vinorelbine on HeLa human cervical cancer cells. HeLa cells incubated in a 37 °C humidified 5% CO2 incubator. Cells were treated with different vinorelbine concentrations ranging from 5µM to 100µM at 24 hours. Effects of vinorelbine on cell viability were assessed using XTT assay. After staining cells with ethidium bromide and acridine orange, viable, apoptotic and necrotic cell ratios were determined by fluorescence microscopy. mRNA expression levels of Cyclin D1 and Bcl-2 genes, which are involved in cell proliferation and regulation of apoptosis, respectively, were analyzed with quantitative real time PCR. All experiments were performed in triplicate. We demonstrated that vinorelbine has an antiproliferative and cytotoxic effects on HeLa cells at 24 hours incubation and IC50 value of vinorelbine was above 100µM. Apoptotic response caused by vinorelbine treatment was found to be dose dependent. There were about 1.5 fold increased mRNA levels for the cyclin D1 (p<0.05) and no significant change was seen for the Bcl-2 mRNA levels at 20µM vinorelbine treatment (p>0.05). On the other hand, 80µM vinorelbine treatment resulted in statistically significant decreased cyclin D1 and Bcl-2 mRNA levels, 7.29 fold and 2 fold, respectively (p<0.05). Our results indicated that high vinorelbine concentrations may have more potent inducer of antiproliferative and apoptotic responses on human cervical cancer cells. In addition to this, decrease in antiapoptotic Bcl-2 and cell cycle regulatory cyclin D1 gene′s expression levels is underlined the importance of the vinorelbine in cancer therapy.

References;

1. Jordan MA, Wilson L: Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr Opin Cell Biol 1998, 10:123-130.2. Jordan MA, Wilson L: Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004, 4:253-265.3. Ngan VK, Bellman K, Hill BT, Wilson L, Jordan MA. Mechanism of mitotic block and inhibition of cell proliferation by the semisynthetic Vinca

alkaloids vinorelbine and its newer derivative vinflunine. Mol Pharmacol. 2001;60(1):225-32.

P295. Apoptotic effects of cisplatin on human colon cancer cell line (HT29)

Venhar Gurbuz, Akin Yilmaz, Ozlem Gokce and Ece Konac

Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Ankara, Turkey

Colon cancer is one of the most common malignancy of the gastrointestinal system. It constitutes approximately 10% of colorectal cancers in men and women (1). Cisplatin is a well-known DNA-damaging agent, and it is cur-rently thought that formation of DNA-platinum adducts is an essential first step in its cytotoxic activity. So far, it is generally accepted that cytotoxic effects of cisplatin are ascribed to its interaction with nucleophilic N7-sites of purine bases in DNA to form DNA−DNA intrastrand and interstrand cross-links and DNA−protein bonds (2). Besides, mitochondrial DNA, RNA and other cellular components, including membrane phospholipids, cytoskeletal microfilaments, and thiol-containing proteins, are potent reactants for the platinum structure (3,4). Cisplatin is effective against various tumors as a chemotherapeutic agent and inhibits cell division and growth. In this study, we aimed to investigate the possible apoptotic and cytotoxic effects of cisplatin on colon cancer cells (HT29) as well as its effects on the mRNA expression levels of BCL2L1, CASP3 and CASP8 genes. For this purpose, HT29 cells incubated in a 37 °C humidified 5% CO2 incubator. Cells were treated with different cisplatin concentrations ranging from 5 to 200 µM at 72 hours. The effects of cisplatin on cell viability was evaluated by XTT assay. Viable, apoptotic and necrotic cell ratios were determined using fluorescence microscopy after staining with ethidium bromide and acridine orange. Moreover, BCL2L1, CASP3 and CASP8 gene expression levels were analyzed with quantitative real time PCR. All experiments were performed in triplicate. We found that cisplatin had a cytotoxic effects in a dose dependent manner and IC50 value was found to be 75µM at 72 hours. Cisplatin was also found to induce apoptosis in HT29 cells. There were statistically significant increased mRNA levels for the BCL2L1 (2,639 fold), for the CASP3 (1.871 fold) and for the CASP8 (4.028 fold) genes in 5µM cisplatin treated cells as compared to control cells after 72 hours drug treatment (p<0.05). When we compared to 50µM cisplatin treated cells and control cells, no significant difference was found for the BCL2L1 mRNA levels (p<0.05). However, CASP3 and CASP8 mRNA levels were detected to be elevated 2.206 and 3.053 fold, respectively (p<0.05). In conclusion, we showed that while cisplatin induced apoptosis in HT29 human colon cancer cells in a dose dependent manner, it also changed the expression levels of BCL2L1, CASP3 and CASP8 genes. Furthermore, effective apoptotic cisplatin concentration was showed to be 50µM at 72 hours incubation period.

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References

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59(4):225-49.Ho YP, Au-Yeung SC, To KK. Platinum-based anticancer agents: innovative design strategies and biological perspectives. Med Res Rev.

2003;23(5):633-55.Bose RN. Biomolecular targets for platinum antitumor drugs. Mini Rev Med Chem. 2002;2(2):103-11.Fuertes MA, Alonso C, Pérez JM. Biochemical modulation of Cisplatin mechanisms of action: enhancement of antitumor activity and circumvention

of drug resistance. Chem Rev. 2003;103(3):645-62.

P296. Acute disruption of bone marrow hematopoietic progenitor cells by benzo(a)pyrene (BP) is reversed by processes dependent on Ah Receptor activation

Alhaji U. N′Jai1, Michele Larsen2, Charles J. Czuprynski3 and Colin R. Jefcoate2

1Pathobiological sciences & Molecular and Environmental Toxicology, University of Wisconsin Madison, Madison, WI, USA, 2Pharmacology, University of Wisconsin Madison, Madison, WI, USA, 3Pathobiological Sciences, Molecular and Environmental Toxicology, and Food Research Institute, University of Wisconsin Madison, Madison, WI, USA

We used colony forming unit (CFU) assays to demonstrate rapid suppression (within 6 h) of bone marrow (BM) lym-phoid (CFU-preB) and myeloid (CFU-GM) progenitor cells in 7, 12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP) treated C57BL/6 mice. The effects of BP are largely reversed with time (48-168 h), whereas those of DMBA increased. The duration of these changes were consistent with the blood levels of DMBA or BP and their metabolites following either IP or oral administration. BP and DMBA treatments both resulted in sustained BM toxicity in mice expressing a PAH-resistant Ah Receptor (AhRd), suggesting that AhR activation is required to reverse the adverse effects of BP. Peripheral blood cell numbers were also reduced following DMBA or BP treatment. Reduction in blood cell numbers did not occur in Cyp1b1 null mice, indicating a requirement for extra-hepatic PAH bioactivation by Cyp1b1. Gene expression responses to DMBA showed constitutive activation of AhR linked genes in the BM adherent (stromal rich) cell fraction, whereas AhR linked genes needed to be induced by PAH treatment in the nonadherent cell fraction. BM adherent cell (stromal rich) gene responses to BP were more extensive than for DMBA, however AhR linked genes (Cyp1a1, Cyp1b1, AhRR, Spint1) responded equally to DMBA and BP. PAH treatment also altered expression of several developmental genes (Spint1, Ankra2, Egr1, Epha5, and Nfatc3) identified as novel AhR targets. These AhR linked responses were reversed in BP treated AhRd mice, consistent with a requirement for AhR activation. BP treatment selectively increased gene expression of inflammatory factors (Cxcl2, Tnf, Cxcl10, Cox2 and IL1β) that are typically induced by oxidant-sensitive transcription factors. The absence of these inflammatory markers in AhRd mice suggests that AhR activation by BP mediates pro-oxidant signaling, that in turn may be required for restoration of CFU progenitor cell functionality.

P297. Association of blood level of methemoglobin with clinical outcome of patients with aluminium phosphide poisoning

Babak Mostafazadeh1, Esmaeil Farzaneh2 and Tahmineh Afsharian3

1Medical Toxicology & Forensic Medicine, Shaheed Beheshty University Of Medical Sciences, Tehran, Iran, 2Medical Toxicology & Forensic Medicine, Ardabil University Of Medical Sciences, Ardabil, Iran, 3Private Physician, Tehran, Iran

Although methemoglobinemia following aluminum phosphide (AlP) intoxication has been reported, probable effect of blood level of methemoglobin (Met-Hb) on outcome of AlP intoxicated patients has not yet been investigated. This study aimed to evaluate this probable association. Methods: This prospective study was carried out at Loghman-Hakim poison hospital from April 2009 to August 2009 All patients aged >12 years who had ingested AlP and were admitted at the hospital were enrolled into the study. Using the co-oximetry, Met-Hb was measured at the time of admission and was repeated 24 hours later if the patient survived. Results: 48 AlP intoxicated cases including 24 males were enrolled into the study. Mean age of the patients was 25.5 ± 9.5 years. There was significant association between Met-Hb blood level

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which measured at the time of admission and mortality (2.4%± 7.1% in survivors versus 15.2%± 13.5% in non-survivors, P<0.001). The same association was found at the 2nd day of admission (2.9%±8.2% in survivors versus 26.5%±19.9% in non-survivors, P=0.02). Conclusions: The present study found an association between blood level of Met-Hb and mortality in AlP intoxicated cases. Whether prophylactic administration of vitamin C and methylene blue can improve outcome of AlP-intoxicated cases should be investigated in future investigations. Reference Shadnia S, Sasanian G, Allami P, Hosseini A, Ranjbar A, Amini-Shirazi N, Abdollahi M. A retrospective 7-years study of aluminum phosphide poisoning in Tehran: opportunities for prevention. Hum Exp Toxicol 2009;28:209-13.

P298. Attenuation of Neurotoxicity Induced by Sub-Chronic Paraquat Administration in Rats by Deprenyl, Quercetin, Green Tea or Malt Extract

Nesrine Salah El Dine El Sayed1 and Hala Fahmy Zaki2

1Pharmacology and Toxicology, Lecturer of Pharmacology & Toxicology, Cairo, Egypt, 2Department of Pharmacology and Toxicology, Associate professor of Pharmacology & Toxicology, Cairo, Egypt

The possible protective potentials of deprenyl (10 mg/kg, i.p.), quercetin (50 mg/kg, p.o.), green tea (1 mg/kg, p.o.) and malt extract (625 mg/kg, p.o.) against sub-chronic paraquat (PQ)-induced neurotoxicity in rats were examined. PQ was administered once weekly as a single injection (20 mg/kg, i.p.) with or without daily pretreatment with any of the chosen agents for 6 successive weeks. Survival rates and changes in total weight gain of animals were assessed. Changes in the enzymatic activities of myeloperoxidase (MPO), superoxide dismutase (SOD) and lactate dehydrogenase (LDH) as well as reduced glutathione (GSH), protein thiols (Pr-SHs) and nitric oxide (NO) contents of the brain were determined. In addition, brain content of thiobarbituric acid reactive substances (TBARS) was measured as an index of lipid peroxida-tion. Sub-chronic PQ administration resulted in 50% mortality and significant reductions in animals′ weight gain as compared to the normal group. On the biochemical level, PQ provoked remarkable brain damage noted by elevation of neutrophils MPO activity accompanied by decreased activities of cytosolic SOD and LDH, depletion of GSH and Pr-SHs contents as well as increased production of NO and TBARS. Daily treatment with any of the chosen agents for 6 weeks significantly ameliorated the PQ-induced reduction in body weight gain of rats and protected against most of PQ-induced brain biochemical changes. It could be concluded that deprenyl, quercetin, green tea and malt extract offered remarkable neuroprotection against PQ-induced brain injury. The most pronounced neuroprotective effects was produced by deprenyl which was the only drug that completely prevented PQ-induced mortality.

P299. Benefit Effect of Naloxone in Benzodiazepines Intoxication: Findings of a Preliminary Study

Babak Mostafazadeh1, Esmaeil Farzaneh2 and Tahmineh Afsharian3

1Medical Toxicology & Forensic Medicine, Shaheed Beheshty University Of Medical Sciences, Tehran, Iran, 2Medical Toxicology & Forensic Medicine, Ardabil University Of Medical Sciences, Ardabil, Iran, 3Private Physician, Tehran, Iran

Background: Naloxone, as a low-priced and available drug, may be useful in improvement of signs and symptoms of benzodiazepines intoxication. The aim of this study was assessment of its effect on benzodiazepines poisoning.

Methods: In this clinical trial study, patients with typical signs and symptoms of benzodiazepines poisoning who referred to a poisoning center in Tehran in 2008 were selected. After recording of patient characteristics, supportive treatment initiated and patients randomly assigned to IV injection of two 0.4 mg naloxone ampules group (case) or control group. Their signs and symptoms were evaluated 0.5 hour later again. Each of diazepam, clonazepam and alperazolam drug group had 30 patients and lorazepam drug group had 26 patients which half of the patients in each drug group received naloxone.

Results: Most of participants were female and the mean age was 28 yr. There were no significant differences between case and control groups in age, sex, time of drug consumption, tablet counts, sings and symptoms and level of conscious-ness in admission time in each drug types. After naloxone injection in case groups, all signs and symptoms significantly improved in all drug tyeps in comparison to control groups except nystagmus. In addition, level of consciousness significantly improved in case groups in all drug types except lorazepam.

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Conclusion: Findings of the study showed that naloxone is effective in management of benzodiazepines poisoning. However, future clinical trials with greater sample size are recommened.

Reference

Seger DL.; Flumazenil-treatment or toxin.; J Toxicol Clin Toxicol. 2004: 42(2):209-16.Seger DL.; Flumazenil-treatment or toxin.; J Toxicol Clin Toxicol. 2004: 42(2):209-16.

P300. Bioactivation of Glafenine by Human Liver Microsomes and Peroxidases: Identification of Electrophilic Iminoquinone Species and Glutathione Conjugates

Bo Wen1, Yanzhou Liu2 and Tian J. Yang3

1Drug Metabolism and Pharmacokinetics, Hoffmann-La Roche, Nutley, NJ, USA, 2Analytical Chemistry, Roche Palo Alto, Palo Alto, CA, USA, 3Dept of Non-Clinical Safety, Hoffmann-La Roche Inc., Nutley, NJ, USA

Glafenine (Privadol; 2,3-dihydroxypropyl 2-[(7-chloro-4-quinolinyl)amino]benzoate) is a non-narcotic analgesic agent widely used for the treatment of pains of various origins. Severe liver toxicity and a high incidence of anaphylaxis were reported in patients treated with glafenine, eventually leading to its withdrawal from market in most countries. It is proposed that bioactivation of glafenine and subsequent binding of reactive metabolite(s) to critical cellular proteins play a causative role. The study described herein aimed at characterizing pathways of glafenine bioactivation and the metabolic enzymes involved. Two glutathione (GSH) conjugates of glafenine were detected in human liver microsomal incubations using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The structures of detected conju-gates were determined as GSH adducts of 5-hydroxyglafenine (M3) and 5-hydroxy glafenic acid (M4), respectively. GSH conjugation took place with a strong preference at C-6 of the benzene ring of glafenine, ortho to the carbonyl moiety. These findings are consistent with a bioactivation sequence involving initial P450-catalyzed 5-hydroxylation of the benzene ring of glafenine, followed by two electron oxidations of M3 and M4 to form corresponding para-quinone imine intermediates that react with glutathione to form GSH adducts M1 and M2, respectively. Formation of M1 and M2 was primarily catalyzed by heterologously expressed recombinant CYP3A4, and to a less extent, CYP2C19 and CYP2D6. We demonstrated that M3 can also be bioactivated by peroxidases, such as horseradish peroxidase and myeloperoxidase. In summary, these findings suggested that bioactivation of glafenine may result in GSH depletion, oxidative stress and covalent binding to proteins, potentially leading to hepatotoxicity.

P301. Cyclopiazonic acid toxic effects on the liver and kidneys: biochemical and histopathological studies

1Malekinejad H, 1Akbari, P, 2Hobbenaghi R, and 1Varasteh S 1Department of Pharmacology & Toxicology and 2 Department of Pathology, faculty of Veterinary Medicine, Urmia University, Urmia Iran

The effect of cyclopiazonic acid (CPA) on biochemical markers in the liver and kidneys of the broilers was studied. Ten-day old male broiler chicks (Ross 308) were assigned to the control and test groups, which received normal saline and 10, 25 and 50 mg/kg CPA, respectively for 28 days. Body weight gain, serum level of ALP, GGT, uric acid, creatinine, and blood urea nitrogen (BUN) were measured after 2 and 4 weeks exposure. Serum levels of bivalent cations including Ca2+, Mg2+, and Fe2+ were also determined. To confirm the biochemical changes, histopathological examinations were conducted on either organ. No significant differences (P>0.05) in body weight gain was found between the control and test groups. While the hepatic weight increased significantly (P<0.05) in animals that received 25 and 50 mg/kg CPA. Both ALP and GGT level in serum were elevated in comparison to the control group. CPA also resulted in uric acid, creatinine and BUN enhancement in broilers. Serum level of divalent cations following exposure to CPA for 2 and 4 weeks declined in a dose- and time-dependent fashion. Among the others reduction of Ca2+ level was more pronounced. Histopathological findings confirmed the biochemical changes in both examined organs characterized by inflammatory

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cells infiltration along with severe congestion and cell swelling suggesting an inflammatory response. These data sug-gest that exposure to CPA resulted in hepatic and renal disorders, which were reflected as biomarkers alteration and pathological injuries in either organ. The biochemical alteration and pathological abnormalities may be attributed to CPA-induced inflammatory reactions and divalent cations changes.

P302. Deuterium insertion approach to reduce metabolism-mediated liabilities: Integration of biotransformation results and structural modification into potential toxicity findings

Amin Kamel, Mithat Gunduz, Natalie Dales, Clayton Springer, Suzie Ferreira, Franco Lombardo and Shawn Harriman

Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, Cambridge, MA, USA

Integration of biotransformation results and structural modification into potential toxicity and DDI findings is a key to better understand/assess metabolism-mediated liabilities. Structural modification via deuterium insertion approach could play a pivotal role in reducing/blocking undesirable metabolic pathways and help in guiding of the back-up program efforts. Compound 1 undergoes several oxidative pathways including oxidative N-dealkylation and subse-quent oxidation of the putative cyclopropyl aldehyde intermediate to form the cyclopropyl carboxylic acid (CPCA). Literature suggests that CPCA may generate unusual acyl-CoA metabolites, which may have direct toxicity, limit cellular CoA availability due to acyl-CoA accumulation, or limit cellular carnitine availability as acylcarnitines are generated from acyl-CoA. In vitro metabolite identification studies were conducted in order to understand the effect of structural modifications on the metabolism of compound 1. The experiments were carried out using human liver microsomes and structure elucidation was carried out on a Thermo-Finnigan LTQ-Orbitrap® with accurate mass measurement and MSn capabilities. Efforts to eliminate the formation of CPCA by inhibiting/blocking the requisite N-dealkylation reaction by removal of the abstractable a-hydrogen and replacement with either a methyl group or a deuterium were successful. Furthermore, the rate of the oxidative N-dealkylation pathway was studied in rat, monkey and human hepatocytes.

Proposed metabolic pathway for the generation of cyclopropyl carboxylic acid (CPCA) from compound 1

Possible mechanism of toxicity associated with CPCA structural alert fragment based on literature

P303. Effect of Exposure to Bile Salts on Urea Formation in Sandwich-Cultured Rat Hepatocytes

Sagnik Chatterjee, Patrick Augustijns and Pieter Annaert

Laboratory for Pharmacotechnology and Biopharmacy, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium

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Sandwich-cultured hepatocytes have been shown to develop functional bile canaliculi after several days in culture. This means that the vectorial bile acid transport and interference of xenobiotics and endogenous compounds with these transport mechanisms can be investigated in this in vitro model. It follows that this model may be used to study hepatotoxicity due to disruption of bile salt homeostasis and disposition (1). The aim of this study was to determine the in vitro toxicity of individual bile salts, based on the evaluation of the urea cycle function. After isolation, rat hepatocytes were cultured in a double collagen gel in sandwich configuration. Twenty four hr after isolation, cholic acid (CA), chenodeoxycholic acid (CDCA), taurocholic acid (TCA) and glycocholic acid (GCA) at 5, 50, 100, 500 µM and 1 mM were included in the culture medium for 18 hr. Deoxycholic acid (DCA) and a mixture of CA: CDCA: DCA (1:1:1), were examined at 5, 50, 100, 250 and 500 µM. After culturing hepatocytes in presence of the bile salts, they were washed twice with PBS, subsequently, they were incubated with arginine-free incubation buffer containing HBSS, 3 mM ornithine, 2 mM glutamine and 10 mM ammonium chloride for 1 hr at 37°C. Finally urea content was determined colorimetrically after reaction with diacetyl monoxime. For this purpose we developed and applied a hitherto unreported 96 well format urea assay. Among the bile salts examined DCA, CDCA and the bile salts mixture showed consistent toxicity in 5 dif-ferent batches of hepatocytes, with mean IC

50 values of 109, 81 and 91µM, respectively. TCA, GCA and CA appeared

to be non-toxic. Our data are consistent with previous reports of bile salt toxicity in different other in vitro and in vivo systems (2). Research is ongoing to investigate the toxicity of those bile salts after 3-4 days in culture, when the bile canaliculi have shown to be fully functional. This will allow us assesing the possible effect of bile canalicular network formation on bile salt toxicity in vitro.

References

1. Tracy L. Marion, et al., Use of Sandwich-Cultured Hepatocytes To Evaluate Impaired Bile Acid Transport as a Mechanism of Drug-Induced Hepatotoxicity. Mol Pharm, 2007. 4(6): p. 911-8.

2. Anabela P. Rolo, et al., Interactions of combined bile acids on hepatocyte viability: cytoprotection or synergism. Toxicol Lett, 2002. 126(3): p. 197-203.

P304. Effect of Phenobarbital on the Cytotoxicity of Valproic Acid and 2-Ene-Valproic Acid in Sandwich Cultures of Rat Hepatocytes

Jayakumar Surendradoss, Stoyan Karagiozov, Thomas K. H. Chang and Frank S. Abbott

Faculty of Pharmaceutical Sciences, Univ of British Columbia, Vancouver, BC, Canada

Therapeutic use of valproic acid (VPA) is associated with a rare but fatal idiosyncratic hepatotoxicity that is character-ized by microvesicular steatosis. Formation of reactive metabolites of VPA is one of the mechanisms suggested to be responsible for this hepatotoxicity. VPA undergoes cytochrome P450-catalyzed terminal desaturation to form 4-ene-VPA, which is then subject to mitochondrial beta-oxidation to form 2,4-diene-VPA. Alternatively, VPA undergoes beta-oxidation to form 2-ene-VPA, which upon cytochrome P450-mediated oxidation, results in the formation of 2,4-diene VPA. The direct administration of 2,4-diene-VPA is cytotoxic to cultured rat hepatocytes. However, inhibit-ing the in situ metabolic formation of 2,4-diene-VPA did not affect the toxicity in hepatocytes treated with VPA. Thus, the role of reactive metabolites in VPA-associated hepatotoxicity is still not clear. Therefore, to further investigate the role of reactive metabolites in VPA toxicity, we compared the extent of oxidative stress, steatosis, and necrosis by VPA and 2-ene-VPA in primary cultures of rat hepatocytes pretreated with phenobarbital, which is known to increase the in situ metabolic formation of 2,4-diene-VPA. Hepatocytes were isolated from adult male Sprague-Dawley rats and cultured in a sandwich configuration with MatrigelTM. Cultured hepatocytes were pretreated with sodium pheno-barbital (100 µM) or culture medium (vehicle control) for 72 h followed by treatment with various concentrations of VPA (the sodium salt), 2-ene-VPA or culture medium (vehicle control) for 24 h. Oxidation of 2′,7′-dichlorofluorescin to 2′,7′-dichlorofluorescein (DCF; a marker of cellular oxidative stress), intracellular accumulation of BODIPY® 558/568 C12 (a marker of steatosis) and release of lactate dehydrogenase (LDH; a marker of necrosis) were measured. Control experiments confirmed that treatment of cultured hepatocytes with phenobarbital increased CYP2B-mediated benzyloxyresorufin-O-dealkylation (BROD) activity. VPA or 2-ene-VPA treatment alone elevated the levels of DCF and BODIPY 558/568 C12, and increased the cellular release of LDH. Pretreatment with phenobarbital enhanced the effect of 2-ene-VPA but not VPA, on DCF (oxidative stress), BODIPY (steatosis) and LDH (necrosis) levels in cultured rat hepatocytes. As expected, control experiments showed that phenobarbital increased the effects of acetaminophen

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on DCF, BODIPY and LDH levels. Thus, phenobarbital pretreatment enhances the cytotoxicity of 2-ene-VPA but not VPA in primary cultures of rat hepatocytes. Studies are currently underway to investigate the mechanism responsible for the selective enhancement of cytotoxicity of 2-ene-VPA by phenobarbital. [This research was supported by the Canadian Institutes of Health Research (Grant MOP-13744) and Michael Smith Foundation of Health Research (senior scholar award to T.K.H.C.)].

P305. Erdosteine has dual effects on haemostasis via its different metabolites in young rats

Süleyman Oktar1, Vefik Arica2, Murat Tutanc2 and Oktay Hasan Ozturk3

1Pharmacology, Medical Faculty of Mustafa Kemal University, Hatay, Turkey, 2Pediatrics, Medical Faculty of Mustafa Kemal University, Hatay, Turkey, 3Biochemistry, Medical Faculty of Mustafa Kemal University, Hatay, Turkey

Erdosteine is a mucolytic and specifically it is a thiol derivative developed for the treatment of chronic obstructive bronchitis, including acute infective exacerbation of chronic bronchitis. In many studies, erdosteine has been suc-cessfully used as a preventive agent against various toxic agents. Therefore, we have performed the effect of erdosteine (10mg/kg, 7 days) on methotrexate induced testicular toxicity in C57BL/6 mice (8 weeks, 20–30 g) (1). We noticed that erdosteine qualitatively accelerated clotting in mice. There was no information in the prospectus of ERDOSTIN® and in the literature on this subject. Erdosteine is often used for 3-5 days in children as the mucolytic. We investigated the effect of erdosteine on platelet function and coagulation cascade in this study. Total 29 Wistar female/male rats (3 weeks old) divided 4 groups. We were peeled capsules of ERDOSTIN® and drug powder was dissolved in sodium bicarbonate. Six rats assigned control, group3 (n=7) was given 3mg/kg erdosteine, group10 (n=7) was given 10 mg/kg erdosteine and group30 was given 30mg/kg erdosteine for 3 days. Rats were killed 24 hours after the last drug administration. Portal vein was cut and 1 ml of blood were collected. It has been examined the Protrombin time (PT), active protromboplastin time (aPTT), international normalized ratio (INR) and clotting process. At the same time, we examined platelet counts in peripheral blood samples. PT/INR values in group3 prolonged compared to the control group (12.53 ± 1.08/0.990.10 vs 11.16 ± 0.81/0.86 ± 0.07, p=0.03). Blood in citrated tubes of group10 quickly became clots compared with control (77% vs 0%) ana so we could not measured PT/INR, PTT values in group10. The platelets were aggregated in peripheral blood of the same group. There was no difference in PTT values among groups. There were no differences in any parameters in group30. It is interesting that erdosteine have dual effects on platelets and coagulation cascade. The cause of this dual effects is unclear. Erdosteine rapidly metabolized into at least 3 active metabolites containing free thiol groups, which tentatively are: N-thioglycolyl-homocysteine (met I), N-acetyl-homocysteine (met II), and homocysteine (met III). N-acetylcysteine causes abnormal haemostatic activ-ity in humans and the inhibition of clotting factor activities may include destabilization of disulphide bonds in the proteins (2). Met I contains a pharmacologically active sulphydryl group and it may be responsible for the prolonged time of PT/INR in treated erdosteine rats. Homocysteine increases platelet aggregation induced by Adenosine diphos-phate (3). We suggest that different metabolites of erdosteine may have different influence on haemostasis. 1. Oktar S, Gokce A, Aydin M, Davarci M, Meydan S, Ozturk OH, Koc A. Beneficial effect of erdosteine on methotrexate-induced testicular toxicity in mice. Toxicology and Industrial Health 2010 (Epub ahead of print) 2. Zanin RF, Campesato LF, Braganhol E, Schetinger MR, Wyse AT, Battastini AM. Homocysteine decreases extracellular nucleotide hydrolysis in rat platelets. Thrombosis Research 2010;125(3):e87-92. 3. Knudsen TT, Thorsen S, Jensen SA, Dalhoff K, Schmidt LE, Becker U, Bendtsen F. Effect of intravenous N-acetylcysteine infusion on haemostatic parameters in healthy subjects. Gut 2005;54:515–521.

P306. Evidences of covalent adduct formation of quinoline compounds and their phase I metabolites with glutathione and N-acetyl cysteine

Bhagwat Prasad1, Amit Garg1, Saranjit Singh1 and Rahul Jain2

1Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Punjab, India, Mohali, India, 2Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Punjab, India, Mohali, India

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Ring-substituted quinolines are effective anti-infective molecules, particularly against malaria and tuberculosis (1-4). In the present study, we investigated toxicity potential of eight such molecules and their phase I metabolites belonging to four different sub-categories, i.e., 8-aminoquinoline, quinolinecarbohydrazide, quinolinecarboxamide and quinoline-carbaldehyde. The assessment was done by looking into the chances of formation of covalent adducts of the molecules or their metabolites with glutathione (GSH) or N-acetyl cysteine (NAC). First, MS and LC parameters were optimized for individual molecules, and their fragmentation pathways were delineated based on Q-TOF (Bruker Daltonics) and MSn (Thermo) studies. The in vitro metabolism studies were performed by incubating the molecules (10 µM) in human and rat liver microsomes (1 mg/ml) supplemented with GSH and NAC, separately. NADPH was used as cofactor in the reactions. The drug or metabolite adducts with nucleophiles were identified using LC-Q-TOF and LC-MSn analyses. Among the tested molecules, primaquine, NP-96, NP-446 and NP-486 were observed to form covalent adducts with the nucleophiles. The presence of adducts with GSH and NAC were detected based on the CNLS of 129 Da in positive and negative modes, respectively. While, the same were confirmed by the unique product ions in negative mode, i.e., fragment of m/z 272 in GSH, and ions of m/z M+32 in NAC adducts. Further, all the four molecules were also found to form reactive metabolites. For example, in case of primaquine and NP-96, conversion of 5-hydroxy products into cor-responding aminoquinones was the possible mechanism. Similarly, for NP-446, two hydroxy products were postulated to form reactive intermediates. Although hydroxylation of NP-486 in the quinoline ring lead to formation aminoquinone intermediate, it also showed possibility to form quinone methide on para-hydroxylation of phenyl ring. The results were supported by the in silico prediction made by DEREK software. The detailed structures and mechanisms of adduct formation will be presented and discussed during the presentation.

References

Vangapandu S, Sachdeva S, Jain M et al., Bioorg Med Chem. 11 (2003) 4557-68.Nayyar A, Jain R. Cur Med Chem. 12 (2005) 1873-86.Nayyar A, Malde A, Jain R, et al. Bioorg Med Chem. 14 (2006) 847-56.Nayyar A, Jain R. Indian J Chem. 47 (2007) 117-28.

P307. Exploiting the Third Dimension for Predicting Drug Induced in vitro Nephrotoxicity

Sreenivasa Ramaiahgari1, Bob van de Water2 and Leo Price3

1Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, Netherlands, 2Div. of Toxicology, LACDR, Leiden University, Leiden, Netherlands, 3Toxicology, Leiden University, Leiden, Netherlands

The epithelial tubular cells of the kidney are highly susceptible to adverse drug reactions, resulting in frequent drug attrition and withdrawal. High throughput cellular assays currently used in preclinical drug safety testing use two-dimensional tissue culture models that are poorly predictive of the effect of compounds in humans. This is principally due to the poor differentiation status resulting in aberrant drug handling and cellular response. To overcome this, three-dimensional cell culture models are being developed that mimic biochemical aspects of a real tissue and can maintain tissue function and homeostasis to a higher extent than monolayer cultures. In 3D cultures, cells form cysts and tubules, which are basic structural units of epithelial organs. We have developed a three-dimensional cell culture model for nephrotoxicity testing using conditionally immortalized mouse proximal tubular epithelial cells (IMPTECs). IMPTECs undergo morphogenesis to form tubular networks in 3D, a process that can be disrupted by nephrotoxicants. Furthermore, the response of established 3D epithelial networks to toxicants differs from responses in 2D. The under-lying mechanisms of these responses are poorly understood and may be determined intrinsically by specific genetic programs and extrinsically by cell-ECM and cell-cell interactions. Exploring the pathways activated in response to cell injury will give us more insights for prediction of toxicity, risk assessment and mechanism of action of various xenobi-otics. We are studying the effect of different classes of nephrotoxic compounds on tissue development, maintenance and function. The assay that we have developed is implemented in a 384 well format for low cost and potential high throughput assays. In contrast to conventional end points of cell apoptosis and necrosis we measure the changes in the organoid morphology in response to compound exposure, which is compared with gene expression data. By analyzing the cellular phenotype of nephrotoxic compounds and by comparing the molecular pathways they regulate with non-nephrotoxic compounds, we hope to establish links between pathways and particular adverse effects. This high content

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analysis approach offers an alternative and potentially more predictive and qualitative means of monitoring effects of compounds in vitro. The availability of state of the art in vivo-like 3D cell culture system, robotics, data process and control software, liquid handling devices, high content image capturing (confocal) microscopes will allow us to identify the compounds that disrupt cellular function on a larger scale.

P308. Human Polymorphisms in the Glutathione Transferase Zeta 1 Maleylacetoacetate Isomerase Gene Predict the Kinetics and Toxicity of Dichloroacetate

A. Larry Shroads1, Taimour Langaee4, Bonnie S. Coats1, Tracie L. Kurtz1, John Bullock1, David Weithorn1, Yan Gong4, David Wagner5, David A. Ostrov2, Julie A. Johnson4 and Peter W. Stacpoole1,3 1Departments of Medicine (Division of Endocrinology and Metabolism), 2Pathology, Immunology and Laboratory Medicine and3Biochemistry and Molecular Biology, College of Medicine and Department of Pharmacotherapy and Translational Research,4Center for Pharmacogenomics, College of Pharmacy, University of Florida, Gainesville, FL; and Metabolic Solutions, NashauThe investigational drug dichloroacetate (DCA) is dehalogenated to glyoxylate by the zeta-1 family isoform of glutath-ione transferase (GSTz1). This enzyme is identical to maleylacetoacetate isomerase (MAAI), the penultimate enzyme of the phenylalanine/tyrosine catabolic pathway. We tested the hypothesis that polymorphisms in the GSTz1/MAAI gene modify the kinetics of DCA and, consequently, the risk of adverse effects from the drug. Twelve healthy adults were genotyped to determine GSTz1/MAAI haplotype and received 5 days of 25mg/kg/d oral DCA. 1, 2- 13C-DCA was administered on days 1 and 5, during which formal kinetic testing was done. Six children who previously participated in a randomized clinical trial of DCA for congenital lactic acidosis were also genotyped and their kinetic data reexamined in light of haplotype variations. Results: GSTz1/MAAI haplotype clearly segregated subjects into fast and slow DCA metabolizers. Those who metabolized DCA slowly showed markedly delayed plasma clearance, increased excretion of unmetaboized drug and increased urinary accumulation of potentially toxic tyrosine metabolites. Atomic modeling of the effect of GSTz1/MAAI polymorphism disclosed that variants associated with slow DCA metabolism induced structural changes in the enzyme homodimer leading to protein instability or abnormal protein-protein interactions. Conclusions: GSTz1/MAAI haplotype predicts the toxicogenetics of DCA. This information can be used prospectively to adjust drug dosing and mitigate risk of adverse events.

P309. Influence of valproate on the levels of tri- and diphosphate nucleotides in rat liver tissues

Paula B. M. Luis1, Lodewijk IJlst2, Jeroen Roelofsen3, André B.P. van Kuilenburg3, Marinus Duran2, Isabel Tavares de Almeida4, Ronald J.A. Wanders2 and Margarida F.B. Silva4

1Metabolism and Genetics group, iMED.UL, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal, 2Laboratory Genetic Metabolic Diseases, Dep. Clinical Chemistry and Pediatrics, AMC, University of Amsterdam, The Netherlands, Amsterdam, Netherlands, 3Laboratory Gentetic Metabolic Diseases, Dep. Clinical Chemistry and Pediatrics, Amsterdam Medical Centre, University of Amsterdam, Amsterdam, Netherlands, 4Metabolism and Genetics group, iMED.UL, Centro de Patogenese Molecular, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal

Background: Valproic acid (VPA) is an effective antiepileptic drug that induces progressive microvesicular steatosis, unequivocally associated with an impairment of mitochondrial function. Objectives: To investigate the influence of VPA treatment in vivo on the levels of hepatocellular tri- and diphosphate nucleotides.

Methods: Two groups of Wistar rats (n=2 × 10) were treated with one single i.p. injection of sodium valproate (100, 500 mg/kg). A third group of rats (n=10) was daily treated for 2 weeks with 100 mg/kg of drug. The respective control groups were treated similarly with 0.9% saline (n=10 single dose, and n=12 subchronic regimen). Adenosine, guanosine, cytidine and uridine triphosphates and diphosphates were determined by HPLC.

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Results: A significant decrease of the ATP/ADP ratio was observed in liver tissues of rats treated with both single doses of sodium valproate as compared with controls. Guanosine, cytidine and uridine tri- and diphosphate levels were not changed in the livers of the same rats. There was no significant difference in the levels of the measured nucleosides in the liver tissues of rats submitted to subchronic treatment.

Discussion: The ratio ATP/ADP was significantly decreased by VPA at the onset of the treatment. However after subse-quent administration of the drug this ratio returned to control levels. Since the remaining tri- and diphosphate nucle-otides were not altered by VPA, the observed effect seems to affect only the cellular energetic status, which may account for the pathogenesis of adverse effects associated either with the start of VPA therapy or with an overdose of VPA.

P310. Mechanism of Hepatoprotective Effect of Tamoxifen Against Drug-induced Liver Injury

Tsuyoshi Yokoi1, Yukitaka Yoshikawa1, Yasuyuki Toyoda1, Satonori Higuchi1, Tohru Tsukui2, Koichi Tsuneyama3, Tatsuki Fukami1 and Miki Nakajima1

1Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan, 2Saitama Medical University, Research Center for Genomic Medicine, HIdaka, Japan, 3Graduate School of Medicine and Pharmaceutical Science for Research, Department of Diagnostic Pathology, Toyama, Japan

Drug-induced liver injury (DILI) is one of the major problems in drug development and clinical drug therapy. However, the underlying mechanism in DILI is not well understood. The administration of ERα agonists, such as estradiol and ethinylestradiol (EE2), is known to cause both aggravate and protect against liver injury, suggesting that ERα is involved in liver injury. In this study, DILI caused by acetaminophen (APAP), bromobenzene (BB), diclofenac (DIC), and thioa-cetamide (TA) was suppressed by tamoxifen (TAM) pretreatment in female mice. DNA microarray analyses suggested that monocyte to macrophage differentiation-associated 2 (Mmd2), whose function was unknown, could be a candi-date gene for liver protection. To investigate the Mmd2 function in DILI, Mmd2 mRNA was knocked down in mice by injection of siRNA for Mmd2 (siMmd2). A significant decrease of Mmd2 mRNA to about 30% was obtained. In Mmd2 knockdown and TAM-pretreated mice, TA-induced liver injury was exacerbated. In addition, administration of TAM to ERα knockout mice did not change the Mmd2 mRNA level, suggesting that Mmd2 was regulated via ERα. In TAM-pretreated mice, amphiregulin (Areg: known as a gene for liver regeneration) mRNA was increased, suggesting that the one of the mechanisms of the liver protective function of Mmd2 was liver regeneration via the increase of Areg. In contrast to mouse, MMD2 mRNA was hardly expressed (0.1% of mouse) in human liver. This is the first report of the involvement of ERα in DILI, and the hepatoprotective mechanism of Mmd2 induction via ERα was also elucidated.

P311. Metabolic Activation of Chlorogenic Acid in vitro and in vivo

Xiaoyan Chen, Cen Xie, Tao Yuan and Dafang Zhong

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Chlorogenic acid (5-caffeoylquinic acid, 5-CQA), is a major active ingredient in some Chinese herbal injections. In recent years, occasional hypersensitivity reactions of these injections have been reported in clinical use. Due to the existence of catechol and a, b-unsaturated carbonyl groups in the molecule, 5-CQA is susceptible to produce reactive metabolites that might link to possible toxicity. The study described herein aimed at characterizing pathways of 5-CQA bioactivation and the CYP enzymes involved. In incubations of 5-CQA with NADPH- and GSH-supplemented human liver microsomes, fifteen GSH conjugates were detected by UPLC/Q-TOF mass spectrometry. Several metabolite stand-ards were synthesized chemically and used to confirm metabolite structures. These conjugates could be mainly classified as two types. GSH conjugates at m/z 658 were formed via 1,4-addition of GSH to the corresponding ortho-quinone, while the site of GSH addition was identified mainly at C-2 of the aromatic ring. GSH conjugates at m/z 660 were formed following GSH react with a, b–unsaturated carbonyl groups of 5-CQA, while the site of GSH addition was determined at C-7. The formation of ortho-quinone intermediate was NADPH-dependent. Isoform profiling with recombinant human P450s suggested that multiple enzymes, including CYP1A2, 2C19, 2D6, 2E1 and 3A4 were primarily responsible for this formation. Inhibition of GSH conjugates formation in human liver microsomes by CYP isoenzyme-selective inhibitors

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supported these findings. O-methylated metabolites were observed as major metabolites in rat urine, feces and plasma following a single i.v. dose of 10 mg/kg 5-CQA. O-methylation occurred at catechol group and could prevent the forma-tion of ortho-quinone intermediate. The predominant metabolites in bile were GSH conjugates of methylated 5-CQA (m/z 674), accounting for approximately 80% of the metabolites excreted in bile. They are formed by Michael addition of GSH to a, b-unsaturated carbonyl group. These results demonstrate that 5-CQA and its ortho-quinone intermediate are highly reactive to nucleophiles such as GSH. This finding indicates that 5-CQA may covalently bind to cellular proteins, leading to toxicity, including hypersensitivity and other immune reactions.

P312. Nephrotoxicity Induced by Fenthion IN ADULT RATS AND THEIR SUCKLING PUPS

Mediha Sefi

Life sciences, Faculty of sciences, Sfax, Tunisia

Environmental pollution by pesticide residues is a serious international concern due to their extensive use in agriculture and in public health programs. Fenthion is an organophosphorus pesticide widely used throughout the world as a broad-spectrum insecticide for numerous crops and also as an ectoparasiticide for farm animals. In addition to ace-tylcholinesterase inhibition, chronic exposure to organophosphates causes′ also oxidative stress leading to generation of free radicals and hence an increased expression of oxygen free radical scavenging enzymes in the target tissues. Soft organs like brain, liver, lung, heart, pancreas and kidney could be affected by organophosphate compounds. The aim of the current study was to evaluate the effects of fenthion on kidney function in adult rats and their progeny. 440 ppm were administered to adult rats by drinking water from the 14th day of pregnancy until day 14 after delivery (this dose represented ¼ of LD50). They caused oxidative stress both in dams and in their suckling pups, as evidenced by an increase level of malondialdehyde (MDA), along with a remarkable rise in kidney superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities and glutathione content (GSH) compared to those of controls. Moreover, plasma creatinine and urea levels were increased.

P313. Paraquat toxic effects are beyond the respiratory system: evidence for hepato- and renotoxicity

Hassan Malekinejad Pharmacology & Toxicolgy, Urmia University, Urmia, Iran, 1177 1 Malekinejad H, 1Rezabakhsh A, 2Rahmani F, 3Hobbenaghi R, and 1Cheraghi H 1Department of Pharmacology & Toxicology, Faculty of Veterinary Medicine, 2Department of Molecular Genetics, Faculty of Basic Sciences, and 3Department of Pathology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Paraquat (PQ) a world wide used herbicide belongs to the family of cationic bipyridylium, which is extremely toxic to mammalians. The lungs are known to be the main target of the toxicant. This study was designed to investigate the effects of PQ sub-acute poisoning on other organs including the liver and kidneys. Twenty adult male rats (150-200 g) were exposed either against saline normal as control group or various doses of PQ (3.5, 7 and 10 mg/kg, s.c.) as test groups for 7 consecutive days. The serum levels of ALP and ALT and the body weight gain were determined. The malondialdehyde, total thiol molecules and nitric oxide contents of the liver, kidney and the lung tissues were measured. Paraquat-exposed animals showed loss of body weight and elevation in serum levels of ALP and ALT in a dose-dependent manner. Moreover, animals in the test groups showed a significant (P < 0.05) increase of malondi-aldehyde and nitric oxide contents in the liver, kidney and lung tissues. By contrast, the total thiol molecules level in all examined organs of the test groups was significantly ameliorated. Histopathological findings confirmed the biochemical alterations as in the lungs severe hemorrhages, marked congestion, alveolar edema, and remarkable infiltration of neutrophils in interstitial tissue was observed. Additionally, in the kidneys in a dose-dependent fashion pathological changes such as congestion, tubular swelling and necrosis, multifocal interstitial nephritis, and the pres-ence of protein casts in renal tubules were manifested. Moreover, histopathological findings in the liver of test groups showed a severe congestion along with swelling of hepatocytes and consequently reduction of sinusoidal spaces. We also observed the fatty degeneration in peripheral zone of classical lobules. This study suggests that the PQ toxic

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effects are not limited to the respiratory system and the other vital organs including the liver and kidneys are affected from PQ intoxication. Moreover, it is also concluded that the biochemical changes such as remarkable elevation of the pro-oxidant production due to PQ-poisoning resulted in severe and life threatening pathological impacts in the lungs, liver and kidneys.

P314. Potential biomarker for the assessment of hepatotoxicity induced by metabolic activation of pyrrolizidine alkaloids

Ge Lin1, Na Li1, Mi Li1, Wood-Yee Chan1, Peter P. Fu2 and Hong-Xi Xu3

1School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, 2National Center for Toxicological Research, Jefferson, AR, USA, 3Hong Kong Jockey Club Institute of Chinese Medicine, Hong Kong

Pyrrolizidine alkaloids (PAs) are phytochemicals that are constitutively produced by plants as secondary metabolites for exerting a defense mechanism against insect herbivores. More than 660 PAs and PA N-oxides have been identi-fied in over 6,000 plants, and about half of them exhibit hepatotoxicity. Toxic PA-containing plants grow in many geographical regions worldwide, rendering it highly possible that PA-containing plants are the most common poison-ous plants affecting livestock, wildlife, and humans. PAs require cytochrome P450-mediated metabolic activation to generate the corresponding pyrrolic metabolites in order to exert hepatotoxicity and genotoxicity. As such, it is timely and important to develop mechanism-based biomarkers for assessing PA-induced hepatotoxicity. Pyrrolic metabolites are chemical reactive and can react with cellular proteins in vivo to form pyrrole-protein adducts lead-ing to toxicity. In the present study, we successfully developed a potential biomarker of PA intoxication in male SD rats, initially involving the use of two hepatotoxic PAs, monocrotaline and retrorsine, and two PA-containing herbs, Crotalaria sessiliflora and Gynura segetum. The developed biomarker was determined by chemical reaction of the pyrrole-protein adducts to release the corresponding pyrrolic analyte followed by LC-MS analysis, which constructed a novel, specific, and sensitive method for the identification and quantification of pyrrole-protein adducts. Using this method, we have, for the first time, unequivocally identified pyrrole-protein adducts in biological samples of rats treated with PAs. The results demonstrated that oral administration of pure PAs or PA-containing herbs caused liver injury in a dose-dependent manner and different PA-containing natural products may cause hepatotoxicity to different degrees depending on structures and contents of PAs present. Furthermore, the results demonstrated that the extent of liver damage correlated well with the level of pyrrole-protein adducts formed. In conclusion, our results demonstrated that the pyrrole-derived protein adduction correlates well with PA-induced hepatotoxicity and has a potential to be developed as a biomarker for the assessment of PA-induced hepatotoxicity. [Supports by Research Grant Council of Hong Kong (Earmarked Research Grant CUHK 2140485) and Hong Kong Jockey Club Charities Trust Fund (JCICM-15-07)].

P315. Precision-cut liver slices as an ex vivo model to study idiosyncratic hepatotoxicity in mouse and human

Mackenzie Hadi1, Yixi Chen1, Harry Emmen2, Miranda Stitzinger2 and Geny Groothuis1

1Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, Netherlands, 2GIT, NOTOX B.V., ‘s Hertogenbosch, Netherlands

Adverse drug reactions (ADRs) related to hepatotoxicity and drug hypersensitivity are responsible for the top two of toxicity-related drug withdrawals and there are no adequate translational strategies to predict safety. Idiosyncratic adverse drug reactions (IADRs) are ADRs that are rare and sporadic, unpredicted by clinical trials, unrelated to pharmacology of the drugs and occur without obvious relation to time or dose. IADRs may arise from drug interaction with inflammatory episodes that renders the liver more sensitive to injury resulting in increased toxicity. With the aim to develop a translational model to unravel the mechanism behind IADRs and to find biomarkers that can detect them, we used human and animal precision-cut liver slices (PCLS) to study the influence of inflammatory reactions on the toxicity of drugs. The technology of PCLS is receiving increased attention as a potential ex vivo toxicological model because PCLS retain the normal tissue architecture of an intact liver with all its cell types in their natural

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environment. PCLS from mouse and human were incubated with paracetamol (APAP)¸ diclofenac (DF), ketoconazole (KT) or clozapine (CZ) alone or in the presence of lipopolysaccharide (LPS), an inflammation inducer. Cell viability (ATP, liver enzyme leakage) and cytokine production were assessed. Both APAP and DF were more toxic in mouse than human. LPS had no influence on APAP and DF toxicity in mouse or human when assessed by viability tests. However, APAP and DF decreased the LPS-induced IL-1β production in both species while IL-6 production was only decreased in mouse PCLS. APAP slightly increased LPS-induced TNF-α production in mouse but strongly reduced it in human PCLS, while in both species TNF-α production was increased due to DF. In contrast to APAP and DF, toxicity of KT and CZ was increased in the presence of a non-toxic dose of LPS in mouse PCLS. Interestingly, the LPS-induced IL-1β production was increased by co-incubation with both KT and CZ, a phenomenon not observed in the cases of APAP or DF. In conclusion, clear species differences were identified in the effect of the drugs on the inflammatory reactions due to LPS. Toxicity of KT and CZ is aggravated by LPS and IL-1β upregulation might be involved in here. Based on these results, PCLS appear to be a promising ex vivo model to study mechanisms behind IADRs particularly in the human liver.

P316. Testosterone influences the antioxidative enzymes activity in benzene treated rats

Yeshvandra Verma Sr.

Department of Zoology/Toxicology, C.C.S.University, Meerut, Meerut, India

Testosterone influences the antioxidative enzymes activity in benzene treated rats It is established that Phase II antioxi-dant enzymes participated in detoxication of benzene and its metabolites. These enzymes include a family of glutathione dependant enzymes viz. Glutathione-s-transferase (GST), Glutathione peroxidase (GXP) and catalase (CAT). Sex steroid hormones regulate the activities of various enzymes of antioxidant defense system. Activity of Phase II enzymes and oxidative stress changes in animals in the presence of physiological sex hormones concentrations and after removal of respective glands. Present paper describes the results on activity of GST, GPX, and CAT in liver and kidney of male rats subjected to benzene treatment and testosterone therapy. Potential of DNA damage by castration and its modulation by using single cell gel electrophoresis also carried out. Our results show that benzene decreases the activity of GST in benzene treated rats. However testosterone stimulated the activity of GST in benzene administered castrated rats. Benzene administration to castrated rats inhibits the activity of GPX. There was no significant change seen in the activity of GPX after the treatment of testosterone in benzene treated castrated rats. Testosterone treatment in benzene treated rats also suppressed the catalase activity but testosterone treatment in benzene plus castrated rats showed no significant change in catalase activity in liver and kidney. Castration induced strand breaks in DNA. Benzene also induced DNA damage in castrated rats. Further treatment of testosterone modulated the induction of strand breaks. Present results establish liver-testis axis in rat considering benzene as the toxin. These investigations are important from occupational health point of view. Benzene exposure affects their reproductive health. Nevertheless, it can be modulated by suitable hormonal therapy. Key Words: Benzene; testosterone; castration; antioxidant enzymes.

P317. TNF-alpha enhances cisplatin-induced renal injury via the JNK and NF-kB pathways

Giulia O.E. Benedetti, J. Meerman, B. van de Water and M. de Graauw

Toxicology, Lacdr, Leiden, Netherlands

Cisplatin is commonly used for the treatment of several solid tumors. However, its clinical use is often limited by renal toxicity, occurring in one-third of the patients treated. Cisplatin causes direct tubular injury through multiple mecha-nisms including direct cytotoxicity with reactive oxygen species (ROS), activating mitogen-activated protein kinases (MAPKs) and inducing apoptosis. Furthermore, in mice, cisplatin induces an impressive production of the cytokine tumor necrosis factor alpha (TNF-α) via the transcription factor nuclear factor κB (NF-κB) and antibody-based depletion of TNF-α or genetic ablation of the TNF receptor (TNFR) inhibits cisplatin-induced nephrotoxicity. So far the intracel-lular signalling pathways activated synergistically by cisplatin and TNF-α in the context of cisplatin-induced renal injury are not well understood. To study this we used conditionally immortalized mouse proximal tubular epithelial cells (IM-PTEC) and observed an increase in cisplatin-induced apoptosis caused by brief TNF-α pre-treatment. Under these conditions the levels of phosphorylated JNK and c-jun were increased and prolonged compared with cisplatin

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or TNF-α alone. Inhibition of JNK decreased cisplatin/TNF-α induced-apoptosis. In addition, inhibition of IKKα and β, downstream Ser/Thr kinases of the TNFR that mediate NF-κB activation, enhanced cisplatin/TNF-α-induced cell death. Transcriptomics analysis revealed a dramatic increase of several cytokines and chemokines compared with cisplatin or TNF-α alone. Interestingly, some NF-κB subunits were differentially expressed in each condition possibly resulting in a shift in target gene expression and, consequently, influencing apoptosis-related programs which would define sensitization to combined cisplatin/TNF-α treatment. In conclusion, TNF-α sensitizes renal proximal tubular cells to cisplatin-induced cell death via the JNK and NF-κB pathways in association with changes in the transcriptional networks. These altered networks are likely determining cisplatin/TNF-α cytotoxicity in vitro and possibly cisplatin nephrotoxicity under in vivo conditions.

P318. Transcriptomics analysis of the Artemisinin stress response in mouse embryonic stem cells

M. Raamsman-Ossevoort, J. Carreras Puigvert and B. Van de Water

Division of Toxicology, Leiden University, Leiden/Amsterdam Center for Drug Research, Leiden, Netherlands

Artemisinin based anti malarial drug combinations are recommended for the treatment of P. falciparum malaria infections throughout all malarial endemic areas of the world. This includes women of child bearing age and pregnant women. However, in preclinical evaluations these drugs are shown to be embryotoxic in several species and even teratogenic in rabbit and rat. We aim to asses the potential hazard posed by these drugs to the developing human foetus and thereby making evidence based recommendations on the risk:benefit of these drugs. To study the mechanisms of embryotoxicity of artemisinins we use mouse embryonic stem (mES) cells as a model. We used artesunate (ARS), dihydroartemisinin (DHA) and a biologically inactive artemisinin metabolite deoxy-dihydroartemisinin (deDHA). While pro-oxidant agents and DNA damage agents caused early onset of apoptosis, both ARS and DHA, but not deDHA, caused apoptosis of mES cells at high concentrations (100 ?M) and at late time points (48 hrs). This apoptosis was preceded by a proliferation block at earlier time points which already occurred at low concentrations (1 ?M). BAC transgenomics-based mES reporter cell lines for DNA damage stress or indirect pro-oxidants demonstrated that artemisinins caused indirect oxidative stress responses in mES cells. Next we performed extensive transcriptomics analysis to unravel the underlying mechanism of both the proliferation block as well as the oxidative damage and apoptosis onset by artemisinins. mES cells were treated with ARS, DHA and deDHA at low, medium and high con-centration for 8, 16 or 24 hrs. mRNA were hybridized on Affymetrix HT MG 430 arrays followed by transcriptomics analysis. These transcriptomics data will be discussed in comparison with our extensive toxicant transcriptomics mES cell database. All data will be used to provide recommendations to the WHO regarding risk:benefit for the use of artemisinins during pregnancy.

P319. Biotransformation Studies of 4-Amino-N-cyclopropyl-8-(3,6-dimethoxypyridazin-4-yl)-7-fluorocinnoline-3-carboxamide, An Orally Active Sub-type Selective GABA-Aá2/3 Receptor Positive Modulator and the Approaches to Remove Its Bioactivation Liabilities

Jian Yu and Maria D. Ribadeneira

Drug Metabolism and Pharmacokinetics, AstraZeneca Pharmaceuticals LLP, Wilmington, DE, USA

4-amino-N-cyclopropyl-8-(3,6-dimethoxypyridazin-4-yl)-7-fluorocinnoline-3-carboxamide (Figure 1, I) is an orally active sub-type selective GABA-Aα2/3 receptor positive modulator for the treatment of anxiety. In this study, the major biotransformation pathways of I were identified in rat and human hepatocyte suspensions and in rat bile and urine using liquid chromatography-high resolution mass spectrometry and radiochromatography. In rat hepatocytes, the major metabolism pathways of I were oxidation at the cyclopropyl group, N-dealkylation and O-demethylation. A GSH adduct, related to the oxidation of C

3H

5NH moiety, was identified in rat hepatocytes. Formation of the GSH

adduct was also observed in vivo in rats, after a single oral administration of [14C]-labeled I (20 mg/kg). The GSH adduct and its degraded products were identified in rat bile and accounted for ∼ 20% of the absorbed dose. Parent drug accounted for ∼3% of the administered dose in bile and urine within 48 hours. In addition, O-demethylation and N-dealkylation products were observed in vivo. The metabolism pathways of I were also evaluated in human hepatocytes. Metabolites identified in human hepatocytes, including GSH adduct, were the same as those found

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in rat, suggesting a potential bioactivation liability may carry over to humans, in vivo. Results in rat and human, showed cyclopropyl ring opening followed by rearrangement and GSH conjugation as one of the major metabolism pathways both in vivo and in vitro. We propose a tentative pathway where, upon oxidation at the C

3H

5NH moiety,

the N-cyclopropyl group may undergo ring opening followed by intra-molecular rearrangement to form a reactive intermediate that reacts with glutathione to form the GSH adduct. Hence, to remove this liaility, the cyclopropyl ring was replaced with an isopropyl side chain (Figure 1 II -4-amino- 8-(3,6-dimethoxypyridazin-4-yl)-7-fluoro-N-isopropylcinnoline-3-carboxamide (Figure 1, II). The results indicated that N-dealkylation, oxidation at the isopropyl group, and O-demethylation were the major metabolic pathways of II in rat and human hepatocytes. Although oxidation at the isopropyl group was observed, no GSH adduct was detected. Our findings provide a viable strategy to remove the bioactivation liability present in compound I and indicated that the bioactivation pathway detected in vitro was predictive of in vivo in rats and was a significant metabolism pathway. In addition, to better assess the bioactivation risk, two other cyclopropyl analogs were also synthesized and analyzed in rat and human hepatocytes. Results from these latter analysis provided a caveat that all cycclopropylamines should not be assumed to be bioac-tivated with the same facility.

P320. Definitive Assignment of Metabolite Structures Using Ion Mobility Mass Spectrometry And Molecular Modeling

John P. Shockcor1, Iain D. Campuzano1, Jayne Kirk1, Gordon Dear2 and Jordi Munoz-Muriedas2

1Dept of Metabolic Profiling, Waters Corp, Milford, MA, USA, 2GlaxoSmithKline, United Kingdom

Definitive Assignment of Metabolite Structures Using Ion Mobility Mass Spectrometry And Molecular Modeling John P. Shockcor 1,3, Jayne Kirk1; Iain D G Campuzano1; Jordi Munoz-Muriedas2; Gordon Dear2 1Waters Corporation, Manchester, United Kingdom; 2GlaxoSmithKline, Ware, Hertfordshire, 3Dept of Biochemistry, University of Cambridge, United Kingdom As an analytical technique, mass spectrometry (MS) cannot separate isomeric species or provide conformational information. Structural information can be gained using tandem MS (MS/MS) techniques, however, there are classes of substituted molecules, such as hydroxylated metabolites with which produce identical MS/MS spectra. Ion mobility (IM) has the ability to separate isomeric species, rapidly ( in msec) based on differences in their collision cross-section (physical shape) in the gas-phase and when combined with mass spectrometry could obvi-ate the need for time consuming metabolite isolation and subsequent NMR analysis. Employing molecular modeling three-dimensional conformations a combination of putative metabolite structures for the compound ondansetron were determined. Using the ten lowest energy conformations for each metabolite the theoretical collision cross section values were calculated for each. The metabolites in biological matrix were then analyzed using a hybrid quadrupole/ion mobility/orthogonal acceleration time-of-flight mass spectrometer. All three hydroxylated ondansetron metabolites (metabolites GR60661, GR63418 and GR90315) had different chromatographic elution times while the MS and MS/MS spectra were all identical, thus precluding the possibility of definitive structural assignment. Theoretically derived cross section values for hydroxylated metabolites GR60661, GR63418 and GR90315 were calculated and yielded the values 111.4, 111.2 and 109.8 square angstroms respectively. Ion mobility measured collision cross-section values are in excellent agreement with the theoretically values, thus allowing definitive structural assignment of the hydroxylated metabolites, even those with a collision cross-section difference of less than 1 angstrom square. This demonstration of the assignment of hydroxylated metabolites shows great promise and could significantly reduce the amount time now required to elucidate metabolite structures.

NH2 NH2

NN

N NN O

O O

O

OOF F

N

N NN NH H

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P321. Detection and Identification of the Metabolites of Daphnetin in Rat Plasma by LC-ESI Tandem Mass Spectrometry

Si-Cheng Liang1, Guang-Bo Ge1, Zhong-Ze Fang2, Peipei Dong1 and Ling Yang3

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China,

3Lab of Pharmaceutical Resource Discover, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Metabolic profiles are pivotal to understand the mechanism of drug biotransformation and elimination, and sequential selection of suitable animal model(s) for pharmacokinetic, toxicological and pharmacological studies. Daphnetin (7,8-dihydroxycoumarin), is an oral medicine for treatment of coagulation disorders and rheumatoid arthritis in China and shows a rapid elimination in rats after intravenous administration with a t

1/2 of approximately 15 min and poor bioavail-

ability [1]. Recently, an in vitro study revealed that glucuronidation may play a role in daphnetin metabolism [2], but the in vivo metabolism of daphnetin remains unreported. Herein, we developed a MS-based method for characterization of the metabolites of daphnetin in rat plasma after oral administration at dose of 50 mg/kg bw. Fifteen minutes after daphnetin administration, seven metabolites but no parent were detected from rat plasma, indicating that the metabo-lism of daphnetin was extensively in rat. These metabolites were identified by comparison of their chromatographic behaviors and ESI-MS/MS spectra to those of daphnetin, an authentic standard 7-O-methyl-8-hydroxycoumarin and three biosynthesized standards, including 7-O glucuronide daphnetin, 8-O glucuronide daphnetin and 7-O-methyl-8-hydroxycoumarin glucuronide. The results showed that glucuronidation played a primary role in metabolic elimina-tion of daphnetin in rat evidenced by the formation of two major monoglucuronides daphnetin (M-1 and M-2) and a diglucuronide daphnetin (M-3). The typical product ions for M-1 and M-2 were m/z 353 and 177, while M-3 showed the fragment ions of m/z 529, 353 and 177. Sulfotransferase(s) was also involved in the metabolism of daphnetin in rat, evidenced by the formation of two trace monosulfates daphnetin (M-4 and M-5) with fragment ions of m/z 257 and 177. Furthermore, glucuronidated (M-6) and sulfated (M-7) derivatives of 7-O-methyl-8-hydroxycoumarin were also detected in rat plasma with the fragment ions m/z 367 and 191 for M-6, and m/z 271 and 176 for M-7, which suggested the overlapping contribution of the conjugation pathways (glucuronidation, methoxylation and sulfation) in daphnetin elimination in rat. These data give us an insight into the mechanism of daphnetin metabolism and elimination, and provide useful information for improving the metabolic stability of this compound.

References

Qu SY, Wu YJ, Wang YH, and Zuo YX. Metabolism and pharmacokinetics of daphnetin. Yao Xue Xue Bao, 1983; 18: 496–500.Liang SC, Ge GB, Liu HX, Zhang YY, et al. Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro

glucuronidation of daphnetin. Drug Metab Dispos, 2010; 38:973-980.

P322. Enhanced Metabolite Identification and Structural Elucidation With a Semi-Automated Software

B. Bonn1, Carina Leandersson2, Fabien Fontaine3 and Ismael Zamora3

1Department of Chemistry, Medicinal Chemistry, University of Gothenburg, Gothenburg, Sweden, 2Biotransformation, Discovery DMPK, AstraZeneca R&D Molndal, Mölndal, Sweden, 3Lead Molecular Design, S.L., San Cugat del Vallés, Spain

The possibility of metabolite identification early in drug discovery is beneficial for a successful design of new chemical entities (NCE). Early identification of liabilities for formation of reactive metabolites will conduce to development of safer drugs. In addition the opportunity to understand and assess structure metabolite relationships (SMR) will guide the medicinal chemists in the design of compound series with desirable metabolic properties. The identification of

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metabolites is almost exclusively done with LC-MS/MS and despite the enormous progress in the development of these techniques and software for handling of data this is a very time-consuming task. Therefore it is a desire to speed up the assay in early drug discovery phases when there is a high throughput of compounds. In this study Mass-MetaSite, a semi-automated software for metabolite identification, was validated. The program is an extension of MetaSite1, which is an in silico tool for site of metabolism (SOM) predictions. Mass-MetaSite combines the information from MS raw data, in the form of collision induced dissociation spectra, with the prediction of SOM in order to assign the structure of a metabolite. The aim of the software is to mimic the rationalization of fragment ions done by a biotransformation scientist in the process of structural elucidation. For this validation metabolite identification in human liver microsomes was performed for 19 commercially available compounds resulting in 92 metabolites. The analysis was made on a Q-TOF instrument and both the use MSE and MS/MS acquired data were tested in Mass-MetaSite. The results were very encouraging and the correct SOM compared to manual inspection of the data was picked in the first rank in ∼80% of the cases. Similar results were achieved both by using MSE and MS/MS data. In conclusion the use of Mass-MetaSite could alleviate the work of a biotransformation scientist and reduce the workload by assigning the structure for a majority of the metabolites. More effort could instead be put on manual interpretation of complex fragmentations and structures that could not be assigned by the software.

References

(1) Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.; Vianello, R. MetaSite: Understanding Metabolism in Human Cytochromes from the Perspective of the Chemist. J. Med. Chem. 2005, 48, 6970-6979.

P323. Evaluation of Active Counting Mode option in LabLogic beta;-RAM Model 5 and Laura software

Huw W. Loaring1, Andrew Williams1, Jonathan Trevor Attwood2 and Brett O′Brien3

1LabLogic Systems Ltd., Sheffield, United Kingdom, 2Development Drug Metabolism and Bioanalysis, AstraZeneca R & D-Charnwood, Loughborough, United Kingdom, 3Development Drug Metabolism and Bioanalysis, AstraZeneca R & D-Charnwood, Loughborough, United Kingdom

LabLogic ßRAM Model 5 radioflow detector includes the option to use variable scintillant flow, under control of Laura 4 software, aiming to reduce the peak broadening observed with the large flow cell volumes used in radioflow detectors. On detection of a peak, the scintillant flow is increased to reduce residence time in the flow cell. The aim of this is to increase signal to noise in order to improve peak resolution and detection over background. This functionality has been evaluated with HPLC and UHPLC. Preliminary data is presented along with recommendations for further evaluation required.

P324. Identification and Screening Major Metabolites of K2-spices in Human Liver Microsome and Human Urine

Hua-fen Liu1, Sumandeep Rana2, Alex Wang1, Andre Schreiber1 and Elliott Jones1

1Application Demo Laboratory, AB Sciex, Foster City, CA, USA, 2Redwood Toxicology, Santa Rosa, USA

Introduction Synthetic marijuana (fake weed), marketed as K2 or Spice is much more potent than naturally occurring THC. JWH-018 and JWH-073 are two main active ingredients of K2. The aim of this study was to identify the main metabolites of JWH-018 and JWH-073 in human urine and to develop an LCMSMS method for routine screening of these compounds. The metabolite identification was challenging because of multiple active ingredients of K2. Also, the positive specimens were from street and there was no control available. To resolve this challenge, JWH-018 and JWH-073 were incubated in human liver microsomes and in vitro metabolite pathway was identified for individual compounds. The metabolites from human urine matrix were then identified based on the structure information and metabolite pathway from in vitro studies. The correlation of in vitro and in vivo metabolite pathway was also discussed.

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Methods: JWH-018 and JWH-073 reference material was obtained from Cayman Chemical Company. Three urine specimens were collected from different individuals claiming to have smoked K2. The specimens were analyzed using a hybrid triple quadrupole linear ion trap system after protein precipitation and centrifugation. The metabolite ID results were also confirmed by a new accurate mass system. An aliquot of each specimen was directly injected on QTRAP® system or subjected to enzyme hydrolysis to cleave the conjugates and subsequently analyzed to get an estimation of the extent of conjugation of various metabolites.

Results:NSimilar metabolite pathways with different distribution were observed for both compounds for the in vitro and in vivo experiments. The most abundance metabolite in vitro was tri-oxidation of either JWH-018 or JWH 073. However, these compounds were extensively metabolized in human urine by mono-or di-hydroxylation and corre-sponding glucuronide conjugation. Parent compound was detected in only one specimen. In general, Hydroxylated N-dealkyl metabolites, carboxy, reduced di- and tri-hydroxy, mono and di-hydroxy metabolites in the side chain as well as both aromatic rings were detected in free and conjugated forms for both JWH-018 and JWH-073. The position of hydroxylation could be elucidated by studying the corresponding fragments for each metabolite. A workflow for K2 routine screening of unknown samples using the QTRAP® system with library confirmation was established based on the study results.

P325. In Vivo Metabolite Identification and Profiling Using a Rapid, Accurate Mass, High Resolution Mass Spectrometer: Quantitative and Qualitative Results from One Injection

James A. Ferguson1, Jeffrey D. Miller1 and Sandeepraj Pusalkar2

1Applications Laboratory, AB SCIEX, Framingham, MA, USA, 2Dmpk, Millennium Pharmaceuteicals, Inc., Cambridge, MA, USA

Most commercially-available accurate-mass instruments must make one or more compromises when collecting data. They typically must to slow their data collection rate to achieve resolution. They also are likely not have linear quantitative capabilities over a large dynamic range, or may not be sensitive enough to look for low-level metabolites and so may not have the ability to follow the concentrations of the parent drug and its metabolites over the full time course of a DMPK study. DMPK data from a high-resolution accurate-mass instrument which does have the ability to do all of the things listed above and has the speed to take advantage of UHPLC peak widths of 2 to 5 s, would be extremely important to investigators. Coupling such an instrument to advanced data acquisition software with on-the-fly multiple mass defect filters or neutral loss scans to trigger accurate mass product ion scans for metabolite confirmation will be presented. Several drugs (verapamil, ketoconazole, and/or tamoxifen) were administered at 1 mg/kg and 5 mg/kg to Sprague-Dawley rats. The plasma and/or urine were collected at appropriate time points. The body fluids were protein precipitated with additional dilution, if necessary, and monitored for the presence of the parent drug and its metabolites. Single-injection workflows showing metabolite ID (qualitative LC/MS and LC/MS/MS data) and metabolite profiling (quantitative data from the TOF survey scan) will be shown. Data has shown quantification to less than 0.1% of the parent drug remaining and linear dynamic range for several compounds at 4 orders of magnitude. The data shows resolutions (m/Dm) of 30,000 or more above m/z 250 and between 20,000 and 30,000 below m/z 250. With this instrument we have seen the resolution (with 10% valley) of two fragments arising from different fragmentation pathways from a metabolite of 1]: [C

14H

8N

4O

3]+• at m/z 280.0591 and [C

14H

10N

5O

2]+ at m/z

280.0829. Mass accuracy of 2 ppm rms or better with external calibration will also be shown, which is sufficient for most applications to disambiguate possible formulae for the metabolites or fragments in the product ion spectra used for confirmation. Standard curves for the parent drugs and a few synthetic metabolites were prepared in the plasma and urine matrices for comparison with the in vivo samples. The quantitative data showed good linear dynamic range and CVs consistent with GLP guidelines.

References

[1] Hopfgartner, Gerard, Data presented at the 58th ASMS Conference on Mass Spectrometry and Allied Topics, May 23-28, 2010, Salt Lake City, Utah, USA.

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P326. Metabolite Detection and Identification Using Fragment Ion Search in Conjunction with Automated Fragment Prediction

Robert Mistrik1, Juraj Lutisan1, Milos Suchy1, Alexej Nikiforov2 and Yingying Huang3

1HighChem, Ltd., Bratislava, Slovak Republic, 2Institute of Organic Chemistry, Univeristy of Vienna, Vienna, Austria, 3Thermo Fisher Scientific, San Jose, CA, USA

Introduction Efficient metabolite identification requires combining modern mass spectrometric techniques with advanced software tools. Here we present a novel software approach for single-run metabolite detection and identifica-tion from LC-MS data using Fragment Ion Search (FISh) in conjunction with automated prediction of fragments from parent structure. In contrast to traditional post-acquisition processing techniques, this approach eliminates the need to predict biotransformations from parent compound. Similar to Mass Defect Filter (MDF), it utilizes the compound structure to filter out matrix-related background ions. However, the method presented here is fundamentally different from MDF and no subjective judgment of expected m/z range and mass defect range is required. The site of biotrans-formation can also be automatically localized using such approach.

Method: With accurate mass data, metabolite peaks can be selectively resolved and identified from background matrix ions by searching ions related with fragmentation products of parent structure. It has been shown1 that parent drug and its metabolites exhibit common fragments ions even if the m/z of their precursor ions are different. In the first step, fragment ions are automatically predicted from parent structure using specialized fragmentation tool. In the next step, a table consisting of accurate m/z values of predicted fragments is generated and subsequently applied as an extraction filter to the high resolution accurate mass LC-MS run. FISh enables selective removal of vast majority of matrix-related background ions. Furthermore, the matching of the predicted fragment ions to the observed fragment ions in the puta-tive metabolites enables the automatic localization of biotransformations.

Results: The performance of such approach was evaluated using in vivo (1mg/kg by IV) rat bile samples. The results from FISh is compared and contrasted to the results from MDF. The inclusion of predicted fragment ions enhanced the number of “signature” fragment m/z values and thus significantly increased detection efficiency for potential metabolites. The base-peak chromatogram resulting from FISh is visually similar to those produced by MDF where vast majority of the matrix-related background ions were removed. The number of metabolites detected was also comparable between the two methods. While MDF only works with the molecular ion of the metabolites thus not useful in suggesting the site of biotransformation, FISh was able to propose the likely structures of the metabolites by drawing the relationship between ions with the matched m/z values and the corresponding fragment structures. Presented approach can be fully automated while the only input data are the LC-MS raw files and the structure of the parent compound.

P327. Rapid Metabolite Profiling of in vivo Metabolites of Escultin in Rat Using Ultra-Fast Liquid Chromatography Combined with Tandem Mass Spectrometry

Guangbo Ge1, Si-Cheng Liang2, Zhong-Ze Fang1 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Esculetin (6,7-dihydroxycoumarin), one of coumarin analogous, has been found to exhibit broaden biological effects, such as anti-tumor, anti-inflammatory, analgesic, anti-arrhythmic, and scavenging activities against reactive oxygen species. Esculetin is a dominant constitute of Ash bark (qinpi), one famous traditional Chinese medicine which has been widely used in Asian countries. In this study, a rapid profiling method by using ultra-fast liquid chromatography combined with tandem mass spectrometry was developed for characterization of trace metabolites of esculetin in rat plasma after oral administration at dose of 50 mg/kg bw. Fifteen minutes after escultin administration, nine metabolites including glucuronidated, methoxylated and sulfated derivatives of escultin were detected from rat

215

plasma, and the escultin can not be measured, indicating that escultin can be metabolized rapidly in rat. All metabo-lites were identified by UFLC-ESI-MS/MS, based on the product ions and typical fragmentation patterns of phase II metabolites. The major metabolite (M1) was prepared by using rat liver microsomes and was fully characterized as 7-O monoglucuronide esculetin by NMR. Two methoxylated metabolites (M2, M3) were assigned as scopoletin (6-methoxylated escultin) and isoscopoletin (7-methoxylated escultin) compared with authentic standards, and these two metabolites can be metabolized rapidly to glucuronidated derivatives (M4, M5) both in vitro and in vivo. M4 and M5 can be rapidly assigned as monoglucuronides of methoxylated escultin according to their typical product ions including m/z 367, m/z 191 and m/z 176. In addition, two monosulfated derivatives of escultin (M6, M7) and two monosulfated metabolites of methoxylated escultin (M8, M9) were also identified based on their informative product ions which are m/z 257 and m/z 177 for monosulfated escultin, as well as m/z 271, m/z 191 and m/z 176 for monosulfated metabolites of methoxylated escultin. In summary, this study revealed three biotransformation pathways of esculetin in rat including glucuronidation, methoxylation and sulfation, while glucuronidation is the major metabolic pathway and the C-7 hydroxyl is the metabolically labile site for glucuronidation. These results are very helpful for understanding the in vivo metabolism and efficacy of escultin, as well as for improving the metabolic stability of this compound.

P328. Simultaneous High Throughput Enzyme Stability Quantitation and Drug Soft Spot Metabolite Analysis using a new style TOF LCMS

Elliott Jones

Application Demo Laboratory, AB Sciex, Foster City, CA, USA

The purpose of these experiments is to investigate the ability of a new type of Hybrid Quadrupole-TOF instrument to perform, metabolite identification as well as drug quantitation within the same LCMS run. The ability to lessen analysis time and to reduce duplicate LCMS runs is a strong motivation to combine these experiments. Unfortunately, a number of classics issues have made this workflow challenging to implement as a routine LCMS ADME screen. First, TOF, Ion Traps and Triple Quad instruments do not perform both qualitative and quantitative experiments at the highest levels of performances simultaneously. Second, cycle rate for high quality quantitation at greater than 10 points over of a narrow fast LCMS profile challenge most instruments. Furthermore, the ability to deliver a true high resolution (>30K) accurate mass (sub 1 PPM) LCMS MSMS spectra along with quantitation has been challenging to achieve to this point for peaks around 2 to 3 seconds base to base. The acquisition rate for data dependent MSMS must be high enough to cover all major metabolites and the vast majority of medium to low-level metabolites. One added experimental criteria would be high sensitivity to allow analysis at 1uM or below in vitro incubation levels most representative of true in vivo biological metabolite profiles. A criterion of fast sub 3 min gradient analysis would also be ideal, using small particle high pressure LC separation. The above listed challenges provide a clear benchmark for the ability of drug development to move to a single LCMS analysis. In this work a new type of hybrid quadrupole TOF instrument will be presented with high acquisition rates (>25 Hz MSMS) and triple quadrupole quality quantitation to enable this workflow. A novel software and generic method workflow will be outlined to begin allowing a user in a HT ADME environment to perform routine analysis of both data streams. In this example two drug compounds Propanalol and Glyburide, were analyzed under the above criteria. LCMS analysis was preformed over a full set of time points between 0 and 4 hours, with replicate injections to test reproducibility. An LCMS experiment consisting of a novel real time multi-MDF filter for enhanced data dependent acquisition was also employed. An acquisition experiment consisting of a single TOF survey scan with 4 TOF MSMS dependent scans was used for sample analysis. The TOF MS experiment was used for quantitation using a 30,000 resolution XIC for parent drug lose and key metabolite formation. This mode allows the investigator to extract unlimited relative quantitation data for a complete picture of metabolism kinetics.

P329. The Simultaneous Collection of MRM and MS/MS Data To Detect and Quantify Drug Metabolites

Joanne Mather, Marian Twohig and Rob Plumb

Pharmaceutical Business Operations, Waters Corporation, Milford, MA, USA

216

The accurate quantification and characterization of drug metabolites provides critical data for DMPK studies in drug discovery and development. In this paper we present a novel approach for the simultaneous detection, characteriza-tion and quantification of drugs and their metabolites using a tandem quadrupole mass spectrometer equipped with a novel collision cell design. Samples from a rat safety assessment study for a novel cancer drug candidate molecule by protein precipitation and analysed in positive ion mode LC/MS/MS using formic acid acetonitrile gradient over 2 minutes and sub 2µm particle LC columns. The MRM transitions for the drug metabolites were automatically gener-ated from the MS/MS spectrum of the dosed compound. The collection of full scan MS/MS data was triggered from the intensity of the MRM peak responses. The average peak width was determined to be 1.5 seconds at the base. The assay was demonstrated to be linear over 4 orders of magnitude and has a LOQ of 100pg/mL from 50µL of processed plasma. The automated method development approach generated MRMs based on the calculated MW of the potential drug metabolites and the from fragment ions from the parent compound; thus generating 2-3 possible MRM transitions per metabolite. Using this MRM triggered MS/MS approach a total of three metabolites were detected and quantified demethylated, dealkylated and two hydroxylated metabolites. It was possible to generate high quality MS/MS spectra of these metabolites at very low concentration. The MS/MS spectral data generated was compared to that obtained from a targeted MS/MS product ion spectra analysis. The results obtained were found to be comparable, giving rise to the same product ions with the same intensity. This approach allowed the metabolites to be detected and quantified with highest sensitivity MRM approach and characterised by the full scan data in one fast analytical run.

P330. An Open-Label Study to Assess the Mass Balance and Metabolic Disposition of an Orally Administered Single Dose of 14C-Labeled Neratinib, an Irreversible pan-ErbB inhibitor, in Healthy Subjects

Richat Abbas-Borhan, Inder Chaudhary, Joel Kantrowitz, Bruce A. Hug, Cathie Leister, Jaime Burns and Daryl Sonnichsen

Pfizer Inc., Collegeville, PA, USA

Neratinib (HKI-272; 2-butenamide, N-[4-[[3-chloro-4-(2-pyridinylmethoxy)phenyl]amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-, (2E)-, (2Z)-2-butenedioate) is a potent, low-molecular-weight, orally administered, irreversible pan-ErbB receptor tyrosine kinase inhibitor in development for the treatment of ErbB2-positive breast cancer. The objective of this open-label study was to characterize the mass balance, general metabolic pathways, and pharmacokinetics (PK) of neratinib and its metabolites in humans after administration of a single oral dose of [14C]-labeled neratinib. Healthy men received a single dose of [14C]-neratinib 200 mg in an oral solution. Blood, urine, and feces were collected predose and up to 216 hours postdose for total radioactivity, metabolite, and PK assessments. Total radioactivity in urine and feces was determined using accelerated mass spectrometry, and mass balance was determined from the total radioactivity recovered in urine and feces. Plasma neratinib and metabolite (M3, M6, and M7) concentrations were determined by liquid chromatography-tandem mass spectrometry (cold assay). For neratinib, M3, and M7, the lower limit of quantitation was and the assay was linear to 250 ng/mL; for M6, the respective values were 1.5 ng/mL and 125 ng/mL. Plasma concentration data were analyzed by a noncompartmental method using WinNonlin. Six men aged 22-48 years (median 35.5 years) were enrolled. In the mass balance analysis, the mean (SD) recovery of radioactivity was 98.2% (8.3%) of the total dose. Fecal excretion accounted for approximately 97.1% and urinary excretion accounted for 1.13% of the total dose. The mean (SD) peak plasma concentration (C

max) for neratinib was

53.7(24.9)ng/mL,theareaundertheconcentration-timecurve(AUC)was1190(573)ng•h/mL,apparentvolumeofdistribution was 4530 (2480) L, apparent oral clearance (CL/F) was 204 (94.3) L/h, elimination half-life (t½) was 16.15 (5.57) hours, and the median (range) time to C

max (t

max) was 7.00 hours (2.00, 12.00). For M3, the mean (SD) C

max, AUC

to the last measurable concentration at time T (AUCT),andt½,respectively,were8.26(5.73)ng/mL,40.9(28.9)ng•h/

mL, and 13.30 (6.49) hours, and the median (range) tmax

was 1.00 (0.50, 4.03) hours. For M6, the mean (SD) Cmax

, AUCT,

andt½,respectively,were17.7(7.93)ng/mL,251(61.7)ng•h/mL,and13.37(2.44)hours,andthemedian(range)tmax

was 2.00 (0.50, 6.00) hours. For M7, the mean (SD) C

max, AUC

T, and t½, respectively, were 8.58 (4.12) ng/mL, 92.9 (77.6)

ng•h/mL,and10.70(4.01)hours,andthemedian(range)tmax

was 2.00 (0.50, 4.03) hours. The mean metabolite ratios for C

max and AUC

T, respectively, were 0.152 and 0.0377 for M3, and 0.363 and 0.262 for M6, and 0.166 and 0.0746 for M7.

The results of this study demonstrated that elimination of [14C]-neratinib-derived radioactivity was rapid and essentially complete, with the feces being the major route of elimination, and confirmed M3, M6, and M7 as major circulating metabolites of neratinib in humans.

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P331. C5-Hydroxylation of Liquirtigenin Is Catalyzed Selectively by CYP1A2

Ying Hu1, Hui-Xin Liu2 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Flavonoids are part of a family of naturally occurring polyphenolic compounds and represent one of the most prevalent classes of compounds in vegetables, nuts, fruits and beverages such as coffee, tea, and red wine as well as medical herbs. Chemically, flavonoids contain a common phenylchromanone structure (C6-C3-C6) with at least one hydroxyl group substituent or a hydroxyl derivative such as a methoxy group. More than 8000 compounds with a flavonoid structure have been identified and categorized into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins, and chalcones. Liquiritigenin (7,4′-dihydroxyflavanone) is found most notably in Glycyrrhizae radix (G. radix, licorice, liquo-rice), which is one of the oldest and most frequently used botanicals in the polyherbal preparations. Liquiritigenin have been shown to have a inhibition of xanthine oxidase activity in vitro, dose related anti-allergic activities, and growth-inhibitory effect on cancer cell. It also has the anti-angiogenic effect, and antioxidant, anti-inflammatory activities. An understanding of the enzymology of the metabolic clearance of a drug, whether by Phase I or Phase II mechanisms, is pivotal to new drug development. The identification of metabolite and metabolic enzymes involved in drug metabolism are important for us to understand which one, parent drug or metabolite(s), is really active and how variations in drug concentrations can lead to differences in drug efficacy and toxicity. In the present study, a novel mono-hydroxylated metabolite was detected in reaction catalyzed by HLMs, and was identified as 4′,5,7-Trihydroxyflavanone by compar-ing the tandem mass spectra and the chromatographic retention time with that of the standard compound. Significant difference in CLint (8-fold) was found between these two oxidative pathway of liquiritigenin, and C5-hydroxylation pathway was identified as the major oxidative metabolism of liquiritigenin. The study with chemical selective inhibi-tor, cDNA-expressed human CYPs, correlation assay, and kinetics study demonstrated that CYP1A2 was the specific isozyme responsible for the C5-hydroxylation metabolism of liquiritigenin in human liver microsomes. Identification of CYP1A2 as being responsible for liquiritigenin C5-hydroxylation will greatly improve future investigations of CYP1A2 interindividual differences associated with liquiritigenin clinical trials and the magnitude of drug-drug interactions.

P332. Characterisation of major human metabolites of JM-1232(-) generated using microbial bi-otransformation techniques

Zbigniew R. Cybulski1, WenHao Zhu1, Brian John2 and Takako Ohkura3

1Drug Metabolism, Huntingdon Life Sciences Ltd, Huntingdon, England, 2Drug Metabolism, Huntingdon Life Sciences, Huntingdon, United Kingdom, 3Dept of Central Rsch Lab, Maruishi Pharmaceutical Co Ltd, Osaka, Japan

JM-1232(-) or (-)-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-phenyl-3,5,6,7-tetrahydrocyclopenta[f]isoindol-1-(2H)-one is a novel compound under development as a potential anaesthetic/sedative. The compound has a strong affinity for the central benzodiazepine binding site of gamma-aminobutyric acid

A (GABA

A) receptors. Metabolism studies in

male and female Sprague-Dawley rats after single intravenous dosing of [14C]JM-1232(-) indicated the presence of several hydroxylated metabolites which could not be fully characterised from the LC-MS/MS data alone. Analysis of samples from a human clinical trial conducted on JM-1232(-) confirmed the importance in both plasma and urine of Phase I hydroxylated metabolites of JM-1232(-) in man, one of which was present at disproportionately higher levels than observed in the rat. In order to further characterise the hydroxylated metabolites, microbial production techniques were used to generate sufficient amounts of each metabolite for NMR analysis. A total of 74 actinomycetes and 30 fungi were screened to identify strains which could produce the hydroxylated metabolites found in human plasma and urine sam-ples. Whilst a number of strains were shown to generate the metabolites, three strains of actinomycetes (Amycolatopsis orientalis, Actinoplanes sp. and one unidentified strain) and one fungus (Curvularia lunata) were selected to produce four hydroxylated metabolites by incubation of JM-1232(-) in a fermentation broth with the appropriate microorgan-ism. After isolation by solvent partitioning, column chromatography and preparative HPLC, low milligram amounts of each purified metabolite were generated for NMR analysis, which confirmed the identity of the major hydroxylated metabolites of JM-1232(-).

218

P333. Characterization of Human UDP-Glucuronosyltransferases responsible for the in vitro Glucuronidation of Armillarisin A

Dongxue Sun1, Guang Bo Ge2, Zhong-Ze Fang3, Liangliang Zhu2 and Ling Yang2

1School of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 3Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Armillarisin A, a new coumarin derivative isolated from the fungus Armillariella tabescens (Scop. ex Fr.) Singer, has been used for treatment of acute cholecystitis, infection of biliary system and pancreatitis in China for several years [1, 2], but its metabolic pathway remains unknown. In the current study, the UDP-glucuronosyltransferase (UGT) conjugation pathway of Armillarisin A was investigated by using human liver microsomes (HLMs) and 12 commercially available recombinant human UGTs. One mono-glucuronide Armillarisin A was identified by liquid chromatography/mass spectrometry (LC/MS), when Armillarisin A was incubated with human liver microsomes in the presence of uridine-5′-diphosphoglucuronic acid (UDPGA). Assays with the recombinant human UGT isoform(s) revealed that human UGT1A1, UGT1A9, UGT1A7 and UGT2B15 involved in the glucuronidation of Armillarisin A. The K

m values of HLM, rUGT1A9, rUGT1A1, rUGT1A7 and

rUGT2B15 for the glucuronidation of Armillarisin A were 799.8 ± 51.5, 134.0 ± 17.2, 124.3 ± 4.8, 399.4 ± 37.8 and 420.2 ± 41.5 μM respectively. The intrinsic clearance of rUGT1A9 (48 ul/min/mg protein) is significantly larger than that of other recombinant UGT isoforms. Furthermore, the glucuronidation of Armillarisin A in HLMs (n=11) had the strong correla-tion (r=0.93, p<0.001) with propofol glucuronidation, a probe reaction for UGT1A9, while the chemical inhibition study using a series of inhibitors to UGTs showed similar inhibitory effects towards HLMs and UGT1A9 mediated glucuronida-tion. These results suggested that UGT1A9 played an important role in hepatic glucuronidation of Armillarisin A.

References

1. J.B. Zhu, L.B. Luan, Q.C. Shi, Acta Pharmacol. Sin. 27 (1992) 231–235.2. http://app1.sfda.gov.cn/datasearch/face3/base.jsp

P334. Characterization of UGT Isoforms Involved in Liquiritigenin Glucuronidation in Human Liver Microsomes

Ying Hu1, Hui-Xin Liu2 and Ling Yang2

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Liquiritigenin (7,4′-dihydroxyflavanone) is found most notably in Glycyrrhizae radix (G. radix, licorice, liquorice), which is one of the oldest and most frequently used botanicals in the polyherbal preparations. For example, 70 of 110 prescriptions in Chinese medicine Shang Han Lun include G. radix. In the United States and European countries (e.g. United Kingdom and Belgium), G. radix products are currently used as flavoring and sweetening agents for tobaccos, chewing gums, candies and beverages and consumed in a large scale (i.e. an annual amount of ∼1.5 kg per person). G. radix exerts anticancer, antitussive, expectorant and antipyrotic actions and is often used to treat cough, pharyngitis, bronchitis, bronchial asthma. Liquiritigenin have been shown to have the anti-angiogenic effect, and antioxidant, anti-inflammatory activities. Glucuronidation is an important pathway in the metabolism of liquiritigenin. However, the metabolites and primary UDP-glucuronosyltransferase (UGT) isoforms responsible for liquiritigenin glucuronidation remain to be determined in human. The identification of metabolites and enzymes responsible for drug metabolism are important for us to understand which one, parent drug or metabolite(s), is really active and how variations in drug concentrations can lead to differences in drug efficacy and toxicity. Here, we characterized liquiritigenin glucuronidation by human liver microsomes (HLMs), human intestine microsomes (HIMs), and 12 recombinant UGT (rUGT) isoforms to identify what kinds of metabolites are present and which human UGT isoforms are involved. One metabolite was

219

detected in reactions catalyzed by HLMs, HIMs, rUGTs and were identified as monoglucuronides by liquid chromatog-raphy mass spectrometry. Among the UGT isoforms investigated, UGT11A9 and 2B7 showed glucuronidation activity for liquiritigenin, with UGT1A9 possessing the higher activity. Glucuronidation of liquiritigenin by rUGT1A9, and rUGT 2B7 showed Michaelis-Menten kinetics with Km values of 35 and 32 µM, respectively. The glucuronidation of liquiritigenin was significantly correlated with morphine 3OH-glucuronidation and 3□Œ-azido-3□Œ-deoxythymidine glucuronidation (r=0.86, p<0.05), a reaction mainly catalyzed by UGT2B7. Propofol inhibited liquiritigenin glucuronidation in HLM with an IC50 value of 189 µM, which is between the IC50 value in recombinant UGT1A9 (106 µM) and UGT2B7 (> 400 µM). In combination, we identified UGT2B7 as the major isozyme responsible for liquiritigenin glucuronidation in HLMs.

P335. Characterization of UGT Isoforms Involved in Naringenin Glucuronidation in Human Liver Microsomes

Ying Hu, Hui-Xin Liu and Ling Yang

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

Flavonoids are part of a family of naturally occurring polyphenolic compounds and represent one of the most prevalent classes of compounds in vegetables, nuts, fruits and beverages such as coffee, tea, and red wine as well as medical herbs. Chemically, flavonoids contain a common phenylchromanone structure (C

6-C

3-C

6) with at least one

hydroxyl group substituent or a hydroxyl derivative such as a methoxy group. More than 8000 compounds with a flavonoid structure have been identified and categorized into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins, and chalcones. Naringenin (4,5,7-trihydroxyflavanone) is thought to be one of the active components in grapefruit juice, which modulates the enzyme activity of cytochrome P-450. Concomitant intake of grapefruit juice alters the pharmacokinetics of a variety of drugs. Citrus flavonoids exert various pharmacological activities, such as the inhibition of selected cytochrome P-450 isoform CYP1A2, and CYP3A4, which may increase oral bioavailability of nifedipine, cyclosporine, nimodipine, and verapamil. Naringenin is a flavonoid that is considered to have a bioactive effect on human health as antioxidant, free radical scavenger, anti-inflammatory. Glucuronidation is an important pathway in the metabolism of naringenin. However, the metabolites and primary UGT isoforms responsible for naringenin glucuronidation remain to be determined in human. The identification of metabolites and enzymes responsible for drug metabolism are important for us to understand which one, parent drug or metabolite(s), is really active and how variations in drug concentrations can lead to differences in drug efficacy and toxicity. Here, we characterized naringenin glucuronidation by human liver microsomes (HLMs), human intestine microsomes (HIMs), and 12 recombinant UGT (rUGT) isoforms to identify what kinds of metabolites are present and which human UGT isoforms are involved. One metabolite was detected in reactions catalyzed by HLMs, HIMs, rUGTs and were identi-fied as monoglucuronides by liquid chromatography mass spectrometry. Among the UGT isoforms investigated, UGT1A1, 1A3, 1A9, and 2B7 showed glucuronidation activity for naringenin, with UGT1A9 possessing the highest activity, followed by UGT2B7. Glucuronidation of naringenin by rUGT1A9, and rUGT 2B7 showed Michaelis-Menten kinetics respectively. The naringenin glucuronidation activity was significantly correlated with UGT2B7 activity rather than with UGT1A9 activity from 15 individual HLMs. The naringenin glucuronidation activity was significantly cor-related with UGT2B7 activity rather than with UGT1A9 activity from 15 individual HLMs. A chemical inhibition study showed that the IC

50 for 3′-azido-3′-deoxythymidine inhibition of naringenin glucuronidation was similar in HLMs

compared with rUGT2B7. In combination, we identified UGT2B7 as the major isozyme responsible for naringenin glucuronidation in HLMs.

P336. Comparative Metabolism of T-2 Toxin by the Fractions of Hepatocytes from Rats, Pigs, Chickens and Carps

Zonghui Yuan1, Qinghua Wu2, Lingli Huang2, Zhaoying Liu2, Min Yao2, Xiaoqin Cao2, Yulian Wang2, Menghong Dai2, Dongmei Chen2, Yuanhu Pan2, Yanfei Tao2, Dapeng Peng2, Xu Wang2 and Zhenli Liu2

1College of Veterinary Medicine,Huazhong Agricultural University, Wuhan, China, 2College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China, Hubei

220

T-2 toxin, the most toxic member of type-A in trichothecenes, is highly concerned by the public for food safety. Its toxicities would be closely related with its metabolic profiles in animals and the liver would be the main metabolic organ for it that would have markedly species differences among animals. To elucidate the metabolic profiles of T-2 toxin in food-producing animals, the metabolism of T-2 toxin incubated with the hepatic microsomes, cytosol and hepatocytes from rats, piglets, chickens, common carps (Cyprinus carpio L.) and grass carps (Ctenopharyngodon idellus) were car-ried out. Metabolites were identified by a mass resolution and highly sensitive liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectrometry (LC/ MS-ITTOF). Metabolic reactions in the microsomal system were stronger than those in other systems. Besides T-2 toxin, HT-2 toxin, neosolaniol (NEO), 3′-hydroxy-T-2, 3′-hydroxy-HT-2, and T-2 triol were found in all the incubation systems. No hydroxyl metabolites were detected in the hepatocytes of rats, chicken and pigs. Hydroxylation was the main metabolic pathway for the carps, whereas hydrolytic products were the major metabolites for rats, chickens and pigs. Significant quantitative and qualitative variations between common carps and grass carps existed as well. The hydroxylation in grass carps was more active, whereas the hydrolytic ability, especially the elimination of C-8 isovaleryl group (neosolaniol), was stronger in common carps. As for livestock and poultry, chicken displayed a stronger hydroxyl ability, whereas pigs showed a more hydrolytic activity. cDNA expressed CYP3A29, a important CYP3A isoform in pigs, was able to efficiently convert T-2 toxin and HT-2 toxin to a higher amount of 3′-hydroxy-T-2 and 3′-hydroxy-HT-2, respectively. The side chains at C-4 and C-8 of T-2 toxin could be removed by isoenzymes of carboxyesterases. CYP450 enzymes were first proved to be involved in the formation of neosolaniol. The present study is very helpful for people to well understand the metabolism of T-2 toxin in different food-producing animals and it provides basic data for the evaluation of T-2 toxin for human food safety.

P337. Compatative in Vitro Metabolism of A Benzylamine Containing Test Drug Using Human, Monkey, Rat, and Dog Plasma – Investigation of Amine Oxidase Involvement in Drug′s Metabolism

Heasook Kim-Kang and Zheming Gu

Drug Metabolism and Biotransformation, XenoBiotic Laboratories, Inc., Plainsboro, NJ, USA

Poor extractability of drug-related compounds from the plasma is an indication of the formation of reactive metabolites, resulting in the covalent binding with endogenous matrix, which in turn becomes a potential cause of adverse reaction or various organ toxicities. The biotransformation of a radiolabeled test article containing a benzylamine moiety ([14C]-TA) was investigated in vitro using dog, human, monkey, and rat plasma. The incubation mixtures were analyzed by HPLC/radio-detection/MS. Amine oxidase involvement in the metabolism of TA in plasma was investigated by using specific inhibitors. Metabolism of TA via oxidative deamination was rapid in dog and monkey, but TA was stable in human and rat plasma. Among the four different amine oxidases evaluated, semicarbazide sensitive amine oxidase was responsible for the metabolism in dog plasma. In addition, dog and monkey plasma yielded a significant amount of unextractable radioactivity in the pellets while rat and human plasma yielded excellent extraction efficiencies, demonstrating that the metabolites in dog and monkey plasma may have been strongly bound to plasma protein. Possible permanent binding of TA-related compounds to plasma protein was investigated by incubation of [14C]-TA with denatured plasma as well as by using Western blot assay. Separated radioactive spots from Western blot assay were visualized by using BAS 1800II Bio-imaging Analyzer System. The binding of the unextractable radioactivity to plasma protein in the pellets was shown to be reversible by Western blot assay, indicating that the metabolites may not pose any toxicity caused by permanent covalent binding.

P338. Driving Efficiency in a High-throughput Metabolic Stability Assay Through a Generic High Resolution/Accurate Mass Method and Automated Data Processing

Yingying Huang1, Mark Sanders2, Wenqing (Angel) Shui1, Song Lin3, Allen Zhang1 and Caroline Ding4

1Thermo Fisher Scientific, San Jose, CA, USA, 2Thermo Fisher Scientific, Somerset, NJ, USA, 3Dept of DMPK/MS 4.2, Chiron Corp, Emeryville, CA, 4Thermo Fisher Scientific, San Jose, CA,

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Improving analytical throughput is strongly desired in many quantitative workflows being developed for early drug discovery. A common practice is to connect ultrahigh performance liquid chromatography (UHPLC) to triple quadrupole mass spectrometry for drug candidate screening. This approach certainly results in short analytical run times, however, in assessing the true throughput one needs to consider all aspects of the workflow, including instrument set-up and the necessity to rerun samples when information is missed. Here we demonstrate a high throughput metabolic stability assay with a simplified instrument set-up which significantly improves overall assay efficiency. Also, as the data collection is non-biased, information on the parent compound and metabolites is gathered at the same time. A series of standard drug compounds were incubated individually with human liver microsomes. Incubation of each drug was quenched at 0, 5, 10, 20 and 30 min. Various UHPLC methods were employed for separation of all the incubation samples to optimize throughput verses quality of data. Mass spectrometry was performed on an Exactive Orbitrap scanning a mass range of 100-1000 m/z at 25,000 or 50,000 resolution. Novel algorithms were used for automated data analysis, including metabolic stability plots of par-ent and potential metabolites. The high resolution/accurate mass approach was validated by comparison with a traditional triple quadrupole SRM method. Described here is a highly efficient metabolic stability assay with sensitivities on par with traditional triple-quadrupole assays, but has significant advantages with regard to overall throughput, data quality and information content. The Orbitrap high resolution/accurate mass (HRAM) approach uses resolutions >20,000 and sub ppm mass accuracies to provide the selectivity and sensitivity required for these analyses. With the UHPLC methods used there were sufficient scans (12-20) across a typical peak (width 2-4 sec) to provide high quality quantitative data. In contrast to triple quadrupole methods where selectivity is provided by a targeted scan specifically tuned to the analyte of interest, the HRAM approach uses an unbiased scan mode. Using the same method for all compounds simplifies the assay set up and reduces data entry errors; no need for developing compound specific MS/MS methods results in significant time savings. Also, because of the unbiased nature of the analysis little information is needed about potential metabolites at the time of the assay. Therefore as knowledge is gained about the compound under study the metabolic stability data can be re-interrogated for the presence or absence of metabolites. In order to increase productivity and mine the information rich data for pertinent metabolite information, automated data processing algorithms were developed to specifically take advantage of the high precision data. These algorithms significantly reduce chemical noise allowing for more efficient detection of both expected and unexpected metabolites. Various UHPLC methods were investigated in order to strike a balance between analytical throughput and data quality. As the goal was to analyze both parent molecule and metabolites, chromatographic separation of potential multiple hydroxylated metabolites was neces-sary. This would require longer analytical run time, but was compensated for by efficiency gains in assay set up and additional information content.

P339. Effect of Oral Administration of Antibiotics on the Pharmacokinetics of Hesperidin

Hye Hyun Yoo1, Ming Ji Jin1, Unyong Kim1, Dong-Hyun Kim2 and Oh-Seung Kwon1

1Doping Control Center, Korea Institute of Science and Technology, Seoul, South Korea, 2College of Pharmacy, Kyung-Hee University, Seoul, South Korea

Hesperidin is a biological active flavanone glycoside found plentifully in citrus fruits and known to be absorbed from the gastrointestinal tract after being metabolized by gut microflora. In the present study, effects of oral administration antibiotics on pharmacokinetics of hesperidin were investigated in rats. For this, after administration of hesperidin to rats, the plasma concentration-time profile of hesperetin was investigated in conventional and antibiotic-treated rats. The C

max values of hesperetin in normal and antibiotic-treated rats were 0.58 and 0.20 μg/mL, respectively, and the

AUC values were 6.3 and 2.8 μg·hr/mL, respectively. Thus, systemic exposure (AUC) of hesperetin as well as Cmax

was significantly higher (P<0.05) in normal rats than in antibiotic-treated rats. The fecal β-glucosidase activities of normal and antibiotic-treated rats were determined to be 0.21 and 0.11 nmol/min/mg, respectively. The fecal α-rhamnosidase activities were found to be 0.37 and 0.12 nmol/min/mg, respectively. The rates of transformation of hesperidin to hesperetin were determined to 6.9 and 2.9 nmol/min/g in fecal samples from normal and antibiotic-treated rats, respec-tively. Taken together, the significant decrease in uptake of hesperidin and the consequent pharmacokinetic alteration was found in antibiotic-treated rats. This phenomenon resulted from the alteration of metabolic activities of intestinal bacteria which is caused by oral administration of antibiotics.

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P340. Erythrocyte Folate LEVELS IN PATIENTS ON Hemodialysis

Saziye Sezin Palabiyik1, Gozde Girgin2, Terken Baydar2, Ramazan Cetinkaya3 and Gonul Sahin2

1Dept. of Toxicology, Ataturk University, Faculty of Pharmacy, Erzurum, Turkey, 2Dept. of Toxicology, Hacettepe University, Faculty of Pharmacy, Ankara, Turkey, 3Department of Nephrology, Faculty of Medicine, Ataturk University, Erzurum, Turkey

ERYTHROCYTE FOLATE LEVELS IN PATIENTS ON HEMODIALYSIS S. Sezin Palabiyik1, Gozde Girgin2, Terken Baydar2, Ramazan Cetinkaya3, Gonul Sahin2 1Department of Toxicology, Faculty of Pharmacy, Ataturk University, Erzurum; 2Department of Toxicology, Faculty of Pharmacy, Hacettepe University, Ankara; 3Department of Nephrology, Faculty of Medicine, Ataturk University, Erzurum; Turkey. Pteridines are natural heterocyclic compounds having vital role in important biochemical pathways. Folates are the conjugated pteridines and play an important role in vital biochemical pathways in which single carbon transfer is required such as purin and pirimidine synthesis, conversion of serin to glycine, histidine catabolism, methionine synthesis and homocycstein regulation [1, 2]. Blood folate concentrations are generally used as bioindicators of folate pool. On the other hand, patients with end-stage renal disease undergo-ing dialysis have an increased risk of cardiovascular morbidity and mortality. This study aimed to assess erythrocyte folate status of patients undergoing hemodialysis. The long-term hemodialysis patient group consisted of 66 patients subgrouped as diabetic nephropathy (n=24), hypertensive nephropathy (n=20), reflux nephropathy (n =14) and inter-stitial nephritis and others (n=14). The samples were collected before the application of hemodialysis. The results of each patient group was also compared with the control group (n = 21). Erythrocyte folate statuses were measured with a modified microbiological assay [4]. In patients on regular hemodialysis folate concentrations were found to be raised and the difference between the patient groups and controls was significant (p< 0.05). As a result; folate is a water-soluble vitamin and removes with urine in the body. This may be related to lower excretion by the kidneys and removal only by dialysis. These results may contribute to renal anemia however this should be supported with future studies. This study was partially granted by Atatürk University Scientific Research Foundation Unit (BAP-2007/223) and also was supported by Toxicology Department, Faculty of Pharmacy, Hacettepe University. Key words: Folate, microbiological assay, renal patients, hemodialysis

References

1. Werner-Felmayer G, Golderer G, Werner ER. Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects. Curr Drug Met, 2002; 3: 159-173

2. Lucock M. Folic acid: Nutritional biochemistry, molecular biology and role in disease processes. Mol Genet Metab 2000; 71: 121-1383. Schaefer RM, Teschner M, Kosch M. Folate metabolism in renal failure. Nephrol Dial Transplant 2002; 17 Suppl: 524–5274. Sipahi H, Baydar T, Ozalp M, Sahin G. A Modified microbiological method for determination of folate levels in erythrocyte and plasma. Pteridines,

2007; 18, 106–113.

P341. Excretion and Metabolism of Forsythiaside in Rats

Qi Chang, Geng-Nan Wang, Yong-Hong Liao, Lei Zhang, Qian Wang and Bin-Xin Xiao

Institute of Medicinal Plant Development, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China

Background Forsythiaside, a phenylethanoid glycoside, is the most abundant component of a well-known traditional Chinese medicine, Lian Qiao, the roots of Forsythia suspense. It is reported that forsythiaside possesses various phar-macological activities including antioxidant, antibacterial, antiviral and vasorelaxant. Our previous study showed that the absolute bioavailability of forsythiaside was only 0.5% after oral administration, indicating that forsythiaside might be suffered from extensive first pass metabolism. Therefore, the present study was carried out to investigate the excretion and metabolism of forsythiaside. These metabolic products might contribute to its pharmacological effects of forsythiaside in vivo. Methods SD rats were received a light surgery for right jugular vein and/or bile fistulas can-nulation with a catheter under anesthesia, and than allowed to recovery and fast overnight. Forsythiaside at 20 mg/kg was given to rats by a single intravenous administration through the jugular vein catheter. After dosing, urine (0-11 h) or bile (0-3 h, rats were at amnesia condition) were collected and analyzed for forsythiaside concentration by LC-MS/

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MS method in negative model (m/z 623/161) (Wang et al, 2010). The metabolites were identified by LC-MS method with full scan of molecular ions from m/z 100-1000 and further confirmed by their product ion scan using LC-MS/MS method. Results The intact form of forsythiaside in the body was mainly excreted from urine at 12.67 ± 4.62% of dose, and slightly excreted from bile at 1.36„b0.31% of dose. Forsythiaside was extensively transferred into its methylated, sulfated or/and glucuronidated metabolites and then excreted from both urine and bile. The main metabolites of forsythiaside were characterized as its monomethylated metabolite M1 at m/z 637; bimethylated metabolite M2 at m/z 651; both methylated and sulfated metabolites M3 and M4 at m/z 717, 731 and their reduced products M5 and M6 at m/z 719, 733; both methylated and glucuronidated metabolites M7 and M8 at m/z 813, 827 and their reduced products M9 and M10 at m/z 815, 829; bimethylated, sulfated and glucuronidated metabolite M11 at m/z 907. Conclusion Renal excretion and conjugated metabolism of forsythiaside are the main routes of its clearance from the body, which biotransformation pathway includes methylation, sulfatation or/and glucuronidation in varied degree.Acknowledgement: Finance support from The Ministry of Science and Technology of the People′s Republic of China (20070463 and 2009ZX09301-003)

P342. Expressions of Hepatic Cytochrome P450 May be Regulated Dynamically and Cooperatively

Akihiro Hisaka1, Yu Sasaki2 and Hiroshi Suzuki2

1Pharmacology and Pharmacokinetics, The University of Tokyo Hospital, Tokyo, Japan, 2Department of Pharmacy, The University of Tokyo Hospital, Tokyo, Japan

In the previous study, we developed a method for simultaneous quantification of multiple species of cytochrome P450 (CYP) in human liver microsome and expressed systems using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [Y Sasaki, et al., Annual meeting of The Japanese Society for The Study of Xenobiotics, 2009]. The analysis was achieved unambiguously by measuring tryptic peptides whose sequence is unique among all human proteins, and all the target peptides were chemically synthesized beforehand, labeled with heavy oxygen (18O) and provided as inter-nal standards. In this study, the method was applied to human liver microsomes from 50 individuals for simultaneous quantification of CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2J2, 3A4, 3A5, and 3A7), enzymes in electron transfer system (CPR; cytochrome P450 reductase, Cb5; cytochrome b5, Cb5R; cytochrome b5-reductase), marker proteins of microsome (MTTP; microsomal triglyceride transfer protein, BiP; major chaperone protein in microsome) and another metabolizing enzyme (UGT1A1). Significant correlations were observed for the expression levels of some of major CYPs and enzymes in electron transfer system (1A2, 2A6, 2B6, 2C8, 2C9, 3A4, CPR, and Cb5), suggesting that these proteins may be regulated cooperatively by a common mechanism. Furthermore, large inter-sample variations of approximately 4-fold of geometric standard deviation were observed for expressions of CYP3A4 and Cb5, and they were apparently larger than typical inter-individual differences observed in drug clearance in vivo (∼2-fold of geo-metric standard deviation). The data implied that expressions of CYPs were regulated dynamically (time-dependently) rather than statically. We developed a mathematical model which explains statistical distribution and correlations of CYP expressions determined in human liver microsome, and considered its mathematical conversion in order to bridge between clearances of in vitro and in vivo situations. This concept may be valuable in the extrapolation of inter-individual differences in pharmacokinetics from in vitro to in vivo. The LC-MS/MS quantification of metaboliz-ing enzymes would be utilized as a basic technology to clarify regulations of their expression and the various factors affecting on pharmacokinetics.

P343. Identification of Metabolites from Maropitant Using a Dual-Pressure Linear Ion Trap and Mass Frontier Software

Yingying Huang, Rose Herbold, David Nakamura

Thermo Fisher Scientific, San Jose, CA, USA, and Elan Pharmaceuticals, South San Francisco, CA, USA

The traditional way of performing metabolite structural elucidation via LC/MS is through the time-consuming process of manual interpretation of tandem mass spectra. Mass shifts of signature fragment ions can be diagnostic of struc-tural soft spots. However, when complex or unusual biotransformations take place, the assignment of the fragment

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ion structure can be challenging. Further fragmentation of mass-shifted fragments may be needed to elucidate and confirm their structural assignments. Literature references may be necessary to confirm fragmentation pathways. All these necessitate a strong background in gas-phase fragmentation mechanisms. Described is an experiment where multi-stage fragmentation information from a LC/MSn run is automatically summarized by software, which then applies known fragmentation mechanism to assign fragment structures, thus facilitating structural elucidation for metabolites. The samples analyzed were dog and mouse microsomal Phase l incubations of Maropitant, a drug commercially available for treatment of emesis in dogs as CereniaTM by PfizerTM. The final concentrations in the one-hour incubation samples were 10µM Maropitant, 1mmol NADPH, and 0.5mg/mL protein. A dual-pressure linear ion trap mass spectrometer with a UHPLC pump and auto sampler were used with a 50X2.1, 1.9µM, C18 column. Multiple stages of fragmentation were performed in the form of MSn (N<5) spectral trees automatically for Maropitant and its metabolites. Mass Frontier software (version 6.0) was used to categorize the multi-stage fragmentation data, identify the metabolites based on the parent structure, and help assign the sites of biotransformation. Spectral trees were generated data dependently, in which the three most intense ions observed in the MS2 spectra were further fragmented to give MS3 spectra. Then MS4 spectra were acquired for the most intense ion found in each MS3 spec-tra. All together there were 8 scan events in each scan cycle, which typically took ∼1 second. Such spectral trees were automatically categorized to each component detected in the LC/MSn run via Mass Frontier. A new function Fragment Ion Search (FISh) was then used to screen for components displaying the same fragments chemically possible to be generated from Maropitant. The resulting components are structurally similar to Maropitant, thus the putative metabolites. The main fragments for Maropitant are m/z=167.05, 177.06, and 276.20. The major Phase l metabolites in the dog microsomal incubation are hydroxylation and demethylation. The single hydroxylation metabolite at retention time 3.19min displays major fragments at m/z=167.05, 193.03, and 276.20. Using the Compare Spectra tool, the mass shift of 16 on the 193 peak revealed the site of hydroxylation. For the demethylation metabolite at retention time 4.23min, there is a major change in the fragmentation pathway resulting in completely different main fragments of m/z=162.95, 167.13, and 293.22. MS4 spectra were acquired to confirm the fragment ion structures which then led to the localization of the demethylation. The putative structures of the metabolites were verified by auto-annotation of their MSn spectra. Once the major metabolites from the dog microsomal incubation were identified, they were saved into a customized library. The Components Editor tool was then used to screen for such metabolites in the mouse matrix.

P344. In Vitro Biotransformations of Chlorinated Bisphenol A by Human and Rat Sub-Cellular Liver Fractions

Anne Riu, Elisabeth Perdu, Emilien Jamin, Vuthy Ty, Sandrine Bruel, Anne Hillenweck and Daniel Zalko

UMR 1089, Xenobiotiques, INRA, Toulouse, France

Bisphenol A (BPA) is widely used (> 3 million tons/year) to manufacture plastics, food can linings, dentistry sealants and thermal paper. Chlorinated BPA analogues are an emerging group of environmental contaminants and are closely related to BPA. Tetrachlorobisphenol-A (TCBPA) has been reported to be used as a flame retardant (<10 000 tons/year). Its presence, as well as the presence of lower chlorinated analogues (mono-, di- and tri-CBPA) has been unequivo-cally demonstrated in environmental samples (1). In addition, recent results have demonstrated the presence of several chlorinated BPA analogues in human fat (2). Given the low levels of production of TCBPA, the origin of most chlorinated BPA in the environment is more likely the chlorination of BPA than the dechlorination of TCBPA. BPA is readily chlorin-ated by reacting with sodium hypochlorite, which is commonly applied as a bleaching agent in paper factories and also as a disinfectant in sewage treatments plants. Moreover, BPA easily chlorinates in aqueous media, and chlorinated BPAs have been found in sea water (3). Contrary to BPA and to the flame retardant tetrabromobisphenol-A (TBBPA), nothing is known about the metabolic fate of chlorinated BPAs either in vitro or in vivo. BPA and TBBPA undergo in vitro oxidative metabolism in addition to conjugation reactions (4, 5). In order to understand the fate of chlorinated BPAs in rat and human, we investigated the in vitro biotransformation of several analogues using rat as well as human liver sub-cellular fractions. 14C-labeled chlorinated BPAs were synthesized and analytical systems enabling the radio-HPLC separation of parent compound and their main metabolites were developed. The structural characterization of metabolites produced through oxidative biotransformation was carried out by LC-ESI-ITMS. Chlorinated BPAs undergo P450-dependant oxidation, which results in a clivage next to the central carbon, and to a subsequent rearrangement

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leading to the formation of hydroxylated chloro-phenols as well as several metabolites of lower polarity and higher MW than the parent compounds.

References

1. Gallart-Ayala H, Nunez O, Moyano E, & Galceran MT (2010) Field-amplified sample injection-micellar electrokinetic capillary chromatography for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers and derivatives in canned soft drinks. Electrophoresis 31(9):1550-1559.

2. Fernandez MF, Arrebola JP, Taoufiki J, Navalon A, Ballesteros O, Pulgar R, Vilchez JL, & Olea N (2007) Bisphenol-A and chlorinated derivatives in adipose tissue of women. Reprod Toxicol 24(2):259-264.

3 Liu H, Zhao H, Quan X, Zhang Y, & Chen S (2009) Formation of chlorinated intermediate from bisphenol A in surface saline water under simu-lated solar light irradiation. Environ Sci Technol 43(20):7712-7717.

4. Zalko D, Prouillac C, Riu A, Perdu E, Dolo L, Jouanin I, Canlet C, Debrauwer L, & Cravedi JP (2006) Biotransformation of the flame retardant tetrabromo-bisphenol A by human and rat sub-cellular liver fractions. Chemosphere 64(2):318-327.

5. Jaeg JP, Perdu E, Dolo L, Debrauwer L, Cravedi JP, & Zalko D (2004) Characterization of new bisphenol a metabolites produced by CD1 mice liver microsomes and S9 fractions. J Agric Food Chem 52(15):4935-4942.

P345. In Vitro Hydrolysis and Trans-esterification of a Novel Ethyl Ester Prodrug, CDP323, in Human Liver and Intestinal Microsomes

Hugues Chanteux1, Maria Rosa2, Claude Delatour3, Celine Jacques-Hespel2, Steven Smith1 and Jean-Marie Nicolas2

1Non-Clinical DMPK, UCB Pharma SA, Braine-L′Alleud, Belgium,2Non-Clinical DMPK, UCB Pharma SA, Braine-l′Alleud, Belgium, 3Research DMPK, UCB Pharma SA, Braine-l′Alleud, Belgium

CDP323 is an ethyl ester prodrug which is hydrolyzed into CT7758, the active carboxylic acid drug. CT7758 is a potent VLA4 antagonist and CDP323 was identified as a development candidate for the treatment of multiple sclerosis. In vitro metabolism data were generated using human intestinal (HIM) and liver microsomes (HLM) in order to charac-terize the enzyme involved in the hydrolysis of CDP323 as well as its propensity to be transesterified in the presence of ethanol. CDP323 was incubated with HLM and HIM (NADPH-free conditions) and the formation of CT7758 was monitored by an LC/MS/MS method. Ethanol, D6-ethanol, and several prototypical esterase inhibitors were tested for their effect on CDP323 clearance and CT7758 formation. In addition, recombinant carboxylesterase-1 (CES1) and carboxylesterase-2 (CES2) were used to further characterize the involved enzyme(s). The hydrolysis rate of CDP323 in HIM was barely detectable when compared to HLM (ie. i-fold difference) and thus, was not further explored. Km and Vmax determination showed that the hydrolysis of CDP323 in HLM was a high affinity (Km of 11.3µM) and high capacity reaction (Vmax of 6.38 nmol/min/mg prot). In addition, assays using recombinant human enzymes showed that only CES1 could hydrolyze CDP323, not CES2. Since CES1 is also known for the transesterification of ester prodrugs, the propensity of CDP323 to be transesterified in presence of ethanol and D6-ethanol was assessed. Addition of ethanol (up to 2%) in the incubation medium decreased the hydrolysis of CDP323 (up to 50% decrease) in accordance with a decrease of the Km and Vmax of the reaction. Assays using D6-ethanol showed that (i) the trans-ester (D5-CDP323) was formed in HLM; (ii) the disappearance rate of CDP323 was similar in incubation medium with or without D6-ethanol; (iii) the formation of the transester directly compensated the decrease observed in the formation of CT7758. The use of chemical inhibitors for arylesterase (DTNB 200µM), acetylcholinesterase (bw284c51 10µM), butyrylcholinesterase (ethopropazine 10µM), CES2 (loperamide 15µM) or CES (paraoxon [100µM], BNPP [1mM], Benzil [5µM]) showed that only non-specific inhibitors of CES (paraoxon, BNPP and benzil) significantly inhibited the hydrolysis into CT7758 and the transesterification of CDP323. Overall, these data showed that the hydrolysis of CDP323 in human is mediated by hepatic CES1 which is in accordance with (i) the tissue distribution of this isoenzyme (not present in intestine), (ii) the structure-activity relationships for the substrate of this isoenzyme (ester with a small alcohol group), (iii) the fact that only CES1 has been reported to catalyze transesterification reac-tions and (iv) with the phenotyping data collected in the present study (recombinant enzyme, chemical inhibitors, tissue difference). In addition, the data showed that CDP323, in presence of ethanol, is able to be transesterified in HLM. However, the impact of the transesterification on the in vivo clearance of CDP323 should be limited regarding the in vivo concentration of ethanol that could be physiologically reached since observed in vitro at relatively high

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ethanol concentrations. In addition, the transesterification of CDP323 with other free hydroxyl group found in vivo (glycerol, …) still needs to be investigated.

P346. Influence of Fluorine Substitution at the C-3 Position on Oxidative Metabolism of N-Ethylpiperidine Analogs

Bianca M. Liederer1, Vikki Dinkel2, John Gaudino2, Sashi Gopaul1, Hoa Le1, Xingrong Liu1, Dan Sutherlin1, Susan Wong1 and Cyrus Khojasteh1

1Genentech, Inc., South San Francisco, CA, USA, 2Array BioPharma, Inc., Boulder, CO, USA

Fluorine is being used in drug design and optimization to improve potency, selectivity and metabolic stability. However, there are limited published reports on the influence of fluorine on piperidine metabolism. Here, we present data that demonstrate the influence of fluorine (mono- and di-) on physicochemical properties (clogP, clogD 7.4, pKa) as well as metabolic stability and metabolites formation from liver microsomes and hepatocytes. Four fluorinated analogs of compound A (N-(4-(3-(1-ethylpiperidin-4-ylamino)-1H-3,4-b]pyridine-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide) and fluorinated analogs of piperidine from a commercial source were used. Physicochemical properties were estimated computationally using Biobyte and MoKa. Sites of oxidative metabolism were assessed by hepatocyte incubations or predicted using Metasite. Metabolic stability studies were performed in four preclinical species (mouse, rat, dog, ) and human using liver microsomes (0.5 mg protein/mL) or hepatocytes (0.5 x 106 cells/mL). As expected, fluorine substitutions decreased basicity as indicated by lower pKa values. The pKa values for A, mono- and di-fluoro substituted analogs were 9.62, 8.07, and 6.34, respectively. In addition, fluorine substitutions increased clogP and clogD 7.4 values from 4 to 5.2 and from 1.8 to 5.2, respectively. The addition of fluorine (decreased basicity and increased lipophilicity) did not significantly change the metabolic stability of the compounds in liver microsomes (< 2-fold change in CL

int), but a trend of increased

int observed with more fluorine atoms. In general, in

hepatocyte CLint

values for A were lower than those in liver microsomes and mono- and/or di-fluorine substitutions resulted in increased CL

int. It is possible that in hepatocytes permeability, protein binding and transporters may play

a role in this difference. The major metabolites in hepatocytes observed are N-deethylation and N-oxide formation. These metabolites were observed regardless of the number of fluorine atoms so no metabolic switching was observed. Although the introduction of fluorine may pose a synthetic challenge, there may be potency/selectivity and ADME considerations to utilize this moiety.

X or Y = H or FCompound A: X and Y = H

N

NN

O

O

O

XY

F

F

NN

N

H

HN

HN

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P347. Investigation of Metabolism in Mice, Rats, Monkeys, Dogs and Humans of a Novel Met Kinase Inhibitor for Species Selection for Preclinical Toxicology Studies

Bianca M. Liederer1, Matthew Baumgardner1, Brian Dean1, Vikki Dinkel2, John Gaudino2, Sashi Gopaul1, Hoa Le1, Xingrong Liu1, Dan Sutherlin1 and Cyrus Khojasteh1

1Genentech, Inc., South San Francisco, CA, USA, 2Array BioPharma, Inc., Boulder, CO, USA

GNE-A (N-(4-(3-((3SHkinasecryopreservedhepatocytesFinniganAccelaGlucuronidation of parent, M2: N-deethylation, M3: Oxidation of N-deethylated parent, M5: Oxidation of parent, M7: -2), M8: Glucuronidation of N-dealkylated par-ent, M9: Oxidation of N-deethylated parent (+14), M10,M11: Glucuronidation of N-dealkylated parent, and M12: N-dealkylation. M2, M9 and M12 have been confirmed by high resolution MS. M2 was compared (retention time and MS/MS data) with synthetic M2. Reduction with TiCl

3 provided supporting evidence that M5 is likely an N-oxide metabolite.

Percent drug-related material was established based on total peak areas of recovered metabolites and parent determined by UV. M2 was the major metabolite in all preclinical species and human (61.7 - 44.7%) except in dog. In dog, M5 (52.1%) was the most significant drug-related component. M9 was more predominant in rat (22.3%) than in other preclinical spe-cies (<1%). In vivo metabolism was assessed in mouse dosed PO at 300 mg/kg and in dog and monkey dosed IV at 1 mg/kg and PO at 3 mg/kg. Metabolite identification of drug-related components in plasma and in urine will be presented.

P348. Kinetics of Bile Acid Sulfation by Human Sulfotransferase 2A1 (SULT2A1)

Yazen Alnouti, Jiangeng Huang and Sai Praneeth Bathena

Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA

Bile acids (BAs) play a variety of physiological functions. However, BAs are also cytotoxic and cancer promoters. In humans, sulfation by sulfotransferase 2A1 (SULT2A1) is the dominant pathway of BA elimination and detoxification. In this study, we have constructed a stable cell line expressing SULT2A1 by transfection into HEK293 cells. The expression system was used to characterize and compare the sulfation kinetics of DHEA and 15 human BAs by SULT2A1. Formation DHEA-sulfate also demonstrated Michaelis-Menten kinetics with apparent K

m and V

max values of 3.8 μM and 130.8 pmol/

min/mg protein, respectively. Sulfation kinetics of BAs demonstrated Michaelis-Menten kinetics with a marked variation in apparent K

m and V

max values between individual BAs. Sulfation affinity was inversely proportional to the number of

hydroxyl groups of BAs. The monohydroxy- and most toxic BA (lithocholic acid) had the highest affinity, whereas the trihydroxy- and least toxic BA (cholic acid) had the lowest affinity to sulfation by SULT2A1, which support the major role of SULT2A1 in the elimination and detoxification of BAs in humans. Intrinsic clearance (CL

int) of ursodeoxycholic

acid (UDCA) was 1.5 and 9 fold higher than that of deoxycholic acid and chenodeoxycholic acid, respectively, despite the fact that all three are dihydroxy-BAs.

P349. Mass Balance and Metabolism of the Taxane Analog [14C]BMS-275183 Following Oral Administration to Humans

S. Nilgün Çömezoglu1, Van T. Ly1, Ihoezo V. Ekhato2, Jinping Gan1, Leila Alland3 and W. Griff Hum-phreys1

1PCO - Biotransformation, Bristol-Myers Squibb, Princeton, NJ, USA, 2Chemical Synthesis, Bristol-Myers Squibb, Princeton, NJ, USA, 3Discovery Medicine and Clinical Pharmacology, Bristol-Myers Squibb, Princeton, NJ, USA

3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxy-paclitaxel (BMS-275183) is a taxane analog that underwent early phase clinical testing in subjects with cancer (1,2). This compound was found to have encouraging antitumor activity but clinical development was later discontinued due to concerns over the pharmacokinetic and safety profiles. In this study, the metabolism and excretion of [14C]BMS-275183 were investigated in humans following oral administration of a single dose of [14C]BMS-BMS-275183 (200 mg/100 uCi). Pooled

228

human urine (0-240 h), pooled feces (0-240 h), and plasma (1, 2, 6, and 12 h) samples from 5 patients were profiled by radio-HPLC. The metabolites in these samples were quantified, and then, identified by liquid chromatography/mass spectrometry (LC/MS). Mean recoveries of the radioactive dose through 240 hours post-dose were 82.6% in feces and 12.6% in urine. Mean total recovery of administered radioactive dose from urine and feces collected through 240 hours post-dose was 95.2%. Maximal mean concentrations in blood (561 ng equiv/mL) and plasma (699 ng equiv/mL) were observed at 1.5 hours post-dose. Mean concentrations of radioactivity were measurable through 12 hours post-dose for plasma. Metabolites were identified by LC/MS. [14C]BMS-275183 was a prominent component in plasma and detected up to 12 h along with various oxidative metabolites. [14C]BMS275183 was extensively metabolized in humans forming mainly oxidative metabolites. Parent drug accounted for approximately 9% of the administered dose in urine and feces. M3, a major component in urine, was identified as hippuric acid. Two prominent metabolites in urine and feces resulted from oxidation of the t-butyl group on the side chain (M24) and cyclization of the oxidized t-butyl group (M21B). M13, an ester cleavage metabolite, was a prominent metabolite in plasma, urine, and fecal samples. Overall, oxidation is the main clearance mechanism of BMS-275183 in humans.

References

1. Bröker EL, de Vos FYFL, van Groeningen CJ, Kuenen BC, Gall HE, Woo MH, Voi M, Gietema JA, deVries EGE, Giaccone G. “Phase I trial with BMS-275183, a novel oral taxane with promising antitumor activity” (2006) Clin Cancer Res, 12(6): 1760-1767.

2. Bröker EL, Veltkamp SA, Heath EI, Kuenen BC, Gall H, Astier L, Parker S, Kayitalire L, Lorusso PM, Schellens JHM, Giaccone G. “A phase I safety and pharmacologic study of a twice weekly dosing regimen of the oral taxane BMS-275183” (2007) Clin Cancer Res, 13(13): 3906-3912.

P350. Mass Balance, Metabolic Disposition, Metabolite Characterization, and Pharmacokinetics of Oral 14C-Labeled Bosutinib in Healthy Subjects

Richat Abbas-Borhan, Inder Chaudhary, Bruce A. Hug, Cathie Leister, Jaime Burns, Sarvesh Vash-ishtha, John C.L. Erve and Daryl Sonnichsen

Pfizer Inc., Collegeville, PA, USA

Bosutinib (SKI-606: 4-[(2-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]- 3-quinolinecarbonitrile monohydrate), a potent inhibitor of Src and Abl tyrosine kinases, is being developed for the treatment of chronic myelogenous leukemia. The objective of this study was to characterize the mass balance, metabolic disposition, and pharmacokinetics of bosutinib after a single oral dose of [14C]-bosutinib in healthy men. In this open-label study, 6 healthy men, aged 22-29 years (median 23.5 years; 3 White, 1 African American, 1 other race), received a single oral dose of 10 nCi [14C]-bosutinib 500 mg (a mixture of radiolabeled and nonlabeled bosutinib) in solution. Blood, urine, and feces were collected predose and up to 216 hours postdose. Radioactivity in whole blood, plasma, urine, and fecal samples was determined by accelerator mass spectrometry (AMS), and the metabolite profiles in urine, fecal, and pooled plasma samples were generated by HPLC with fraction collection followed by AMS (HPLC-AMS). The metabolites in radioactive peaks were collected as fractions and characterized by nanospray mass spectrometry. Plasma samples were analyzed for bosutinib, oxydechlorinated bosutinib (M2), and Ndesmethyl bosutinib (M5) by a validated LC/MS/MS assay. The quantitative ranges of the LC/MS/MS assay were 1 to 500 ng/mL for bosutinib, 0.5 to 500 ng/mL for M2, and 0.5 to 200 ng/mL for M5 using 0.1 mL of plasma. Plasma concentration data were analyzed by a noncompartmental method using WinNonlin. For nonradiolabeled bosutinib, mean (SD) peak plasma concentration (C

max, 87.9 [30.8] ng/

mL) was reached at a median (range) time (tmax

) of 3.5 (2.00, 6.00) hours, the area under the concentration-time curve (AUC)was2190(661)ng•h/mL,apparentvolumeofdistributionwas14300(6060)L,apparentoralclearance(CL/F)was 248 (80.9) L/h, and elimination half-life (t½) was 39.70 (8.49) hours. For M2, the mean (SD) C

max, AUC, and t½,

respectively,were18.0(6.33)ng/mL,423(110)ng•h/mL,and33.55(9.24)hours,andthemedian(range)tmax

was 5.00 (2.00, 8.00) hours. For M5, the mean (SD) C

max,AUC,andt½,respectively,were25.0(7.21)ng/mL,545(162)ng•h/mL,

and 80.51 (38.35) hours, and the median (range) tmax

was 2.50 (2.00, 3.00) hours. The mean metabolite ratios for Cmax

and AUC, respectively, were 0.21 and 0.20 for M2, and 0.29 and 0.25 for M5. The mean (SD) recovery of radioactivity was 94.6% (7.49%) of the total dose, with the majority (72.5%) of the dose recovered within 96 hours. Fecal excretion accounted for 91.3% of the total dose and urine accounted for 3.29%. In plasma, as the radioactivity concentration was below the lower limit of quantitation, metabolite profiles could not be obtained. However, extraction of a large volume of plasma pooled near C

max led to the detection of bosutinib, M2, M5, and oxydechlorinated bosutinib sulfate (M11)

229

by mass spectrometry. Bosutinib and M5 were the major drug-related components in feces, while bosutinib and M2 were the major components in urine. In conclusion, elimination of [14C]-bosutinib-derived radioactivity was rapid and essentially complete, with the feces being the major route of elimination.

P351. Mechanism Studies for Determining the Loss of Nitrile Moiety and Formation of Primary Alcohol by CYP

Hoa Le, Susan Wong, Jim Driscoll, Cornelis Hop and Cyrus Khojasteh

Genentech, Inc., South San Francisco, CA, USA

Objective: Here we report the mechanism of oxidation accompanied by loss of nitrile and formation of an alcohol metabolite for a model compound.

Methods and materials: The test compound was incubated in liver microsomes of mouse, rat, and human (1 mg/mL) at 37 °C for 1 hr in the presence of NADPH (1 mM), alamethicin (100 μg/mL), UDPGA (5 mM) and GSH (5 mM) to determine and compare the extent of metabolism. Samples were processed and analyzed by LC-MS/MS (ES+) using LTQ XL and LTQ Orbitrap.

Results: Cross-species differences in CLint were observed: mouse > rat > human. There were 3 major metabolites detected that were all oxidative. The most interesting metabolite was metabolite M that has a molecular ion ([MH+]) at m/z 304 and the accurate mass measurement indicated that the elemental composition was C

15 H

22 O

2 N

5.

Confirmation of M: Compared to the active drug, [MH+] was 9 amu lower, which combined with the elemental composi-tion, suggests loss of CN and addition of OH. The product ion spectrum of m/z 304 produced an informative fragment ion at m/z 274 which is consistent with the loss of H

2CO. The other fragment ions are consistent with observation of no

changes to the rest of the molecule.

Reaction phenotyping: The formation of M is mediated by CYP3A4.

Incubation with 18O-water: We observed 96.3% incorporation of H2

18O from water in M.

Incubation with D2O: One D displaced the H on a-C in M (R-CHDOH).

Trapping with methoxylamine: The aldehyde formed was trapped by methoxylamine. Proposed mechanism: carbon oxidation takes place by P450 that results in loss of nitrile group and formation of an aldehyde. The aldehyde is mostly in the hydrated form prior to reduction most likely by P450. In the process of reduction the source of addition of H to the a-carbon is from water.

P352. Metabolic Activation of Strychnine to Reactive Intermediates by Human Liver Microsomes and CYP3A4

Yuxi Mao1, Zhong-Ze Fang2, Guang-Bo Ge1 and Ling Yang1

1Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

H OH

HO

H H

HCN

H H

HH

N N

N

O

R R

RR

R

DOH

−H2O

OH*OH

H2*O

D2O+H2NOCH3

R

230

Strychnine is one of the main active components of Semen Strychni which has been used as a traditional Chinese medi-cine for the treatment of nervous diseases, vomiting and arthritic and traumatic pains. It is highly toxic to humans and most domestic animals and acts as the poison curare originating from South America. Chemically reactive metabolites may induce toxicities and adverse side reactions, which might lead to withdrawing of the drug from market. There is exiting metabolic pathways in which Strychnine is oxidized to hydroxyl metabolites which might undergo a two-electron oxidation leading to formation of an electrophilic quinine imine intermediate. It is of significant interest to detect and characterization of reactive intermediates generated in the disposition of strychnine. Bioactivation potential of strychnine in human liver microsomes and recombinant CYP isoforms were performed. LC-MS/MS analysis of extracts of human liver microsomal incubations containing strychnine (200 μM), NADPH and GSH revealed a GSH adduct of the metabolite of strychnine was detected. Among tested recombinant CYP isoforms, CYP3A4 had the highest activity to catalyze the bioactivation of strychnine. Selective chemical inhibition investigation demostrated that ketoconazole dramatically inhibited the formation of GSH adduct. In the further stage of this investigation, GSH adduct is to be chemically synthesized to examine whether strychnine or GSH adduct plays a role in the toxicity of strychnine.

P353. MICROBIAL PRODUCTION of PHASE I and PHASE II METABOLITES of Propranolol

Cyrille Marvalin1, Christine Soustelle1, Robert Azerad2 and Xavier Morge1

1Molecular Biodiversity, Bertin-Pharma, Montigny-le-bretonneux, France, 2Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris Descartes, Paris, France

(R,S)-Propranolol is a classical b-adrenoreceptor antagonist which is often prescribed in its racemic form as an anti-hyperten-sive agent. S(-) propranolol is the most active enantiomer (about 100 times more potent than its optical antipode). Both R(+)- and S(-)-enantiomers are metabolized in the human liver by CYP2D6 (4- and 5-ring hydroxylation and N-deisopropylation) and CYP1A2 (N-deisopropylation). Conjugation of propranolol and 4-hydroxypropranolol by several uridine 5-diphosphoglu-curonosyl transferases (UGTs) has been shown to produce the corresponding O-b-D-glucuronides in rat or human liver microsomes. Currently it does not exist a quantitative method to produce substantial amount of propranolol phase I and phase II derivatives that could be used as standards for developing bioanalytical methods (LC-MS). A large number of natural micro-organisms (fungi and bacteria) from the Bertin-Pharma collection was screened in order to select the most adapted strains to produce substantial amounts of the metabolites of propranolol, including hydroxylated and glucuronidated species. In the same time, we have evaluated a possible enantioselectivity in the formation of these metabolites, as occasionally observed in animal metabolism of propranolol. After an exhaustive screening, the fungal strains selected to convert (R,S)-propranolol to its phase I and phase II metabolites, regio- and stereoselectively were incubated with substrates (0.1-1 g/L) in liquid cultures at +28°C and 200 rpm orbital shaking during 48-168 hours. The bioconversion was followed by analysis (HPLC-UV/MS) of the culture samples taken at different times. Then, the metabolites were extracted (with organic solvents or adsorbed on XAD-16) and purified using RP preparative HPLC methods. All metabolites were characterised by MS, 1H- and 13C-NMR. Purity was controlled by HPLC-UV/MS. When necessary, chiral chromatography was used to determine the enantioselectivity of the reac-tions. The follo-up of the cultures shows that the strain M50002 transforms (R,S)-propranolol into 4-hydroxy-(R)-propranolol (20% total yield in 72 h, >98% purity). The strain M50036 transforms (R,S)-propranolol into 5-hydroxy-(R,S)-propranolol (70% total yield in 72 h, >98% purity). Hydroxy derivatives are obtained in multimilligram amounts by scaling up the incuba-tions. When (R,S)-propranolol is incubated with strain M52104, the 10-O-b-D-glucuronide diastereomers are slowly formed in a 1:3 (S/R) ratio. Separation on semipreparative reverse phase HPLC allows to obtain in multimilligram amounts pure diasteromeric glucuronides (>98% purity). Similarly, 4- or 5-(R,S)-hydroxypropranolols are quantitatively glucuronidated by strain M52104 (100% yield in 48 h) to give the corresponding 4- or 5-O-b-D-glucuronides (>98% purity) as a 1:1 mixture of unseparated diastereomers in each case. Propranolol is commonly used as a typical substrate for hydroxylation by CYP2D6 and for O-glucuronidation by various UGTs. Owing to the poor commercial availability of most of these derivatives, particularly pure glucuronide conjugates, this method, compared to chemical synthesis, presents an outstanding interest to obtain, in a one step microbial reaction, such compounds in good yields, affording necessary analytical standards.

P354. Oxidative ipso-substitution of 2,4-difluoro-benzyl-phthalazines - Identification of a rare stable quinone methide and subsequent glutathione conjugate

Mithat Gunduz, Upendra Argikar, Franco Lombardo and Shawn Harriman

Novartis Institutes for Biomedical research, Metabolism and Pharmacokincetics, Cambdrige, MA, USA

231

Lead optimization efforts within the benzylphthalazine series led to identification of compound 1. This compound 1 was designed to include two fluorine atoms on benzyl-phthalazine moiety in an effort to block the potential formation of a quinone methide, a reactive intermediate. In vitro metabolite identification and glutathione trap-ping studies were conducted in order to understand the bioactivation potential of compound 1. The experiments were carried out using human liver microsomes fortified with NADPH and glutathione, as per protocols reported in the literature. Structure elucidation was carried out on a Thermo-Finnigan LTQ-Orbitrap® with accurate mass measurement and MSn capabilities. The results revealed the formation of a rare, stable quinone methide metabolite (M1) via ipso substitution of the fluorine and subsequent formation of a glutathione adduct (M2). Furthermore, these results provided a direct evidence for the formation of an ipso substituted stable quinone methide – M1, as a precursor to the glutathione conjugate M2. Collectively, these results also indicated that a fluoro-subtituion to benzylphthalazine moiety was insuffecient to block the oxidation leading to formation of a quinone methide metabolite. CYP phenotyping reactions were carried out with CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP1A2 and CYP2E1 in order to understand the enzyme responsible for the formation of M1. The formation of both, M1 and M2 was observed to be NADPH dependent and primarily catalyzed by CYP3A4. Interestingly, other CYP isoforms - CYP2C9, CYP2C19, CYP2D6, CYP1A2 and CYP2E1 did not form M1 and the subsequent glutathione adduct M2.

P355. Polymorphisms of Flavin-Containing Monooxygenase 3 (FMO3) Gene in Healthy Turkish Population

Pinar Topal and Gül Özhan

Department of Pharmaceutical Toxicology, Istanbul University, Istanbul, Turkey

Flavin-containing monooxygenases (FMOs), one of the most important monooxygenase systems, are involved in xenobiotics metabolism and variability in drug response. FMO gene catalyzes the monooxygenation of numerous N-, P- and S-containing xenobiotics. Substrates of FMO include many drugs (morphine, cocaine, nicotine, chlorpromazine, imipramine, tamoxifen, cimetidine etc), chemicals (phorate, aldicarb etc) and endogenous substrates (cysteamine, methionine, trimethylamine etc). FMO3 is associated with the major-ity of FMO-mediated adult hepatic metabolism. In the adult human liver, FMO3 is the prominent form and other FMOs are not expressed or are expressed at very low levels. 13 of totally 40 genomic FMO3 variants are Single Nucleotide Polymorphisms (SNPs) which cause low function. Allele frequencies of three func-tional SNPs (E158K, V257M, and E308G) vary greatly among different ethnic populations with distinct genetic backgrounds. The present study aimed to determine frequencies of FMO3 polymorphisms and haplotypes in Turkish population. We genotyped in 120 healthy volunteers using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. For statistical analysis, it was used the computer software SPSS version 13.0. Haplotype frequencies were estimated from genotype data by PHASE version 2.0 software based on Bayesian algorithm. The results were compared with those obtained from different populations. The study on alleles of FMO3 gene could be useful in understanding the variant and haplotype distributions of FMO3 alleles in Turkish population because FMO3 alleles, which can be observed on various races and even on sub-populations, have not been researched in Turkey even though it has been researched on different populations.

232

P356. PRODUCTION of PHASE I and PHASE II METABOLITES of Diclofenac and Chlorzoxazone by Bioconversion USING NATURAL and Recombinant MICRO-Organisms

Christine Soustelle1, Cyrille Marvalin1, Robert Azerad2 and Xavier Morge1

1Molecular Biodiversity, Bertin-Pharma, Montigny-le-bretonneux, France, 2Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris Descartes, Paris, France

In preclinical studies, the availability of large amounts of phase I and phase II drug metabolites for the drug metabo-lism studies as analytical standards (drug-drug interactions, efficiency, and toxic effects,…) is often a limiting factor. We describe here a bioconversion method, as an alternative to the microsomes and chemical ways for the produc-tion of phase I and phase II drug metabolites. This methodology uses the bioconversion of drugs by screening our platform comprising more than 200 natural and recombinant micro-organisms. We present the results of the produc-tion of hydroxylated metabolites of Diclofenac and Chlorzoxazone by recombinant yeast strains, over-expressing human CYPs (CYP2C9 and 2E1, respectively) and the production of phase II metabolites (glucuronidation) from these phase I metabolites by bioconversion with a natural micro-organism strain of our platform. For the production of phase I metabolites, Diclofenac and Chlorzoxazone (0.2 g/L) were incubated with recombinant yeast strains in liquid cultures and bioconverted in human hydroxylated metabolites during 5 days at +28°C, with an orbital shak-ing of 170 rpm. For the production of the phase II metabolites, 4′-OH-Diclofenac and 6 OH-Chlorzoxazone were incubated (0.2 g/L) with a natural strain (M52104) at +27°C during 3 to 7 days, with an orbital shaking of 200 rpm. In both cases, the bioconversion is followed by analysis of culture samples taken at different times using RP HPLC and mass spectrometry. The metabolites were extracted from the culture media by liquid-liquid or XAD-16 adsorp-tion extraction and purified by RP preparative HPLC. Their structure is characterised by NMR analysis. During the incubations, the bioconversion levels reach 10 to 50% for the phase I metabolites and 60 to 90% for the phase II metabolites (ratio of UV peak areas). The NMR analyses show that the phase I metabolites correspond to the human 4′OH Diclofenac and 6 OH Chlorzoxazone as expected after metabolisation by the CYP2C9 and CYP2E1 human CYP450 isoforms and the glucuronidated metabolites correspond to the 4′hydroxydiclofenac-O-b-D-glucuronide and to the 6 hydroxychlorzoxazone-O-b-D-glucuronide (human metabolite). Using a biological way of metabolism, we are able to produced phase I and subsequently phase II drug metabolites in amounts compatible with a use in preclinical studies. This method is validated as a quantitative alternative for the production of drug metabolites since several dizaines of mg to 1g could be extracted and purified from the incubations with the micro-organisms of our bioconversion platform.

P357. Proportionality Assessment of Metabolites from Microdose to High Dose in Rats Using LC-MS

Jinsong Ni1, Hui Ouyang1, Carmai Seto2, Takeo Sakuma2, Robert Ellis2, Josh Rowe1, Andrew Ache-ampong1, Devin Welty1 and Gabriella Szekely-Klepser1

1Drug Safety Evaluation, Allergan, Irvine, CA, USA, 2Product Application Lab, AB Sciex, Concord, ON, Canada

Background: To evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to characterize metabolites in plasma and urine at microdose level in rats and to investigate proportionality of metabolite exposure from the microdose to high dose in rats

Methods: Four drugs (atorvastatin, ofloxacin, omeprazole and tamoxifen) were individually administered orally to male Sprague-Dawley rats at doses from 1.67 mg/kg (microdose) to 5000 mg/kg. Plasma samples collected at different time points were extracted using protein precipitation and analyzed by LC-MS/MS using multiple reaction monitoring and information dependent data acquisition (MRM-IDA) functionality.

Results: Several competing metabolic pathways were found for ofloxacin, omeprazole and tamoxifen at the 5000 mg/kg dose. However, only glucuronide metabolite of ofloxacin, hydroxylation metabolite of omeprazole and hydration metabolite of tamoxifen were detected and characterized in plasma as well as in urine at the microdose level. It appears that the exposure of detected metabolites of omeprazole and tamoxifen increased in non-proportional manner with increasing doses even though exposure of parent drugs of omeprazole and tamoxifen increased proportionally with the doses. For atorvastatin, although exposure of ortho- and para- hydroxy atorvastatin increased with increasing doses

233

from 1.67 to 5000 mg/kg with near proportionality, exposure for atorvastatin or atorvastatin lactone does not increase proportionally with increasing doses.

Conclusions. LC-MS/MS has demonstrated its usefulness for detecting and characterizing the major metabolites in plasma and urine at microdosing levels in rats. Current study suggest that the exposure of metabolites at microdose level following oral dosing might not simply be used to predict its exposure at higher dose level in rats.

P358. Quantification of Glutathione Adducts in Human Liver Microsomes using Ultra Performance Liquid Chromatography and Inductively Coupled Plasma Mass Spectrometry

Cathy MacDonald1, Christopher Smith2, Richard Weaver1 and Ian Wilson2

1Discovery DMPK, AstraZeneca R&D Charnwood, Loughborough, United Kingdom, 2CPD, AstraZeneca, Cheshire, United Kingdom

Introduction Reactive electrophilic metabolites have been implicated in idiosyncratic drug reactions that have lead to the withdrawal of drugs from the market. Electrophilic intermediates can be detected by trapping with nucleophiles such as glutathione (GSH) and identified by mass spectrometry. A limitation of this approach is that the data are qualitative rather than quantitative unless the parent molecule or nucleophile are radio-labelled. Here we desribe a method for quantification of GSH adducts using Ultra Performance Liquid Chromatography and Inductively Coupled Plasma Mass Spectrometry (UPLC-ICP-MS) which quantifies the elemental sulphur associated with the GSH adduct without the need for labeling.

Methods: GSH adducts were generated in human liver microsomes for clozapine and 5 AstraZeneca compounds (AZ1-5) by incubating 10 μM drug, 1 mg/mL human liver microsomes, 1 mM NADPH and 2 mM GSH in 5 mL of 0.1M phosphate buffer at 37°C for 60 min. The reactions were terminated with 5 mL 0.1% formic acid and prepared for analysis by SPE (final volume 150 μL). Chromatography was performed on a Waters Acquity UPLC™ system using 0.1% formic acid in methanol and 0.1% formic acid (aq) as mobile phases A and B respectively. The LC gradient was: 0-1 min 5% A, 1-7 min 5-80% A, 7-7.5 min 80% A, 7.5-7.6 min 80-5% A, 7.6-13 min with an Acquity UPLC 1.7 μm C

18 100 mm x 2.1 mm ID column

maintained at 60°C with a flow rate of 0.25 mL/min. The UPLC system was coupled either to an ICP-MS (Elan DRC) for detection of sulphur or TOF-MS (Micromass® q-TOF Micro) for confirmation of the m\z of the sulphur response.

Results: Sulphur responses were detected in clozapine samples and for 4/5 AstraZeneca compounds and these were quantified against a sulphur calibration curve. The m\z of the sulphur responses were detected using UPLC-TOF-MS and in all cases the m\z was consistent with the formation of GSH adducts from parent compound. Total GSH adduct formed for each compound ranged from 0.4 – 0.9 μM and this accounted for between 4 and 26% of total metabolites. Clozapine generated the highest proportion of GSH related metabolite and the amount of clozapine GSH adduct measured in this study was consistent with that reported previously (1).

Conclusions: We have demonstrated the utility of UPLC-ICP-MS to detect and quantify GSH adducts generated in human liver microsomes circumventing the need to use labeled material to quantify responses.

Reference

(1) Gan J, Harper TW, Hsueh MM, Qu Q, Humphreys WG (2005) Dansyl Glutathione as a Trapping Agent for the Quantitative Estimation and Identification of Reactive Metabolites. Chemical Research in Toxicology 18: 896 – 903.

P359. Reactive metabolites of thiophenic compounds: A new trapping method for thiophene sulfoxides

Patrick M. Dansette, Stéphanie Thébault, Lucie Durand-Gasselin, Gildas Bertho and Daniel Mansuy

Cnrs Umr 8601, Université Paris Descartes, Paris, France

We have shown in the past that the thiophene ring can be metabolized trough two primary cytochrome P450 catalyzed oxidation pathways: C-hydroxylation leading to a 2-hydroxy-thiophene (tautomeric a thiolactone) and S-oxidation

234

leading to thiophene-S-oxides which are generally unstable and dimerize through Diels Alder cycloaddition. In presence of glutathione (GSH), S-oxide adducts (S-glutathionyl-dihydrothiophene S-oxides) are often obtained (1-3). Chemical results from the Thiemann′s group (4) show that thiophene S-oxides react well with dienophiles.

This poster describes that maleimides are dienophiles compatible with metabolic bioactivation and are able to trap metabolically formed thiophene S-oxides as stable Diels Alder cycloaddition products. This is demonstrated in the case of several thiophene containing drugs known to form S-oxides: Ticlopidine (5), Clopidogrel (6), Tienilic acid isomer (1) and Methapyrilene (6).

Thus N-ethyl- N-methyl or N-phenylmaleimide at 1 mM was found to give thiophene S-oxides Diels Alder adducts for the four above drugs as shown by HPLC-MS. In addition the structure of the ticlopidine S-oxide (5) adduct of N-methylmaleimide was fully characterized after chemical synthesis by a procedure adapted from that of Thiemann, by mass spectrometry (MS) and 1H and 13C NMR spectroscopy. The present finding show that thiophene S-oxide can be trapped by two complement-ary methods, either by Michael addition with a thiol which may be sometomes complex (1) or by metabolically compatible dienophiles as maleimides.

References

1) Valadon, Pet al; (1996). Chem Res Toxicol, 9(8), 1403-1413.2)Dansette, P. M.et al. (1992). Biochem Biophys Res Commun, 186, 1624-1630.3) Treiber, A., et al. (1996) J AmChem Soc, 119(7), 1565-1571.4) Li Y et al.(1997) J Org Chem. 62(23):7926-7936.5) Ha-Duong et al. Biochemistry 2000 40(40): 12112-12122.6) Dansette et al. Chem ResToxicol. 2009 Feb;22(2):369-73.7) Graham et al. J Pharmacol Exp Ther. 2008: 26(2):657-71.

P360. Separation and Identification of Norcantharidin Metabolites in vivo

Wei Chunmin

Institute of Clinical Pharmacology,, Qilu Hospital of Shandong University, Ji′nan, China

Objective: To separate and identify the metabolites of norcantharidin after oral administration in vivo.

Methods: To investigate the biotransformation and the polarity characteristics of 3H-norcantharidin transfor-mation products in mice by radio tracing combined with thin layer chromatogram technology, and separate norcantharidin and its metabolites from the impurities in rat serum and bile samples by derivatization GC-MS method, and then conjecture the chemical structure characteristics of norcantharidin metabolites by mass inter-pretation for new peak or area-increasing peak followed by comparing the chromatograms of blank with incurred bio-samples.

Results: There were seven possible metabolites at retention time of 14.782, 17.057, 17.406, 17.569, 19.004, 19.988 and 24.210 min were identified.

S SN

N

S

O

O

O

O

O

O

R2 R2

R4

R4

R2

R1 R1

R1

R3 BF3 éthérate,CH2Cl2, mCPBA

R3 R3

O

S SS

ClCl

OO O

O

O

O N R

CO-Ar CO-Ar

NEMOH

P450, NADPH, O2

235

Conclusion: It can offer scientific proof for proper evaluation of dose-effect relationship, individually clinical adminis-tration, and new derivatization or new formulation development of norcantharidin to initially confirm the metabolites and possible metabolism pathway.

P361. Significant Species Difference of Amide Hydrolysis of GDC-0834, a Novel Potent and Selective Bruton′s Tyrosine Kinase Inhibitor

Lichuan Liu1, Jason Halladay1, Young Shin1, Susan Wong1, Melis Coraggio1, Hank La1, Matthew Baumgardner1, Hoa Le1, Peter Kuebler1, John C. Davis Jr.1, X. Charlene Liao1, Joseph Lubach1, Alan Deese1, C. Gregory Sowell1, Kevin S. Currie2, Wendy B. Young1, Cyrus Khojasteh1, Cornelis Hop1 and Harvey Wong1

1Genentech, South San Francisco, CA, USA, 2CGI Pharmaceuticals, Branford, CT, USA

Bruton′s tyrosine kinase (BTK) plays a critical role in the development, differentiation and proliferation of B-lineage cells, making it an attractive target for the treatment of rheumatoid arthritis. GDC-0834 is a potent and selective inhibitor of BTK. Early metabolite identification studies of GDC-0834 suggested significant species differences in the extent and type of metabolism. The objective of this study was to characterize the species differences in the metabo-lism of GDC-0834. Metabolism identification studies using pooled cryopreserved hepatocytes from mouse, rat, dog, monkey and human revealed predominant formation of an inactive aniline metabolite (M1) via amide hydrolysis in vitro in human. The formation of M1 appeared to be NADPH independent as GDC-0834 was equally unstable in human liver microsomes (LM) with and without NADPH. Consistent with the in vitro hepatocyte data, M1 was found in only minor (rat and monkey) to moderate (dog) quantities in plasma from preclinical species dosed with GDC-0834. Predictions of human clearance using various methodologies resulted in clearances ranging from low to high in human. Based on the high interest in a BTK inhibitor for clinical evaluation but the considerable risk of poor human pharmacokinetics, a single dose IND was filed and GDC-0834 was advanced to a single dose human clinical trial. In plasma from the first 2 cohorts (35 mg and 105 mg), substantial concentrations of M1 were observed, whereas GDC-0834 level was below the limit of quantitation (< 1ng/mL) in most plasma samples. Metabolite identification studies with human plasma and urine detected only the aniline metabolite (M1) and its derivatives suggesting that amide hydrolysis was the major metabolic pathway in vivo in human. To further investigate species differences, an enzyme kinetics study, in which M1 formation was monitored, was performed in rat, dog, monkey and human LM without NADPH. The maximum rate of M1 formation (V

max) was substantially higher in human compared with

other species. In contrast, the Michaelis-Menten constant (Km

) was comparable among species. Intrinsic clearance (V

max/K

m) of aniline formation in human was 23 - 169 fold higher than that in rat, dog and monkey. The preliminary

enzyme identification showed cholinesterases may play a role in the amide hydrolysis of GDC-0834. To the best of our knowledge, this study is the first study showing direct in vitro-in vivo correlation of amide hydrolysis in different species.

P362. The Intermolecular Disulfide Bonds of UGT1A Splice Products with Active UGT1A Enzymes May Determine their Ability to Inhibit Glucuronidation Activity

Mélanie Rouleau, Mario Harvey, Judith Bellemare and Chantal Guillemette

Pharmacogenomics Laboratory, CHUQ Research Center and Faculty of Pharmacy, Laval University, Quebec, QC, Canada

The UDP-glucuronosyltransferases (UGTs) are pivotal membrane proteins of the endoplasmic reticulum (ER) play-ing a role in xenobiotic metabolism and maintaining homeostasis of a variety of small endogenous molecules. The current knowledge of protein-protein interactions among UGTs is limited. However, experimental evidence supports their existence as homodimers or higher form of oligomers covalently linked by disulfide bonds. Recently, we uncovered novel spliced products derived from the human UGT1A gene, so-called UGT1A_i2 isoforms. The 45 kDa i2 proteins observed in drug metabolizing tissues lack the transmembrane domain and glucuronic acid

236

transferase activity but are localized in the ER along with active 55 kDa UGT1A_i1 enzymes. Our previous works using endogenous knockdown and recombinant proteins support an inhibitory effect of i2 species on UGT1A activity likely through formation of inactive heteromeric i1+i2 assemblies. In this study, we examined whether the forma-tion of intermolecular disulfide bonds is implicated in the oligomerization process between UGT1A splice species. We used microsomal recombinant UGT1A1 i1 and i2 proteins as prototypes as well as microsomal protein extracts from human cells expressing both types of endogenous splice products. SDS-page analyses using an anti-UGT1A antibody revealed protein complexes of high molecular weight under non-reducing conditions, which are not detected upon treatment with a reducing agent. These observations are consistent with a covalent association between UGT1A products through intersubunit disulfide bonds. Preliminary analyses of protein-protein interac-tion by co-immunoprecipitation assays using selected recombinant truncated UGT1A1_i1 proteins support the involvement of domains that would be distinct for interaction occurring between i1+i1 and i1+i2. For instance, the truncation of the peptide signal and the transmembrane domain of UGT1A1_i1 lead to loss of interaction with itself (i1mutant + i1 wild type) but did not affect oligomerization of truncated i1 with its splice form i2. Further point mutations of specific cysteines (C223Y and C127Y) previously reported to be critical for dimerization and UGT activity did not prevent homo- or hetero- oligomerization between UGT1A1 splice species. From these initial experiments, we conclude that intermolecular disulfide bond formation is involved in formation of inactive oli-gomers between active UGT1A1 isoform 1 and its i2 splice product. Our results also suggest that the formation of homo- or hetero-oligomers would depend on different protein domains. Further studies concerning the effect of specific protein domains and cysteine mutagenesis upon function and disulfide bond formation between human UGT1A splice products is ongoing.

P363. Time Dependent Changes of Enzymatic Phase I and Phase II Activites In Plated Human Hepatocytes

Roberto Tolando1, Scott Heyward2, Micheal Chesebrough3, Ji Young Lee1 and Timothy Moeller1

1Sales and Marketing, Celsis In Vitro Technologies, Halethorpe, MD, USA, 2Research and Development, Celsis In Vitro Technologies, Halethorpe, MD, USA, 3BioAnalytical, Celsis In Vitro Technologies, Halethorpe, MD, USA

It is well known that cytochrome P450 activities decrease over time in cultured hepatocytes. Studies have measured these activities using cultured rat hepatocytes and human liver slices. Other reports have included P450 mRNA losses in human cultured hepatocytes but lacked associated P450 enzymatic activities. Here, we report measuring the P450 enzyme activi-ties along with UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) activities in cultured human hepatocytes. Plateable cryopreserved human hepatocytes were used to determine the enzymatic activity of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4, as well as UGT and SULT, over a time course of 36 hours. There was an increase in most P450 activities in most donors within the first four hours after attachment. The P450 activities trended downwards at the 12-, 18-, 24- and 36-hour time points. The raw activities did vary from donor to donor; however, the percent loss was similar in most P450s. UGT activity increased over the first four hours, then remained stable out to 36 hours. SULT activity varied from donor to donor with an increase in the first four hours and then differences in maintaining the activity to 36 hours. One donor lost SULT activity from the four hour value but maintained approximately the initial value measured. The other donors retained their initial increases in SULT activity. In all, we confirmed the reported loss of P450 activities in human hepatocytes and the stability of UGT activity. Retention of SULT activity in cultured cryopreserved human hepatocytes was in contrast to the reported loss in human liver slices and rat hepatocyte cultures.

P364. Ultra-performance liquid chromatographic–electrospray mass spectrometric determination (UPLC-ESI-MS) of hydroxylation metabolite of liquiritigenin in vitro: Assay development, human liver microsome activities and species differences

Ying Hu, Hui-Xin Liu and Ling Yang

Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China

237

Drug metabolic profile plays an important role in discovering and developing the novel drug from the metabo-lites possessed the pharmacological activities. An understanding of the enzymology of the metabolic clearance of a drug, whether by Phase I or Phase II mechanisms, is pivotal to new drug development. Early knowledge of the potential biotransformations of drug in the target species is of great interest. Traditionally, rodents such as the rat and nonprimate species such as the dog have been used as animalmodels in studies aimed at evaluating the pharmacodynamics, metabolism, pharmacokinetics, and safety of new chemical entities. The identification of metabolite and metabolic enzymes involved in drug metabolism are important for us to understand which one, parent drug or metabolite(s), is really active and how variations in drug concentrations can lead to differences in drug efficacy and toxicity. In addition, knowledge of species differences in drug metabolism will help us to select appropriate animal models to study the drug in question. A simple and sensi-tive method for determination of the hydroxylation activity of rat, dog, minipig, and humanliver micrsomes toward liquiritigenin using ultra-performance liquid chromatography with mass detection (UPLC-MS) has been developed. The method uses chemically synthesized mono-hydroxylated metabolite of liquiritigenin (4′,5,7-trihydroxyflavanone, THF) as a standard for method validation. Validation was done with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separa-tion was achieved on a UPLC BEH C18 column (50mm x 2.1mm i.d., 1.7 μm), with phase of acetonitrile–water (ratio 30:70). Selective ion reaction (SIR) monitor was specific for liquiritigenin, THF and I.S. Themethodwas specific since therewere no interference peaks fromthe reaction matrix. The calibration curve for THF was linear from 0.5–100 μm with r2 = 0.9999. The newly developed method has good precision and accuracy. The method was successfully used to determine the kinetics of THF activities toward liquiritigenin in liver microsomes from different species. Dog liver microsomes (DLMs) were the most active in liquiritigenin hydroxylation (709.7 pmol/min/mg protein) followed by rat liver microsomes (RLMs) (579.6 pmol/min/mg protein), and then minipig liver microsomes (PLMs) (417.3 pmol/min/mg protein). The developed method was appropriated for rapid screening liquiritigenin hydroxylation activity in liver microsomes from different species.

P365. Chromium Levels in Pulmonary Tissues from Lung Cancer and Control Subjects

Sevtap Gülgösteren1, Görkem Mergen2, Mahmut Gülgösteren3, H.Esra Özaydın4, Vugar Aliyev2, Tülin Söylemezoğlu2 and Cebrail Şimşek1

1Department of Chest Diseases, Atatürk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey, 2Institute of Forensic Medicine, Ankara University, Ankara, Turkey, 3Department of Chest Surgery, Atatürk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey, 4Department of Pathology, Atatürk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey

Chromium (Cr) is considered to be an essential metal for maintenance of normal physiological functions. On the other hand, excess of Cr is harmful to health; it is a known human carcinogen, particularly in lung cancer. It is revealed that occupational exposure to Cr causes higher incidence of lung cancer and Cr contaminant is involved in human lung tumorigenesis. In this study, chromium levels of pulmonary tissues with and without tumor were investigated and com-pared to display the effect of Cr in formation of lung cancer. For this purpose, pulmonary tissue samples were collected during the surgeries of 35 patients with diagnosis of lung cancers in Atatürk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey. Bronchi and lung parenchym tissues from 64 patients with no-carcinoma were also included as control. Excised fragments of pulmonary tissues were classified as tumors and tissues without tumors. Samples were dried at 75°C for 24 hours for weight stability. Following the analytical weighting, dried samples were dissolved in 10 mL of nitric acid, transferred to PTFE tubes and microwave digestion was applied. Digested tissue solu-tions were diluted with ultra pure water (1:1) before introducing to flame atomic absorption spectrometry for analysis of Cr. When the levels of Cr for tissues with carcinoma and without carcinoma were compared, the results were found as 3.54 ± 0.7 5µg/g

238

P366. Dose-dependant effect of chronic contamination to depleted uranium on xenobiotic metabolizing enzymes in rat

Yann Gueguen1, Caroline Rouas2, Audrey Monin3, Stéphane Grison3, Patrick Gourmelon3 and Isabelle Dublineau3

1Drph / Srbe, IRSN, Fontenay-aux-Roses, France 2Drph / Srbe, IRSN, France, 3Drph / Srbe, IRSN, France

The extensive use of depleted uranium (DU) in both civilian (nuclear industry, mill tailings, phosphate fertilizers) and military applications results in the increase of the number of human beings exposed to this compound. Xenobiotic-metabolizing enzymes (XME) has been previously identified has a possible target of DU-exposure [1-4]. To pursue this work, a dose-effect study from environmental concentration found around uranium mine (0.2 mg/L) to supra-environ-mental concentration (120 mg/L) were undertaken. XME were studied in two metabolizing organs: the liver (first organ exposed to xenobiotics after absorption), and the kidney (primary target of uranium) of rats exposed to DU through drinking water. Plasma biochemical parameters were measured in all groups and didn′t reveal major toxicologicity of the liver (transaminases, albumin, PA, bilirubin) or the kidneys (creatinin, urea). According to previous studies done on rats exposed to 40 mg/L of DU [1,3,4], expression and activity of CYP3A, the major phase I enzyme of the xenobiotic metabolism, is decreased in the liver of rat exposed to 120mg/L of DU. On the contrary, cytochromes P450 enzymes studied (CYP1A1, 2B1, 2C11, 2E1, 3A) are poorly altered in the kidney whatever the concentration of DU. The second important result concerned the phase II enzymes and especially ST1A1 (sulfotransferase 1A1) in the kidney. Among the four enzymes of the phase II considered (GSTA2, UGT2B1, UGT1A1, ST1A1), only ST1A1 is modified; the gene expression of this enzyme dose-dependently increased (2 to 4-fold) with the concentration of DU in the drinking water (from 10 to 120 mg/L). Finally, transporters MRP2 and MDR1 were not altered in the liver and kidney of rats contaminated to DU. Overall, this study confirms that some XME, CYP3A in the liver and ST1A1 in the kidney, could be used as biomarkers of chronic contamination to DU; the effects observed being enhanced when the exposure to DU increase.

P367. Oral administration of lead induced hepatic and renal damage in wistar rats

Missoun Fatiha IV1 and Slimani Miloud2

1Biology Department, UNIVERSITY OF MOSTAGANEM, Mostaganem, Algeria, 2Biology Department, UNIVERSITY OF ORAN, Oran, Algeria

Lead is hazardous substance to human and animals. The present study was carried out to investigate the toxic effect of lead on liver and kidney in rats. Fourty male Wistar rats were divided into 2 groups; group 1 was given a tap water diet and group 2 was given 2000 ppm lead acetate in drinking water for 4 weeks. Lead concentration in blood and organs was determined by atomic absorption. Hepatotoxicity was monitored in the rats as a function of changes in serum levels of aspartate transaminase (AST), alanine transaminase(ALT), alkaline phosphatase (ALP), total serum albumin, bilirubin level and glycemia. The renal function of this metal was monitored by changes in urea and creatinine in urine and serum. Group two when compared with the control, there was significant (p<0.05) increase in serum (ALT),(AST), (ALP),creatinine, urea and bilirubin level, but significant decrease in urine levels of urea and creatinine. histolpathologi-cal observations showed severe damage in the liver and kidneys of treated rats.These observations may be suggestive of deranged membrane structures and functions of liver and kidneys in rats intoxicated by lead.

P368. Abstract Withdrawn.

P369. The effect of various metals on polyphenol oxidase and peroxidase activities from goosefeet plant (Chenopodium anthelminthicum)

Gulnur Arabaci and Ayse Usluoglu

Chemistry, Sakarya University, Sakarya, Turkey

239

Peroxidases [EC 1.11.1.7] are heme proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group. These are a group of oxidoreductases that catalyse the reduction of peroxides, such as hydrogen per-oxide and the oxidation of a variety of organic and inorganic compounds. Peroxidases play important roles in plants, participate in the lignification process and in the mechanism of defence in physically damaged or infected tissues. [1] Polyphenoloxidase (PPO) is a cupric enzyme that catalyzes the hydroxylation of monophenols to o-diphenols, and the oxidation of o-diphenols to o-quinones. Quinones are very reactive compounds which strongly interact with other molecules, leading to a large variety of dark colored compounds [2]. It is responsible for the undesired browning reac-tions during handling, storage and processing of damaged tissues of fresh fruits and vegetables, as well as some animal products. Enzyme in plants can be affected by some metals. Iron, copper are important for survival, defense system and enzyme activities of plants however heavy metals can inhibit enzyme activities which is responsible for defense system of plants. In this study, the effects of some metals on partially purified POD and PPO activity from goosefoot plant (Chenopodium anthelminthicum) were investigated. Goosefoot plant were consumed as food and also used for medicinal purposes by local population. Goosefoot was harvested fresh from the region of Sakarya, in Turkey. Plant was homogenized in 0.1 M of phosphate buffer (pH 7.0) and all steps were carried out at 4°C. Then, Ca(II), Co(II), Cu(II), Fe(III), Na(I), Mg(II), Mn(II), Zn(II), Sn(II), at (1-10 mM) on the goosefoot PPO activity was assayed with 4-Methyl cathecol and POD activity was assayed with H2O2 and 4-Methyl Catechol as substrate at pH 7.0. The results showed that goosefoot PPO enzyme activity was increased by ten to fifty percent at 10 mM of Ca(II), Cu(II), Mg(II), however PPO enzyme activi-ties markedly inhibited by Mn(II), Zn(II), Sn(II) at 10 mM. The metals Fe(III), Co(II), Na(I) had no significant effect on the enzyme activities at 10 mM. Results for POD enzyme; Ca(II), Fe(III) increased the enzyme activity nearly ten percent at 10 mM, however POD enzyme activity markedly inhibited by Co(II), Cu(II), Sn(II), Mn(II) at 10 mM. The metals Zn(II), Na(I), Mg(II) had no significant effect on the enzyme activitiy at 10 mM. Thus, Sn(II) plays very important inhibitory role for PPO and however Sn(II) and Mn(II) both inhibitory effect on POD activity of goosefoot plant.

References

[1] Mohsina Hamid, Khalil-ur-Rehman. Potential applications of peroxidases, Food Chemistry 115, 1177–1186, 2009.[2] Goulart, P.F.P., Alves, J.D., Magalhães, M.M., Lima, L.C.O., Meyer, L.E. Purification of polyphenoloxidase from coffee fruits. Food Chemistry

83, 7–11, 2003.

P370. Adaptive Response to Reactive Aldehydes in Mouse Liver: Role of Aldehyde dehydrogenases

Russell A. Prough1, Ngome L. Makia1 and Daniel J. Conklin2

1Biochemistry & Molecular Biology, U. Louisville School of Medicine, Louisville, KY, USA, 2Medicine/Cardiology, U. Louisville School of Medicine, Louisville, KY, USA

Propene-2-al (acrolein) and 4-hydroxy-2-nonenal (4-HNE) are alpha,beta-unsaturated aldehydes generated during the peroxidation of lipids and are implicated in several pathophysiological conditions, including atherosclerosis and steatohepatitis. These aldehydes are potent electrophiles that undergo Schiff base and Michael addition reactions with nucleophilic groups on protein and DNA leading to impaired cellular homeostasis, DNA damage and cell death. In an attempt to identify cytoprotective genes in mice liver that participate in this protective response against cytotoxic aldehydes, we performed microarray with interest on aldehyde detoxification enzyme genes. Mice on normal chow diet were administered either saline (control) or acrolein (5 mg/kg) by gavage daily for 7 days. Significant induction (2-fold) of Aldh1a1 gene expression was observed in acrolein-fed mice 24 h after the last treatment, and confirmed by quantitative real-time PCR. The aldehyde dehydrogenases (ALDH) are a superfamily consisting of NAD(P)-dependent enzymes capable of detoxifying these reactive aldehydes. Hepatic Aldh1a1 mRNA level was also induced »2.5-fold when mice were fed diet containing 0.45% butylated hydroxyanisole (BHA) for 7 days. Acrolein and BHA are activator of redox-sensitive transcription factors such as nuclear factor-erythroid 2 related factor 2 (Nrf2) and the activator protein 1 (AP-1). Moreover, cytosolic fractions isolated from the livers of acrolein and BHA-treated mice showed 3.5- and 4.5-fold increases in Aldh1a1 activity, respectively, compared to control. Kinetic studies demonstrate that the reactive aldehydes, acrolein (K

m 727 µM), HNE (K

m 95 µM) and MDA (K

m 78 µM) are possibly physiological substrates of Aldh1a1. We also showed

by MTT assay that Aldh1a1 deficient cells are more sensitive to acrolein-induced cell death compared to control cells. Therefore, we hypothesize that induction of Aldh1a1 gene by acrolein in mice liver is an adaptive response to reactive aldehydes. To decipher the mechanism of electrophile-induced transcription of Aldh1a1, we analyzed the mRNA levels

240

of hepatic Aldh1a1 in C57BL6 (WT) and Nrf2−/- null mice exposed to BHA. BHA exposure resulted in »2-fold increase in mRNA levels of Aldh1a1 in both WT and Nrf2−/- mice compared to control, while expression of known Nrf2 target genes was not induced upon BHA administration in Nrf2−/- mice. Transient transfection experiments were conducted in HepG2 cells with Aldh1a1 5′-flanking luciferase reporter vectors. While co-transfection with Nrf2 expression plasmid had no effect, over-expression of c-Jun resulted in ≈4-fold induction in Aldh1a1 transcriptional activity. We propose that electrophile-induced transcription of hepatic Aldh1a1 is mediated by c-Jun/AP-1. Over-expression of a dominant nega-tive c-Jun (TAM67) in HepG2, and treatment with 50 mM JNK (SP600125) and MEK1 (PD98059) inhibitors significantly abrogate the c-Jun mediated transcription of Aldh1a1. We further showed that c-Jun induces Aldh1a1 promoter activity as a homodimer and not as c-Jun/c-Fos heterodimer. Deletion and mutagenesis studies established that two AP-1 sites at position -758 and -1069 relative to Aldh1a1 transcription start site mediate c-Jun-induced transcription of Aldh1a1. We propose that increased expression of Aldh1a1 in mice may serve as a protective mechanism against toxic electrophiles and oxidative stress in the liver and cardiovascular tissues. Supported by NIH Grants P01ES011860 and P30ES014443.

P371. Arylacetamide deacetylase is a determinant enzyme for the difference in hydrolase activities of phenacetin and acetaminophen

Tatsuki Fukami, Akinobu Watanabe, Shiori Takahashi, Yuki Kobayashi, Nao Nakagawa, Miki Nakajima and Tsuyoshi Yokoi

Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

Phenacetin was withdrawn from the market because it caused renal failure in some patients. Many reports indicated that the nephrotoxicity of phenacetin is associated with the hydrolyzed metabolite, p-phenetidine. Acetaminophen (APAP), the major metabolite of phenacetin, is also hydrolyzed to p-aminophenol, which is a nephrotoxicant. However, APAP is safely prescribed if used in normal therapeutic doses. This background prompted us to investigate the difference between phenacetin and APAP hydrolase activities in human liver. In this study, we found that phenacetin is efficiently hydrolyzed in human liver microsomes (HLM) (CLint: 1.08 ± 0.02 μl/min/mg), whereas APAP is hardly hydrolyzed (0.02 ± 0.00 μl/min/mg). To identify the esterase involved in their hydrolysis, the activities were measured using recom-binant human carboxylesterase (CES) 1A1, CES2, and arylacetamide deacetylase (AADAC). Among them, AADAC showed similar Km values (1.82 ± 0.02 mM) with HLM (3.30 ± 0.16 mM), and the highest activity (Vmax: 6.03 ± 0.14 nmol/min/mg). In contrast, APAP was poorly hydrolyzed by the three esterases. The large contribution of AADAC to the phenacetin hydrolysis was demonstrated by the prediction with a relative activity factor. In addition, the phenacetin hydrolase activity by AADAC was activated by flutamide (5-fold) as well as that in HLM (4-fold), and the activity in HLM was potently inhibited by eserine, a strong inhibitor of AADAC. In conclusion, we found that AADAC is the principal enzyme responsible for the phenacetin hydrolysis and the difference of hydrolase activity between phenacetin and APAP is largely due to the substrate specificity of AADAC.

P372. Baeyer-Villiger Oxidation Specifically Catalyzed By Human Flavin-Containing Monooxygenase 5 (FMO5)

W. George Lai1, Nadia F. Misiaszek1, George A. Moniz2 and Y. Nancy Wong1

1DMPK-Andover, Biopharmaceutical Assessments, Eisai Inc., Andover, MA, USA, 2Process Research-Andover, Eisai Inc., Andover, MA, USA

E7016 is an inhibitor of poly (ADP-ribose) polymerase developed for anticancer therapy. One of its major metabolites identified in preclinical animal studies is the product of an apparent oxidation and ring-opening of the 4-hydroxypipe-ridine. In vitro, this oxidative metabolite cannot be generated by incubating E7016 in liver microsomes. Further studies reveal the formation of this unique metabolite in hepatocytes. In a NAD(P)+-dependent manner, this metabolite was also generated in liver S9 fractions and in recombinant human FMO5, fortified with liver cytosol fractions. In liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), and by a brief heat treatment of the liver S9 at 50 °C. Based on the results, the overall metabolic pathway involves a two-step oxidation process, which includes dehydrogenation of the secondary alcohol in liver cytosol and a FMO5-mediated

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Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps are coupled via regeneration of NAD(P)+ and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In a NAD(P)H-dependent manner, the ketone intermediate was metabolized to the same ring-opening metabolite in liver microsomes. The metabolic pathway was inhibited by BNPP and by a brief heat treatment of the liver microsomes at 50 °C. Methimazole, the substrate/inhibitor of FMO1 and FMO3, showed no inhibition of this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the ketone intermediate in recombinant enzymes of FMO1, FMO3, and FMO5.

P373. Competitive Inhibition of CYP1A1 by Flavonoids, Quercetin and Rutin: Promising Agents for Cancer Prevention

Duygu Yilmaz1, Azra Bozcaarmutlu2 and Emel Arinç3

1Biochemistry Graduate Programme and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey, 2Department of Chemistry, Abant Izzet Baysal University, Bolu, Turkey, 3Graduate Programme in Biochemistry and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

Quercetin and rutin are the main flavonoids consumed in human diet and show anticancer activity in laboratory ani-mals. Most of the chemical carcinogens are activated by cytochrome P450 enzymes in order to exert their genotoxic and carcinogenic effects. Hence, one possible mechanism is that quercetin and rutin may prevent the carcinogenic effect of chemicals through inhibition of cytochrome P4501A1 (CYP1A1) activity. CYP1A1 is a Phase I enzyme which is known to be involved in the activation of procarcinogens. In order to clarify the mechanism of cancer preventive effects of quer-cetin and rutin, we investigated, for the first time, the in vitro modulatory effects of these flavonoids on liver microsomal CYP1A1 associated 7-ethoxyresorufin O-deethylase (EROD) activity of leaping mullet caught from the highly polluted sites of Izmir Bay. Microsomes were prepared from mullet liver by differential centrifugation and kept in liquid nitrogen until used. EROD activity was measured spectrofluorometrically, and CYP1A1 protein level was determined by Western blotting. The effects of flavonoids on CYP1A1 activity were studied by adding various concentrations of these compounds into the reaction mixture. IC50 values, inhibitor concentration to produce 50% inhibition in percent of control, were determined by linear regression against logarithmic inhibitor concentrations. After the calculation of IC50 values, three different flavonoid concentrations were chosen to study the inhibition mechanisms. The results indicated that fish caught from highly polluted sites of Izmir Bay contaminated with PAHs, PCBs and related organic compounds, showed a highly induced EROD activity and CYP1A1 protein level. Both quercetin and rutin were found to inhibit CYP1A1 associated EROD activity in a competitive manner with IC50 values of 1.32 µM and 0.64 mM, respectively. Inhibition constant (Ki) values of quercetin and rutin were calculated from Dixon plots as 0.12 µM and 100 µM, respectively. Ki values of quercetin and rutin indicated that quercetin was a more potent inhibitor than rutin. However, rutin, being a glycoside of quercetin (quercetin-3-O-rutinose), is also hydrolyzed enzymatically to quercetin in small intestine of humans. In conclusion, this study has indicated that quercetin and rutin are strong inhibitors of CYP1A1 associated EROD activity. These results support the hypothesis that mechanism of cancer prevention of quercetin and rutin are related to the reduction of the for-mation of carcinogens through inhibition of CYP1A1 activity which is known to be involved in carcinogen activation.

P374. A novel approach for the management of malathion-induced toxic stress in rat blood and brain mitochondria

Mohammad Ali Rezvanfar, Mohammad Amin Rezvanfar and Mohammad Abdollahi

Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Centre, Tehran University of Medical Science, Tehran, Iran

Background and objectives: Malathion toxicity has been related to the inhibition of acetylcholinesterase and induction of oxidative stress, while Rolipram has been shown to possess neuroprotective effects in experimental and clinical studies. Methods: This study investigates the effects of subacute exposure to organophosphate insecticide malathion by measuring the activities of the brain mitochondrial and plasma peroxynitrite (ONOO−), glutathione peroxidase (GPx),

242

superoxide dismutase (SOD), Mn-SOD, catalase (CAT), and lipid peroxidation (LPO) in rats. Malathion (200 mg/kg/day) and rolipram (200 µg/kg/day) were administered alone or in combination for 7 days by intraperitoneal injection. At the end of the experiment, the brain mitochondria and plasma of the animals were separated. Results and discussion: In the brain cells mitochondria and blood plasma, the LPO, ONOO− and GPx were higher in the malathion group as compared with controls. Rolipram ameliorated all of malathion-induced changes. Plasma CAT decreased in malathion-treated animals while it increased in brain mitochondria comparing with controls. Coadministration of rolipram with malathion improved CAT in both brain mitochondria and plasma. Malathion and rolipram did not alter total SOD or Mn-SOD in the plasma while both caused a significant elevation in brain mitochondria. Conclusion: Based on these results the following hypothesis can be presented: the toxic effect of malathion may be related, among others, to the impairment of antioxidant enzymes as an important non-cholinergic effect of malathion, which can be rescued by Rolipram treatment.

P375. Antiapoptotic Effect of Aminoguanidine on Doxorubicin Induced Apoptosis

Suna Sabuncuoglu Jr.1 and Pierre Hainaut Sr.2

1Department of Pharmaceutical Toxicology, Hacettepe University, Faculty of Pharmacy, Ankara, Turkey, 2Molecular Carcinogenesis Group, International Agency for Research on Cancer, Lyon, France

Doxorubicin (DOX) is a broad-spectrum anthracycline that has cardiotoxicity as a major side effect. ROS and nitrogen species (RNS) generation have been proposed to be an important mechanism of DOX induced cardiotoxicity and cardiomyocyte apoptosis, a processes that may be mediated by the p53 protein. Aminoguanidine (AMG) is an effective antioxidant and free radical scavenger which has long been known to protect against ROS formation. A549 lung cancer cell line were incubated different concentration of AMG (100 to 1000μM) in the presence or absence of 0.25 μM DOX for 24 hours. The expresssion of p53 and of its transcriptional target p21 were analysed by western blot. Apoptosis was analysed with Annexin V assay. JC1 and H2ax immunofluorescence were used to assess mitochondrial and nuclear DNA damage, respectively. Results demonstrate that AMG has a dose-dependent antiapoptotic effect on doxorubicin-induced apoptosis. Thus, these data further identify AMG as a chemopreventive agent with great potential to reduce ROS and NOS damage generated by DOX.

P376. Antioxidant Effect of Amaranthus lividus L. Against CCl4-induced Hepatotoxicity in Rats

Tugba Yilmaz1, Ayse Can1, Zeliha Pala2 and Alper Okyar2

1Biochemistry, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey, 2Pharmacology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey

Oxidative stress, a disbalance in favor of the prooxidants and disfavoring the antioxidants, potentially leading to cell damage [1]. Natural dietary antioxidants are extensively studied for their ability to protect cells from various dam-age. Amaranthus plants are widely distrubuted throughout the world and they are able to produce grains and leafy vegetables. Amaranthus lividus L. (Family:Amaranthaceae) is used as popular vegetable in West Black Sea Region of Turkey. In vitro antioxidant potential of the plant has been investigated in our earlier study [2]. The objective of the present study was to investigate the antioxidant activity of A. lividus L. against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. The stems with leaves and flowers of A. lividus were collected from Bartin, Turkey (ISTE 83401). A crude water extract was prepared by heating powdered A. lividus (10 g) with 100 mL distilled water for 30 min. The extract was filtered and evaporated to dryness under reduced pressure and controlled temperature (40-50 oC) in a rotary evaporator. Twenty-six Wistar male albino rats were randomly divided into four groups. Grup 1 (n=5) served as control and received water extract of A. lividus (500 mg/kg p.o.) once daily for 9 days. Grup 2 (n=7) received CCl4 (1.5 ml/kg as a 50 % olive oil i.p.) on the 10th day. Grup 3 and 4 animals (n=7) were treated with two different doses water extract of A. lividus (250 and 500 mg/kg p.o., respectively), once daily for 9 days and CCl4 (1.5 ml/kg as a 50 % olive oil i.p.) on the 10th day. Animals were sacrificed 24 h after CCl4 administration, the livers were removed, washed with saline and 10% (w/v) homogenate prepared in PBS (phosphate buffer saline, pH 7,4). The homogenates were centrifuged and the supernatants used for the measurement of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-R) and glutathione-S-transferase (GST) activities. Pretreatment of rats with dif-ferent doses of plant extract (250 and 500 mg/kg) significantly increased antioxidant enzyme activities compared with

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CCl4 treated rats.The present study suggested that the treatment with A. lividus L. extract enhance the recovery from CCl4 hepatotoxicity due to its antioxidant and hepatoprotective property.

References

[1] Sies, H., Strategies of antioxidant defense, European Journal of Biochemistry, 215: 213-9, 1993. [2] Ozsoy, N., Yilmaz, T., Kurt, O., Can, A., Yanardag, R., In vitro antioxidant activity of Amaranthus lividus L., Food Chemistry, 116: 867-872, 2009.

P377. Assesment of Antioksidan Capacity of Metformin and Insulin on Treatment of Type II Diabetes Mellitus

Gulsen Akalin1, Ipek Erdogan2, Aysen Akalin3 and Ozkan Alatas2

1Biochemistry, University of Anadolu, Eskisehir, Turkey, 2Biochemistry, Eskisehir Osmangazi University, Eskisehir, Turkey, 3Endocrinology, Eskisehir Osmangazi University, Eskisehir, Turkey

Hyperglycemia increases oxygen-reactive species generation and reduces the protective capabilities of antioxidant defense systems. In patients with type 2 diabetes mellitus (DM), the increased production of oxygen free radicals may be linked to the development of chronic complications of diabetes. In vitro studies have demonstrated that oral antidiabetic drugs have antioxidant effects that might be secondary to an inhibiting role in lipid peroxidation. The purpose of this study was to determine the effects of metformin and insulin terapies on oxidative stress which induce Cardiovascular Disease risk of Type II Diabetes Mellitus patients. The study included 59 patients (35 female and 24 male; mean age 59 ± 14 years) who were diagnosed as type 2 DM and used either metformin or insulin. Control group was healthy individuals. Blood samples were collected after overnight fast. The plasma concentrations of MDA levels were measured by spectrophotometric method. PON activity was measured by Rel Assay PON kit. Plasma concentrations of MDA for control group, metformin group and insulin group were respectively mean (SD) 21.88 (3.65), 34.03 (12.00), 36.90 (5.58) µmol/L, control MDA levels were significantly different from other groups.( p<0,001). PON levels of control, metformin and insulin groups were respectively mean (SD) 250 (29,5), 224 (26.30), 178 (27,7) U/L. Control PON levels were sig-nificantly different from metformin and insulin groups ( respectively p<0,01, p<0,001). Metformin PON levels were significantly different from insulin groups (p<0,001). In conclusion, we have demonstrated that metformin treatment of Type II Diabetes Mellitus Patients have better effects on HDL antioxidant function than insulin treatment.

P378. Effects of caffeic acid phenethyl ester on isoproterenol-induced myocardial infarction in hy-pertensive rats

Süleyman Oktar1, Nigar Yilmaz2, Selçuk Ilhan3 and Engin Sahna3

1Pharmacology, Medical Faculty of Mustafa Kemal University, Hatay, Turkey, 2Biochemistry, Medical Faculty of Mustafa Kemal University, Hatay, Turkey, 3Pharmacology, Medical Faculty of Firat University, Elazig, Turkey

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives and has also antioxidant features. We investigated that may CAPE protect the heart from isoproterenol (ISO)-induced myocardial infarction in hypertensive rats. NG-nitro-L-arginine (L-NNA) was given orally at 25mg/kg/day in drinking water to rats during 14 days to produce hypertension. To induce myocardial infarction, ISO was given intraperitoneally to rats (150mg/kg) once a day for 2 consecutive days. Control group: untreated, LNNA group: rats were treated with L-NNA (25 mg/kg/day), LNNA+ISO group: L-NNA treated rats were given ISO (150 mg/kg) for 2 consecutive days in the 12th and 13th days of L-NNA treatment, LNNA+ISO+CAPE group: L-NNA treated rats were also given CAPE (10 µmol /kg/day) during 7 days after first week and treated with ISO (150 mg/kg) for 2 consecutive days at the 12th-13th days of L-NNA treatment. Systolic blood pressure (SBP) in L-NNA treated groups increased (145 ± 2 vs 110 ± 1; day 14th vs initial in LNNA rats). ISO reversed SBP to basal levels in LNNA+ISO group (133 ± 2 vs 107 ± 2 mmHg; day 7th vs 14th in LNNA+ISO rats). CAPE did not change SBP (136 ± 2 vs 102 ± 2 mmHg; days 7th vs 14th in LNNA+ISO+CAPE rats). AST and LDH levels in LNNA+ISO group were significantly increased compared to control (125 ± 5 vs 105 ± 2 U/l

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for AST, 861 ± 154 vs 571 ± 46 U/l for LDH). CAPE reversed the enhanced AST and LDH levels in LNNA+ISO+CAPE group compared LNNA+ISO (125 ± 5 vs 106 ± 9 U/l for AST, 861 ± 154 vs 372 ± 46 U/l for LDH). There were no statisti-cally significant difference in the levels of CK-MB among all group. SOD activity in LNNA and LNNA+ISO groups decreased. The activity of CAT in LNNA+ISO group were significantly increased compared to control (0.263 ± 0.01 vs 0.196 ± 0.01 k/mg protein). CAPE more enhanced activities of SOD and CAT in LNNA+ISO+CAPE group. MDA levels in LNNA+ISO group were significantly increased compared to control (3.22 ± 0.25 vs 2.50 ± 0.22 nmol/g protein) and CAPE treatment reversed the enhanced MDA levels (3.22 ± 0.25 vs 2.55 ± 0.19 nmol/g protein). We demonstrated an increase in lipid peroxidation and a decrease in SOD and CAT activities in heart tissue of L-NNA treated rats given ISO, and the weakening of antioxidant enzymes and lipid peroxidation-mediated myocardial injury was prevented by CAPE treatment. Indeed, the authors showed that CAPE inhibits lipid peroxidation and suppresses oxidative stres (1). In our previous study, we have detected an association between myeloperoxidase activity and cardiac damage but we did not measured myeloperoxidase activity in this study (2). Our results suggest that CAPE may be an avail-able agent to protect the myocardium from infarction via induction of the antioxidant enzymes and the inhibition of lipid peroxidation.

References

1. Sud′ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 1993;329: 21-24.

2. Oktar S, Aydin M, Yonden Z, Alcin E, Ilhan S, Nacar A. Effects of caffeic acid phenethyl ester on isoproterenol-induced myocardial infarction in rats. Anadolu Kardiyoloji Dergisi 2010 (Epub ahead of print).

P379. Evaluation of Oxidant/Antioxidant Status in H1N1 Infected Children

Ali Asci1, Can Samet Atas2, Pinar Erkekoglu1, Murat Kizilgun3, Ates Kara4, Ali Bulent Cengiz4, Meh-met Ceyhan4 and Belma Giray5

1Department of Toxicology, Hacettepe University Faculty of Pharmacy, Ankara, Turkey, 2University of London, School of Pharmacy, London, England, 3Ankara Children′s Health and Disease, Hematology, Oncology Training and Research Hospital, Ankara, Turkey, 4Department of Pediatric Infectious Diseases, Hacettepe University, School of Medicine, Ankara, Turkey, 5Department of Toxicology, Hacettepe University Faculty of Pharmacy, Ankara, Turkey

It has been demonstrated that oxidative stress and reactive oxygen species (ROS) may be implicated in the pathogen-esis of several viral infections including hepatitis, influenza and HIV. On the other hand, the decline in selenoenzyme expressions can elevate the susceptibility to infections in consequences of impaired immune response. This study has been undertaken to investigate possible alterations in antioxidant defense systems including selenoenzymes and lipid peroxidation levels in children infected by pandemic 2009 H1N1 influenza. Erythrocyte antioxidant enzyme activi-ties [glutathione peroxidase (GPx), thioredoxin reductase (TrxR), superoxide dismutase (SOD), catalase (CAT)] and plasma malondialdehyde (MDA) levels as a measure of lipid peroxidation have been determined in the patient group (n=11) and compared with healthy control children (n=11). Significant decreases in the activities of selenoenzymes [GPx (∼45%) and TrxR (∼30%)] in H1N1 group were observed and the activities of SOD (∼40%) and CAT (∼40%) also decreased significantly compared to control group. Although MDA concentrations in H1N1 group elevated (∼15%), the differences between groups were found not to be significant statistically. The results indicate possible role of oxidant/antioxidant imbalance in the development of pandemic H1N1 influenza and suggest the importance of antioxidant supplementation in the treatment and prevention of disease.

P380. Glutathione and related enzyme activities in human lung cancer patients

Tulay Coban1, Fatma Sener2 and Huseyin Cakmak3

1Faculty of Pharmacy, Ankara University, Ankara, Turkey, 2Turkish Social Security Institution, Sivas, Turkey, 3Department of Chest Surgery, Demetevler Oncology Hospital, Ankara, Turkey

245

Lung cancer is a common disease having the features of late diagnosis, and high mortality. The human lungs have a large surface area, and they contact with oxygen in the air directly. Because of that, they are the most effected organs from the toxic effects of environmental free radicals and reactive oxygen species (ROS). In order to protecting against the toxic effects of these substances, the lungs have the antioxidant molecules like glutathione (GSH) and antioxidant enzymes like glutathione peroxidase (GSHPx). GSH and GSHPx play crucial role in cell defense against ROS, which are implicated in tumor disease. The aim of this study was to determine if neoplastic diseases of lung may influence blood GSH level and its dependent enzyme activity. For that reason, in this study, 35 lung cancer patients who are in different grades of lung cancer were used. GSH levels and GSHPx activities in the erythrocytes of these patients were measured. These results were compared with the results obtained from 34 healthy persons and the association between lung cancer and oxidative stress was investigated. The findings obtained from this study showed that, GSH levels and GSHPx activity in the lung cancer patients′ were significantly lower than the activity in healthy persons. When the patients were divided into two groups as the disease′s grade, GSH levels and GSHPx activity were lower in the advanced grade patients than the early grade patients′. But these results weren′t statistically significant. In according to these findings it would be possible to say that in lungs, the alterations in antioxidative balance may lead the development of cancer and GSH and glutathione system enzymes could be useful biomarkers in lung cancer prognosis because of their involvement in the metabolism of a wide range of chemotherapeutic agents.

P381. Hepatoprotective and antioxidant effects of silymarin against manganese-induced hepatotox-icity in rats

Yassine Chtourou1, Hamadi Fetoui1, Mohamed Barkallah1, Tahia Boudawara2 & Najiba Zeghal1 1Animal Physiology Laboratory, Sciences Faculty of Sfax, Sfax, Tunisia, 2Histopathology Laboratory, CHU Habib Bourguiba-Sfax, Tunisia

Manganese (Mn) is an essential nutritional element and the occupational hazard factor and environmental pollutant. It is also widely employed in industry (ferro-alloy plants), in agriculture (fertilizers and pesticides) and it has recently been introduced as antiknock agent in unleaded gasoline. The mechanism of toxic effects of Mn is not understood, involving probably an inhibition of energy metabolism and formation of free radicals. In the present study, Mn (20mg/ml) was administered to adult rats through drinking water for 30 days. Silymarin (SIL), a flavonoid complex extracted from the fruit of (a Silybum marianum), was evaluated for its protective effect against manganese-induced hepatic dysfunction. Co-administration of SIL (100 mg/kg body weight i.p daily) significantly prevented Mn induced hepatic damage as indicated by the serum marker enzymes activities such as alanine transaminase (ALT), serum aspartate transaminase (AST), lactate dehydrogenase (LDH), gamma glutamyl transferase (GGT) and total bilirubin. Parallel to these changes, silymarin improved Mn-induced oxidative stress in the rat liver by inhibiting lipid peroxidation and protein carbonyla-tion, and restoring the levels of antioxidant enzymes (SOD, CAT and GPx,) and glutathione. The biochemical changes were consistent with histopathological observations suggesting a marked hepatoprotective effect of silymarin.

P382. Abstract Withdrawn.

P383. Hypercholesterolemia Alters Oxidative Balance in Rat Liver

Saadet Gumuslu1 and Yesim A. Gocmen2

1Biochemistry Department, Akdeniz University, Medical Faculty, Antalya, Turkey, 2Biochemistry Department, Bozok University, Medical Faculty, Yozgat, Turkey

Hypercholesterolemia (HC) is a common health problem that significantly increases risk of cardiovascular disease. This study was conducted to evaluate how hypercholesterolemia effects on the main indicators of hepatic oxidative status. The rats were divided into 2 groups as following; (1) control (C), (2) hypercholesterolemic (HC). Control group were fed standard diet for 80 days and hypercholesterolemic group was given 5% cholesterol diet for 80 days. The total cholesterol levels were found to be increased in HC group when compared to controls. In the end of the feeding period, rats were

246

sacrificed for the collection of liver and blood. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and the levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were studied. Particularly the activities of hepatic GPx and CAT were found to be reduced in hypercholesterolemia while GR and GST activity was elevated. Dietary supplementation cholesterol increased hepatic GSH and GSSG levels. On the other hand GSH/GSSG ratio was significantly decreased. In conclusion, we can propose that Hypercholesterolemia could make alterations in the oxidative balance in favor of the oxidants.

P384. Impacts of Methylsulfonylmethane association with Carbenoxolone on cancer cell lines

Hossein Hamzeiy Pharmacology and Toxicology, (1)Research Center for Pharmaceutical Nanotechnology, (2) School of Pharmacy, Tabriz University, Tabriz, Iran

Impacts of Methylsulfonylmethane association with Carbenoxolone on cancer cell lines Hossein Hamzeiy1, 2, Jaleh Barar1, Yadollah Omidi1, Eghbal MA2, Masoud Asadi1, 2, Sadollah Mohammadi1, 2 1Research Center for Pharmaceutical Nanotechnology, 2 School of Pharmacy, Tabriz University of medical sciences, Tabriz, IRAN ABSTRACT: Gap junctions play an important role in the cell proliferation in mammalian cells. However, there are controversial issues about cell communications in neoplastic states. This study was designed to evaluate the cytotoxic effects of Carbenoxolone as a prototype of inter- cellular gap junction blocker in association with Methylsulfonylmethane (MSM). On the basis of specific and general roles of oxidative systems in different kinds of cells, three cancerous cell lines (A549, BT20 and MCF7 cells) were cultivated, and at designated confluency cell monolayers were treated with Carbenoxolone (150 micro Molar) and MSM. Cellular cytotoxicity was examined using standard colorimetric assay associated with cell viability tests. MCF7 and BT20 cells were significantly affected by Carbenoxolone and MSM. Treated MCF7 and BT20 cells with MSM and Carbenoxolone (alone or as combination) showed significant toxicity. Based upon this investigation, it can be concluded that there exists relationship between cell-cell communications and intercellular cross talking, perhaps via gap junctions, at which we propose interestingly orchestrated biochemical machinery of cells may be related to oxidative stress induced regulatory systems which may play a role in development of cancer. Keywords: gap junctions, Methylsulfonylmethane, Carbenoxolone, cancer cell line

P385. Inducible nitric oxide synthase genetic polymorphism and risk of asbestosis: the role od gene-gene and gene-environment interactions

Alenka Franko1, Metoda Dodič-Fikfak1, Niko Arnerić1 and Vita Dolžan2

1Clinical Institute of Occupational Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia, 2Institute of Biochemistry, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia

Asbestosis is one of the most frequently studied diseases associated with inhalation of asbestos fibers. Reactive oxygen and nitric species (ROS and RNS) are involved in the pathogenesis of asbestosis. We have previously shown that in addi-tion to asbestos exposure also genetic factors influencing defence against ROS influence the risk for asbestosis. Nitric oxide (NO) synthesized by inducible NO synthase (iNOS) was also suggested to play a role in asbestos-induced lung injury. Transcriptional activity of iNOS gene has been shown to increase with the number of CCTTT repeats ((CCTTT)

n)

in the promoter region. The aim of this study was to investigate whether iNOS (CCTTT)n polymorphism is associated

with an increased risk of asbestosis in workers occupationally exposed to asbestos. The study cohort consisted of 262 cases with asbestosis and 265 controls with no asbestos-related disease. Cases and controls were matched by gender and age. Data on cumulative asbestos exposure and smoking were available for each subject. The number of iNOS CCTTT repeats was determined using PCR-based methods. Alleles with 11 or less repeats were defined as short (S) and alleles with 12 or more repeats as long alleles (L). Logistic regression was performed to estimate asbestosis risk. iNOS LL genotype was found in 132 cases and 121 controls, SL genotype in 83 cases and 96 controls and SS genotype in 43 cases and 42 controls. The OR of asbestosis was 1.20 (95 % CI=0.85–1.69) for the LL genotype compared to the combined SL and SS genotypes and 1.26 (95 % CI=0.86–1.85) for the LL genotype compared to the SL genotype. This risk remained practically unchanged after adjustment by gender, age, cumulative asbestos exposure and smoking. The observed association was modified by catalase (CAT) polymorphism as higher asbestosis risk for iNOS LL genotype

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was observed only among carriers of CAT -262TT (OR=5.14, 95 % CI=1.30–20.36), but not CAT -262CT or CC genotype (OR=1.08, 95 % CI=0.75–1.55). iNOS (CCTTT)

n polymorphism also modified the association between asbestos exposure

and asbestosis, as well as the association between smoking and asbestosis. In conclusion, our results suggest a possible role of iNOS (CCTTT)

n polymorphism in the risk of asbestosis, and indicate that gene-gene as well as gene-environment

interactions play a role in the development of asbestosis. Further studies including a larger number of subjects are needed to clarify the observed associations.

P386. Induction of Reactive Oxygen Species, p53 and p21 in DEHP- and MEHP-Treated LNCaP Cells and Possible Protection by Selenium Compounds

Pinar Erkekoglu1, Ozge Gursoy Yüzügüllü2, Walid Rachidi3, Belma Giray4, Mehmet Ozturk2 and Filiz Hincal1

1Department of Toxicology, Hacettepe University Faculty of Pharmacy, Ankara, Turkey, 2Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey,3Inac/Scib/Lan, CEA Grenoble, France, 4Department of Toxicology, Hacettepe University Faculty of Pharmacy, Ankara, Turkey

It has been demonstrated that oxidative stress and reactive oxygen species (ROS) plays important roles in many physi-ological processes. The tumor suppressor protein, p53, controls the integrity and correctness of all processes in each individual cell. Activated p53 binds DNA and activates expression of several genes including WAF1/CIP1 gene encoding for p21. Selenium is an essential cofactor in the key enzymes involved in cellular antioxidant defense; plays a critical role in testis and reproduction, and regulates DNA damage within the prostate. Phthalates are ubiquitous environmental contaminants that cause alterations in endocrine and spermatogenic functions in animals. Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. The aim of this study was to investigate whether the toxic effects of DEHP (3 mM) and its main metabolite mono(2-ethylhexyl)phthalate (MEHP, 3 µM) are mediated through the activation of ROS production, and whether the phthalates cause activation of p53 and p21 in LNCaP cells. We also examined whether the two selenium compounds, sodium selenite (SS, 30 nM) and selenomethionine (SM, 10 µM), have beneficial effects against ROS production and induction of p53 and p21 in DEHP- and MEHP treated LNCaP cells. Both DEHP and MEHP exposures caused strongly amplified production of ROS, and MEHP exposure caused significant increases in p53 and p21 expressions. Selenium supplementation with either SS or SM forms reduced the ROS production and the expressions of p53 and p21. The overall results of this study, thus, demonstrated that DEHP, and particularly MEHP caused increases in intracellular ROS production, and MEHP seemed to affect cell cycle progression through the induction of p53 and subsequently p21 in LNCaP cells, indicating that one of the important mechanisms underlying the toxicity of these compounds is the induction of oxidative stress. Generated data also emphasized the critical role of selenium in the modulation of ROS, and cell cycle via p53 and p21 induction.

P387. Linking exposure to environmental pollutants with oxidative stress in Bangkok bus drivers

Krongtong Yoovathaworn1, Katesiri Satethasilnuchai2, Jintana Sirivarasai3 and Sming Kaojarern3

1Department of Pharmacology and Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand, 2Graduate Program in Toxicology, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand, 3Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Exposure to air pollution has been associated with health problems especially in occupational exposed population such as professional bus drivers. This study aimed to determine oxidative stress status in 100 Bangkok bus drivers and 100 controls. After institutional ethical approval, the oxidative stress was determined by measuring oxidative stress status which are glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and malondi-aldehyde (MDA). The internal PAHs exposure was also estimated by measuring urinary 1-hydroxypyrene (1-OHP). Bus drivers had significantly lower plasma GSH level with higher SOD and CAT activity as well as MDA and urinary 1-OHP levels than those of the control subjects. Confounding factors associated with oxidative stress such as cigarette smoking

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and age were also taken into consideration. Cigarette smoking had a significant association with urinary 1-OHP in both the bus drivers and the controls. Only smoker bus driver had an increase in GPx activity with a decrease in GSH level, an effect not observed in the control group. At the age >40 years, a significant increase in GPx activity was observed in bus drivers. At the same age range, increased SOD and CAT activity with decreased GSH level were observed in the control group. PAHs exposure did not seem to be a major influence of oxidative stress status, as no significant correlation between 1-OHP and MDA in bus drivers was observed. The results of the present study indicated that exposure to air pollutants is one of the major factors contributing to oxidative stress in Bangkok bus drivers. Cigarette smoke and aging are significant confounding factors affecting oxidative stress in both the bus driver and the control groups.

P388. Mitochondrial Membrane-bound Glutathione Transferase Can Form Mitochondrial Permeability Transition Pores

Naoki Imaizumi, Ryosuke Katayama and Aniya Yoko

School of Health Sciences, University of the Ryukyus, Okinawa, Japan

Mitochondrial permeability transition (MPT) pore is opened by oxidative stress in which thiol groups in mitochondrial proteins play a key role. However component or structure of MPT pore is still obscure. Previously we showed that mitochondrial membrane-bound glutathione transferase (mtMGST1) has critical thiol and contributes to MPT pore opening (Lee et al., 2008). In the present study we investigated how mtMGST1 contributes to the MPT pore using peroxynitite (PON).

(Methods): Mitochondria from rat liver were incubated with PON followed by measuring MPT pore opening and GST activity. MPT pore opening was evaluated by mitochondrial swelling (absorbance at 540nm). MPT regulator pro-teins such as adenine nucleotide translocator (ANT) and cyclophilin D (CypD) as well as mtMGST1 were analyzed by immunoblotting.

(Results and Discussion): PON induced mitochondrial swelling which was prevented by GST-inhibitors (S-hexylglutathione, cibacron blue, tannic acid), MPT-inhibitors (cyclosporin A, bongkrekic acid, ADP) and by a disulfide bond reducing agent, dithiothreitol (DTT). In addition, when mitochondrial fraction was analyzed with electrophoresis followed by immunoblotting with anti-mtMGST1antibody, the protein with high molecular weight (HMP) was detected in PON-treated mitochondria, not in control, and this HMP formation was prevented by these swelling inhibitors. When the HMP was extracted and analyzed by immunoblotting with various antibodies in reducing conditions, mtMGST1, ANT and CypD were detected. These results clearly show that the HMP is a complex consisting of mtMGST1, ANT, and CypD in which each protein binds, at least, through a disulfide bond. The HMP was detected in the mitochondrial inner and outer membranes although ANT and CypD are known to be localized in the inner membrane and in matrix, respectively. Since mtMGST1 is distributed in both inner and outer membranes, these results suggest that thiols in ANT, CypD and mtMGST1 in the inner membrane are oxidized by PON resulting in ANT/CypD/mtMGST1 complex, and also ANT and CypD translocate into the outer membrane followed by formation of ANT/CypD/mtMGST1complex. Taken together, it could be concluded that oxidant PON oxidizes thiols among mtMGST1, ANT and CypD resulting in formation of a disulfide-linked mtMGST1/ANT/CypD complex, which may act as MPT pores.

P389. Occurrence of Cyclopiazonic acid in broiler′s diet and its effect on oxidative and nitrosative stress

Malekinejad H, Akbari P, Alizadeh A, Taheri-Brojerdi Meysam and Tabatabaie SH

Department of Pharmacology & Toxicology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

The excessive generation of reactive oxygen/nitrogen species (ROS/RNS) leads to severe organ damages. Thirty six broiler diet samples including maize, soy bean, and mixed diet were collected from 3 broiler chicks producing sites in northwest of Iran and the primary mycological tests were conducted. To detect the Cyclopiazonic acid (CPA) contami-nation, the collected samples were subjected to mycotoxin analyses by means of HPLC. To study the toxic effects of CPA in broiler chicks, ten-day old male broiler chicks (Ross 308, n = 40) were assigned to the control and 3 test groups (n=10), which received normal saline and 10, 25 and 50 mg/kg CPA, respectively for 28 days. The total thiol molecules

249

(TTM), malondialdehyde (MDA) and nitric oxide (NO) contents of the liver and kidneys were assessed. The myco-logical analysis of diet samples revealed that 34.46 and 23.07 % of the isolated fungi were As. flavus and P. cyclopium, respectively. HPLC analyses showed that the highest level of CPA (0.95 ± 0.35 ppm) was found in maize samples and the lowest concentration (0.05 ± 0.01 ppm) was found in mixed diet samples. The MDA and NO content of the liver and kidneys showed significant (P<0.05) increase in a dose-dependent manner. By contrast the TTM levels in the liver and kidneys were significantly (P<0.05) attenuated. This study suggests that one of the poultry diet contaminations might be CPA and CPA producing fungi. Moreover, it is also concluded that CPA via oxidative and nitrosative stress in broiler chicks may cause severe hepatic and renal disorders.

P390. Protection of small molecule against oxidative damage by inducing HO-1 expression mediated by Nrf2 signaling

Sung Su Yea and Nam I Kim

Department of Biochemistry, College of Medicine, Inje University, Busan, South Korea

This study investigated the protective effect of plant-derived small molecule IJ112 against oxidant-mediated injury and its mode of action in RAW264.7 macrophages. MTT assay showed that H

2O

2 treatment reduced cell viability, whereas

IJ112 protected cells from H2O

2-mediated cytotoxicity. FACS analysis showed that lipopolysaccharide-induced reactive

oxygen species production was also decreased by the treatment of IJ112. Because heme oxygenase-1 (HO-1) is known to protect cells against oxidative damage, we investigated the effect of IJ112 on HO-1 gene expression. IJ112 induced HO-1 mRNA and protein expression, as determined RT-PCR and Western blotting, respectively. IJ112-mediated HO-1 induction was abrogated in the presence of specific inhibitors for phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2), or c-Jun N-terminal kinase (JNK), indicating that PI3K and two MAP kinases were involved in the up-regulation of HO-1. IJ112 also increased the nuclear levels of Nrf2, a transcription factor governing anti-oxidant response elements. Collectively, these results suggest that IJ112 has anti-oxidative activity that is mediated, at least in part, via the activation of PI3K-ERK/JNK-Nrf2 and the subsequent induction of HO-1 gene expression.

P391. Protective effects of food factors on ischemia-reperfusion injury in rat small intestine

Shirou Itagaki

Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Small intestine acts as a barrier against xenobiotics. Many kinds of metabolic enzymes and transporters are expressed in the small intestine. Importance of cooperation of phase II metabolism and efflux transport has been accentuated in the xenobiotic detoxification. Intestinal ischemia-reperfusion (I/R) is a common clinical problem in small bowel transplantation, circulatory shock, and strangulation ileus. The intestinal mucosa is damaged structurally and func-tionally after intestinal I/R. The intestinal mucosa is extremely sensitive to reactive oxygen species (ROS) and ROS are responsible for intestinal I/R injury. Dietary antioxidants can enhance cellular defense and help to prevent oxidation damage to cellular components. There has been considerable public and scientific interest in therapeutic use of natural antioxidants. In this study, we used a rat mesenteric I/R injury model and investigated the protective effect of some kinds of food factors on I/R injury of small intestine. Upper villous cells were sloughed by intestinal I/R. Moreover, The amount of lipid peroxide in the small intestine and vascular permeability in the small intestine was increased by intestinal I/R. We found that intestinal I/R injury was attenuated by caffeic acid (constituent of coffee), chlorogenic acid (constituent of coffee), ferulic acid (constituent of rice bran), epigallocatechin gallate (constituent of green tea) and rutin (constituent of buckwheat).We then investigated the in vitro antioxidant properties of these food factors. We focused on superoxide anion-scavenging activity, xanthine oxidase inhibition activity and chain-breaking activity. We found that chain-breaking activity may play a contributory role in the protective effect of these food factors on intestinal I/R injury. (Supported in part by Grant-in-Aid for Young Scientists (A), Kieikai Foundation, The Skylark Food Science institute, Takano Life Science Research Foundation, Fuji Foundation for Protein Research, The Nisshin Seifun Foundation and The Ichiro Kanehara Foundation)

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P392. Protein oxidation after acute spinal cord injury in rats and the effect of Erythropoietin(EPO)

Yasemin Erdogan Doventas1, Servet Yigit1, Macit Koldas1 and Bulent Ozdemir2

1Clinical Biochemistry, Haseki Education and Research Hospital, Istanbul, Turkey, 2Neurosurgery, Haseki Education and Research Hospital, Istanbul, Turkey

Introduction: Oxidative stress leading to protein oxidation is a major cause of secondary injury following spinal cord injury (SCI). The objectives of this study were to determine the levels of protein oxidation following acute SCI and the efficacy of low-and high dose - administration of erythropoietin on increasing protein oxidation. Traumatic injury to the spinal cord triggers several secondary effects, including oxidative stress and compromised energy metabolism, which play a major role in biochemical and pathological changes in spinal cord tissue. Erythropoietin(EPO) is a hematopoietic growth factor that stimulates proliferation and differentiation of erythroid precursor cells and is also known to exert neurotrophic activity in the central nervous system. The purpose of this study was to investigate the effectiveness of recombinant human EPO in attenuating the severity of experimental SCI.

Material and method: A total of 32 spraue-dawley rats underwent clip-compression induced SCI. They were opereted on with posterior laminectomy.Spinal cord trauma produced by extradural placement of the anevri-ysm clip, for 1 min.Animals were divided into four groups.The first group only were operated on with posterior laminektomy,second group were underwent clip-compression induced SCI. The third group received a low total dose.(EPO –L).(2 doses of 1000 IU each i.p) The fourth group received a high total dose (EPO-H) early post-op 5000iu/kg i.p., in postop 24 h. 3000iu/kg ip and in post-op 48 h.1000iu/kg i.p. (3 doz-9000iu/kg). Follow-up was for 6 weeks. Estimation of the functional progress of each rat was calculated using the locomotor rating scale of Basso et al, with a range from 0 to 21.

Results: After surgery the animals suffered paraplegia with urinary disturbances. Rats that received EPO demonstrated statistically significant functional improvement compared to the Control group, throughout study interval. On the last follow-up at 2 weeks, the EPO-L rats AOPP levels were found 56,6+/-16,1, the EPO-H 43,6+/-8,1, and the control group 25,8+/-12,6 Comparison between the two EPO groups reveals superior final outcome of the group treated with higher dose.

Conclusion: Our study supports current knowledge, that EPO administration has a positive effect on functional recovery after experimental ASCI. These data reflect the positive impact of EPO on the pathophysiologic cascade of secondary neural damage. However, we observed a dose-related effect on functional recovery. Interestingly, large doses do not seem to favor the neurological recovery as lower doses doour results suggest that i.p. EPO applied after spinal cord injury significantly attenuated the oxidative damage of spinal cord injuries in rats.

P393. The effect of acetyl-l-carnitine on sperm characteristics, oxidant-antioxidant system and histologic structure in methotrexate treated male wistar rat

Mohammad Amin Rezvanfar1, Mohammad Ali Rezvanfar2 and Mohammad Abdollahi2

1Toxicology and Pharmacology, 1.Osvah Bio/Pharmaceutical Company, R&D Department; 2. Faculty of Pharmacy, and Pharmaceutical Science Research Centre, Tehran, Iran, 2Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Science Research Centre, Tehran University of Medical Sciences, Tehran, Iran

Background and objectives: L-Carnitine (LC) and acetyl-L-carnitine (ALC) are highly concentrated in the epididymis and play a crucial role in sperm metabolism and maturation. They are related to sperm motility and have antioxidant properties. The aim of this study was to investigate the possible protective role of acetyl-l-carnitine (ALC) on testicular toxicity induced by methotrexate (MTX) in rats.

Methods: 28 male rats were divided into four groups as follows: group1, control; group2, MTX-treated; group3, ALC-treated and group4, MTX+ALC-treated. In the second day of experiment, a single dose of methotrexate was intraperito-neally administered to groups 2 and 4, although a daily single dose of ALC was intraperitoneally administered to group 3 and 4 for 7 days. At the end of the experiment, the testes of the animals were removed and prepared for assessment of oxidative stress parameters and histopathological analysis.

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Results and discussion: In the testes tissue, the level of lipid peroxidation as malondialdehyde and activities of super-oxide dismutase, catalase and glutathione peroxidase were higher in the methotrexate group than in the control group. ALC ameliorated all of MTX-induced impairments in enzymatic antioxidant defense system. Also the histopathological study revealed that MTX induced marked reductions in sperm′s content of epididymis, obstruction in seminiferous tubules, hypospermia and dilated congested blood vessels in testicular sections. Co-administration of ALC returned sperm′s content of epididymis to normal and histological abnormalities were markedly improved in ALC + MTX -treated rats.

Conclusion: In conclusion, the administration of MTX causes elevation of oxidative stress leading to a marked testicular dysfunction and ALC ameliorates these effects by antioxidant mechanisms suggesting a protective role against male infertility.

P394. The effect of Omega-3 and Selenium on Methotrexate-Induced Hepatotoxicity

Bassim Irheim Mohammad1, Najah Raiesh Hadi2 and Asma Swadi Thwainee1

1Pharmacology and Therapeutics, College of Medicine/ Al Qadisiyah University, Diwaniyah, Iraq, 2Pharmacology and Therapeutics, Kufa College of Medicine, Kufa, Iraq

Background: Methotrexate (MTX) associated hepatotoxicity is a significant clinical problem that affect the compliance with MTX-containing treatment regimens.

Materials and methods: A total of 28 New Zealand White Male Rabbits were used in the study, after two week accli-matization period, the animals were randomized in to four groups (7 rabbits each), all groups were maintained on standard chow diet throughout the experiment (8 weeks). Group 1 was treated with normal saline (control), group 2 with MTX, (hepatotoxic control), group 3 with MTX plus Omega-3 and group 4 with MTX plus Selenium (Se). Induction of hepatotoxicity was carried out by treating the rabbit with MTX (0.25 mg/kg /day i.m) for 8 weeks. Liver function markers, oxidative stress parameters and histological examination were performed.

Results: Treating rabbits with MTX for eight weeks resulted in significant changes in serum liver enzymes, as compared to baseline group; SGOT, SGPT, ALP, and bilirubin were significantly increased (p< 0.001), while total serum protein was significantly decreased. Similarly, eight weeks of MTX treatment produced significant (p< 0.001) prolongation in PT. It was found that serum MDA levels and SOD activity were significantly increased (p< 0.001), while serum GSH levels were significantly decreased (p< 0.001). Adding Omega-3 and Se to MTX was found to cause significant (p< 0.001) reduction in GOT, GPT and ALP. Furthermore, these additive drugs caused a significant (p< 0.001) reduction in serum bilirubin levels, shortening of PT and significant rise in TSP (p< 0.001).

Conclusion: Addition of Omega-3 and Se to MTX regimen restored the altered liver function parameters, improve oxidative stress and produced significant improvement in liver histopathology. Therefore, these additive drugs posses hepatoprotection against MTX induced hepatotoxicity

P395. THE EVALUATION OF MN AND SCE FREQUENCIES AND OXIDATIVE STRESS STATUS OF HOSPITAL STAFF OCCUPATIONALLY EXPOSED TO LOW DOSES OF IONIZING RADIATION. DOES THE LOW DOSE STIMULATE DEFENSE STATUS?

Ahmet Aydin1, Ayse Eken2, Onur Erdem2, Cemal Akay2, Bugra Soykut2, Ahmet Sayal2 and Ibrahim Somuncu3

1Department of Toxicology, Yeditepe University, Faculty of Pharmacy, Istanbul, Turkey, 2Department of Toxicology, Gulhane Military Medical Academy, Ankara, Turkey, 3Department of Radiology, Gulhane Military Medical Academy, Ankara, Turkey

Radiation with higher energy levels called ionizing has enough energy to direct affect the structure of atoms of the materials. Ionizing radiation is known to induce oxidative stress through generation of reactive oxygen spe-cies (ROS) resulting in imbalance of the pro-oxidant and antioxidant activities ultimately resulting in cell death. Cellular DNA can be damaged by direct and indirect effect of ionizing radiation. The aim of this study was to

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evaluate the oxidative stress status and the genotoxic effects in occupational radiation exposure in radiology unit staff. Antioxidant enzyme activities and malondialdehyde (MDA) levels were measured as oxidative stress parameters. The cytokinesis-blocked micronucleus (CBMN) and sister chromatid exchanges (SCE) techniques were used as biomarkers of genetic damage. This study included 40 exposed individuals together with 30 from the same hospital without occupational exposure to radiation, which served as controls. The activities of eryth-rocyte copper-zinc superoxide dismutase (CuZn-SOD) (2038 ± 288 U/g Hb) and erythrocyte selenium dependent glutathione peroxidase (Se-GPx) (26.31 ± 5.20 U/g Hb) observed for the exposed groups were statistically very sig-nificantly higher than in the control group (1389 ± 368 U/g Hb and 20.61 ± 5.82 U/g Hb, respectively, p<0.001). The activities of erythrocyte catalase (CAT) (331.02 ± 59.72 KU/g Hb) and erythrocyte MDA levels (76.13 ± 21.83 nmol/g Hb) in the exposed groups were found statistically significantly lower than in the control group (370.02 ± 83.63 KU/g Hb and 114.87 ± 26.27 nmol/g Hb, respectively; p<0.05, p<0.001). It was found that the mean values of MN frequency observed for the exposed group were significantly (p<0.05) higher than in the control group (6.88 ± 2.54 and 5.50 ± 2.0 respectively). There was no significant change found in the frequencies of SCE in exposed group versus controls (p>0.05). In conclusion, it has been found a stimulant effect of chronic low-dose radiation in antioxidant protection of exposed individuals and an enhanced resistance to oxidative stress. However, it is concluded that it is necessary of following doses recorded by Thermoluminescence Dosimetry (TLD), taking regularly antioxidant supplementation for protective prevention by exposed individuals. Our findings showed that the increased frequencies of MN in radiology staff indicate the cumulative effect of low-level chronic exposure to ionizing radiation.

P396. The Uremic Solute Indoxyl Sulfate Has Both Pro- and Anti-oxidant Properties Depending on Its Physiological Concentration

Hiroshi Watanabe1, Yohei Miyamoto1, Yasunori Iwao2, Yuka Tasaki1, Keizo Sato3, Yu Ishima1, Daisuke Kadowaki1, Toru Maruyama1 and Masaki Otagiri1

1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan, 2School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan, 3School of Pharmacy, Kyushu University of Health and Welfare, Miyazaki, Japan

Indoxyl sulfate (IS), an uremic toxin, is derived from dietary protein including tryptophan and contains an indole ring. It is present at high levels in the serum of patients with chronic kidney disease (CKD) (the serum concentration of CKD patients is 100 μM∼1 mM versus 0.1∼10 μM in healthy subjects). Recent findings indicate that IS is likely involved in both the progression of CKD and the development of complications. In fact, IS causes tubulointerstitial injuries in rodent models and produces oxidative stress in various cell systems, such as renal proximal tubular cells, mesangial cells and endothelial cells. On the other hand, from a structural perspective, it is possible that endogenous indole derivatives have the potential to function as an antioxidant against various reactive oxygen spe-cies (ROS), attributed to the electron-attracting nitrogen atom (-NH) in the pyrrole ring. Therefore, we hypothesize that, although under high serum levels, IS functions as a pro-oxidant due to an enhancement in oxidative stress, it may also function as an anti-oxidant under normal-physiological conditions. In this study, the effect of IS in scavenging superoxide anion radicals (O

2•-) generated from both the xanthine/xanthine oxidase (X/XO) system

and activated neutrophils treated with LPS was investigated using electron paramagnetic resonance spectroscopy, combined with a 2-ethoxycarbonyl-2-methyl-3, 4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1∼10 μM) resulted in a decreased the formation of an EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived O

2•- generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals, suggesting

IS acts as a novel endogenous antioxidant under normal-physiological conditions. The findings were compared with the data for other antioxidant compounds, such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD, an O

2•- scavenger)

and allopurinol (a xanthine oxidase inhibitor), and were also compared with that for other uremic toxins. When the normal-physiological concentration of IS at 1 µM was added to human umbilical vein endothelial cells (HUVECs), intracellular ROS was significantly decreased. In contrast, IS at levels in CKD conditions such as 100 or 500 µM sig-nificantly enhanced the intercellular ROS. Theses results suggest that IS has both pro- and anti-oxidant properties, depending on its physiological concentration.

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P397. Affymetrix DMET™Plus Genotyping Technology: Evaluation of Cell Lines and Healthy Volunteer Samples

Tracy Mills1, Mark Pilling1, Glynis Nicholls2, Tommy B. Andersson3, Petter Björquist4, Anders Aspegren4 and Peter Sartiby4

1R&D Genetics, AstraZeneca, Macclesfield, United Kingdom, 2CPD, AstraZeneca, Macclesfield, United Kingdom, 3AstraZeneca R&D Mölndal, Clinical Pharmacology and DMPK, Mölndal, Sweden, 4Cellartis AB, Göteborg, Sweden

We have conducted a pilot study to assess the robustness of SNP genotyping by the Affymetrix DMET™plus platform in the hands of Beckman Coulter Genomics to develop data handling, statistical analysis and modelling methods and to characterise six pre-clinical cell lines & tissues and 72 samples from a healthy volunteer study. The Affymetrix DMET™ Plus Panel represents 1936 genetic variants (1931 SNPs or In-Dels and five copy number region markers) in 225 genes of importance to ADME. These include the 32 core genes of importance to the PharmADME consortium of academic & pharmaceutical researchers. One hundred and sixty-nine of these variants are located in genes that are currently classified as known valid biomarkers according to the FDA (http://www.fda.gov). The six human pre-clinical samples consisted of HepaRG, Caco2, two cryopreserved hepatocytes and two embryonic stem cell lines. A further six control samples representing blinded triplicates were included for QC purposes. Thorough QC has shown that the quality of the data generated was very high; successful genotype data (for over 98% of the SNPs on the panel) in 82 of the 84 samples tested was generated. Two samples (both cell-lines) fell below this threshold even after repeat analysis. In addition, 98% of SNPs analysed produced genotype calls for >95% of the 84 samples. Genotype concordance >99.9% was observed from two control samples triplicated and 100% genotype concordance was observed in 20 SNPs genotyped by Taqman. Hardy-Weinberg Equilibrium testing of the 72 healthy volunteer samples further suggested that there were no gross genotyping errors. Approximately 65% of the variants of the DMET™ Plus Panel are reported to have a minor allele frequency of less than 5% and this was reflected in our observation that 41.2% of the SNPs were polymorphic in the 72 Caucasian volunteer samples. Data will be presented describing the information obtained for these cell lines.

P398. Allopurinol –induced severe cutaneous adverse drug reactions in a Thai population is strongly associated with HLA-B*5801

Wongwiwat Tassaneeyakul1, Somsak Tiamkao2, Napat Prabmeechai3, Thawinee Jantarar-oungtong4, Pei Chen5, Pao-Yu Lin5, Pansu Chumworathayi6, Usanee Khunarkornsiri7, Parinya Konyoung7, Pachadaporn Chucherd8, Suda Vannaprasaht3, Charoen Choonhakarn3, Alisara Sang-viroon9 and Wichittra Tassaneeyakul3

1Department of Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand, 2Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 3Pharmacology, Khon Kaen University, Khon Kaen, Thailand, 4Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 5Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 6Pharmacy, Khon Kaen University, Khon Kaen, Thailand, 7Pharmacy, Udornthani Hospital, Udornthani, Thailand, 8Pharmacy, Surin Hospital, Surin, Thailand, 9Pharmacy, Police General Hospital, Bangkok, Thailand

Allopurinol, a uric acid lowering drug commonly used for hyperuricemia and gouty arthritis, has been reported as a common cause of severe cutaneous adverse drug reactions (SCADR) including Stevens–Johnson syndrome (SJS), Toxic epidermal necrolysis (TEN) and Hypersensitivity syndrome (HSS). A strong association between allopurinol-induced SCADR and HLA-B*5801 was observed in a Han Chinese population, but only a moderate association was observed in other populations (i.e. European and Japanese). The present study aimed to investigate the association between allopurinol-induced SCADR and HLA-B*5801 in a Thai population. Thirty-seven allopurinol-induced SCADR and 62 allopurinol-tolerant patients were enrolled in the study. The presence of HLA-B*5801 allele in these patients

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were analyzed using a PG5801 DNA detection kit. All of the 37 (100%) allopurinol-induced SCADR patients carried HLA-B*5801 while only 8 out of 62 (12.90%) of the control patients had this allele. The risk of allopurinol-induced SCADR was significantly higher in patients with HLA-B*5801 when compared with those that did not carry this allele. The sensitivity and specificity of the HLA-B*5801 allele for prediction of allopurinol-induced SCADR were very high. Therefore, the HLA-B*5801 is a good and useful pharmacogenetic marker for screening of allopurinol-induced SCADR in a Thai population.

P399. Alteration of DNA methylation as an early event for non-genotoxic carcinogens

Sibel Ozden1, Tao Chen2, Wolfgang Dekant3, J. Kevin Chipman4 and Angela Mally3

1Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey,2School of Biosciences, The University of Birmingham, Birmingham, United Kingdom, 3Department of Toxicology, University of Wuerzburg, Wuerzburg, Germany, 4School of Biosciences, The University of Birmingham, Birmingham, United Kingdom

Non-genotoxic carcinogens are a diverse group of chemicals, inducing tumors by mechanisms other than direct modifica-tion or damage to DNA. There is increasing evidence to suggest that altered expression of tumor suppressor genes and oncogenes, which in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogenesis. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and the male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG promoter DNA methylation in their respective target tissues of rats. No significant dose-related effects on global DNA hypomethylation in target tissues of rats compared to controls by using LC-MS/MS. While these data suggest that global DNA hypomethylation, which in tumors, may require sustained exposure, determination of 5-methylcytosine levels may not be sufficiently sensitive to detect epigenetic changes due to gene-specific methylation and subsequent silencing of tumor suppressor genes. Thus, CpG island methylation in promotor regions of key tumor-suppressor genes (connexin-32, E-cadherin, p16, VHL, Igfbp2 and p15) and oncogene (c-myc) was analysed by methylation specific PCR (MSP) following bisulfite DNA modification. For the investigated genes, only p16 gene methylated in the liver treated with HCB and TCDD by using MSP. Further studies and other methods might provide genotoxicmethylation.

P400.NAssociation of Estrogen Receptor α Gene Polymorphism with Bone Mineral Density and Body Mass Index in Turkish Postmenopausal Women

Ozlem Kurt1, Hulya Yilmaz Aydogan2, Turgay Isbir3, Mehmet Uyar4 and Ayse Can1

1Biochemistry, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey, 2Molecular Medicine, Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey, 3Medical Biology, Medical Faculty, Yeditepe University, Istanbul, Turkey, 4Phsycial Medicine and Rehabilitation, Uskudar State Hospital, Istanbul, Turkey

Estrogen receptor alpha (ERα) gene has been suggested as possibly involved in endocrine-related diseases such as osteoporosis and obesity. In the present study we investigated the relation of ERα PvuII and XbaI polymorphisms with the bone mineral density (BMD) and the body mass index (BMI) in postmenopausal women from Turkey through RFLP-PCR technique. BMD was measured at the lumbar spine and hip (femoral neck, trochanter and Ward′s triangle) by dual-energy X-ray absorptiometry (DXA). 53 healthy control, 112 osteopenic and 81 osteoporotic postmenopausal women were recruited. The BMD and BMI values were found to be lower in osteopenic and osteoporotic women as compared to healthy women (p<0.001). Distribution of ERα genotypes was similar in the three groups (p>0.05). ERα PvuII PP genotype was associated with low values of femoral neck (p<0.05 versus Pp/pp genotype and p allele) and total hip BMD (p<0.05 versus pp genotype and p allele) in osteopenic women. ERα XbaI X allele was associated with low values of BMD at the femoral neck (p<0.05 versus xx genotype), and Xx genotype and X allele were associated with low BMI values (p<0.05 versus xx genotype). ERα PvuII and XbaI genotypes have no significant effect on BMD and BMI in control and osteoporotic groups (p>0.05). Our data indicate that PvuII and XbaI polymorphisms of the ERα gene may be potential determinants of BMD and BMI in Turkish postmenopausal women.

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P401. Association of HLA-B*1502 and severe cutaneous adverse reactions caused by aromatic antie-pileptic drugs

Wichittra Tassaneeyakul1, Somsak Tiamkao2, Napat Prabmeechai1, Thawinee Jantararoungtong3, Pei Chen4, Shu-Yi Lin4, Wei-Hsuan Chen4, Pansu Chumworathayi5, Usanee Khunarkornsiri6, Par-inya Konyoung6, Alisara Sangviroon7, Patcharee Kanjanawat8, Arkhom Arayawhichanont9, Cha-roen Choonhakarn10 and Kongkiat Kulkantrakorn11

1Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 2Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 3Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 4Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 5Pharmacy, Srinagarind Hospital, Khon Kaen University, Khon Kaen, Thailand, 6Pharmacy, Udornthani Hospital, Udornthani, Thailand, 7Pharmacy, Police General Hospital, Bangkok, Thailand, 8Pharmacy, Sappasitthiprasong Hospital, Ubonratchatani, Thailand, 9Medicine, Sappasitthiprasong Hospital, Ubonratchatani, Thailand, 10Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 11Medicine, Faculty of Medicine, Thammasart University, Prathumthani, Thailand

Aromatic antiepileptic drugs such as carbamazepine (CBZ) and phenytoin (PHT) have been reported as common culprit drugs for severe cutaneous adverse drug reactions (SCADR) in several Asian countries including Thailand. These severe cutaneous adverse drug reactions include Stevens–Johnson syndrome (SJS), Toxic epidermal necrolysis (TEN) and Hypersensitivity syndrome (HSS). A strong association between HLA-B*1502 and CBZ-induced SJS/TEN has been reported in Han Chinese but not in Caucasian and Japanese populations. Moreover, it has been demonstrated in Thai patients that 6 out of 6 PHT-induced SJS/TEN patients carried HLA-B*1502. A case-control study was therefore conducted to determine whether HLA-B*1502 is a valid pharmacogenetic test for SCADR caused by CBZ and PHT in a Thai population. Among 49 CBZ-induced SJS/TEN patients, 45 (89.58%) patients carried the HLA-B*1502 while only 5 (10.42%) of the CBZ-tolerant controls had this allele. For PHT, only 5 out of 18 (27.8%) PHT-induced SCADR patients and 7 out of 36 (19.4%) PHT-tolerant control patients carried this allele. Contrast to CBZ-induced SJS/TEN, the risk of PHT-induced SCADR was not significantly associated with HLA-B*1502. Results from this study suggest that HLA-B*1502 is a good marker for CBZ-induced SJS/TEN but not for PHT-induced SCADR in a Thai population.

P402. Association of serotonin transporter gene (SLC6A4) polymorphisms with alcohol dependence in a Turkish population

Ozgur R. Karaca1, Melih O. Babaoglu1, Umit Yasar1, Yavuz Ayhan2, Can S. Gurel2, Nesrin Dilbaz3, Basaran Demir2, Berna Ulug2 and Atilla Bozkurt1

1Department of Pharmacology, Hacettepe University, Faculty of Medicine, Ankara, Turkey, 2Department of Psychiatry, Hacettepe University, Faculty of Medicine, Ankara, Turkey, 3The First Psychiatry Clinic, Ankara Numune Education and Research Hospital, Ankara, Turkey

The central serotonergic system contributes to rewarding mechanisms in development of alcohol addiction. An inser-tion/deletion polymorphism and a single nucleotide substitution (A/G, rs25531) in the promoter region of serotonin transporter gene (SLC6A4) have been shown to influence transcriptional activity. Previous studies associating these polymorphisms with alcoholism have been inconclusive. This study aimed to investigate whether these genetic poly-morphisms were associated with alcohol dependence in Turkish population. Alcoholic patients (n=186) and a healthy control group (n=127) were examined. After DNA extraction from peripheral blood lymphocytes, genotyping for long (L

A and L

G) and short (S) variants was performed using polymerase chain reaction and endonuclease digestion. The

subjects were divided into three genotype groups according to their estimated functional transcriptional activity as high (L

AL

A ), intermediate (SL

A or L

GL

A) and low (SS, SL

G or L

GL

G) activity groups. The genotypic distributions between

alcoholic and control groups were analyzed for statistical difference by using chi- square test. The frequencies of allelic variants were L

A (41.2%), L

G (5.1%) and S (53.7%) within the study group. The numbers and frequencies of subjects in

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high, intermediate and low activity genotype groups were 89 (47.8%), 31 (16.7%) and 66 (35.5%), respectively in alcoholic subjects and 63 (49.6%), 22 (17.3%) and 42 (33.1%), respectively in control subjects. The genotypic distributions were similar both in alcoholic and control groups (χ2 = 0.19, p= 0.90). In conclusion, we did not find an association between alcohol dependence and the insertion/deletion polymorphism or the A/G substitution (rs25531) in serotonin trans-porter gene. Our results are not in agreement with previous studies reporting an association between alcoholism and serotonin transporter gene polymorphisms.

This study was supported by a grant from Hacettepe University Research Fund (04.01.101.012) and Dr. Babaoglu is supported by Turkish Academy of Sciences, Young Scientist Achievement Award Program (TUBA-GEBIP/2007-06).

P403. Association of XPD Asp312Asn polymorphism with cisplatin-related toxicities and treatment outcome in malignant mesothelioma

Nina Erculj1, Vita Dolzan2 and Viljem Kovac3

1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia,2Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 3Institute of Oncology, University of Ljubljana, Ljubljana, Slovenia

Platinum-based drugs are among the most active and widely used agents in treatment of different cancers. The inter-individual variability in both outcome and toxicity of platinum-based chemotherapy may be a consequence of genetic variability in nucleotide excision repair (NER) enzymes, involved in repair of cisplatin-induced DNA adducts. The aim of our study was to evaluate the association of selected single nucleotide polymorphisms (SNPs) in NER pathway with the risk of cisplatin-related toxicities and treatment outcome in malignant mesothelioma patients. The study included 128 patients with malignant mesothelioma treated with (n=81) or without (n=47) cisplatin. All individuals were genotyped for XPD Asp312Asn, XPD Lys751Gln, ERCC1 C8092A, and ERCC1 Asn118Asn using a fluorescence-based competitive allele-specific PCR system (KASPar). All the clinical data, including treatment toxicity were available from patients′ files. Tumor response, overall (OS) and disease-free (DFS) survival were assessed to evaluate the treatment outcome. No associations of XPD Lys751Gln, ERCC1 C8092A, and ERCC1 Asn118Asn with treatment-related toxicities and treat-ment outcome were found. In cisplatin-treated patients, variant allele XPD 312Asn was significantly associated with decreased risk of neutropenia (OR=0.147, 95% CI=0.026-0.834, P=0.020) and alopecia (OR=0.057, 95% CI=0.009-0.354, P=0.003). We did not find any associations of XPD Asp312Asn with tumor response and OS. However, multivariate Cox regression analysis revealed significantly worse DFS probability in cisplatin-treated patients with XPD312 Asn/Asn genotype compared to patients with wild-type genotype (OR=3.200; 95% CI=1.341-7.640; P=0.009). In contrast, in patients who were not treated with cisplatin, polymorphism XPD Asp312Asn influenced neither toxicities nor treatment outcome. Our data suggests that XPD Asp312Asn polymorphism may influence the risk of toxicities as well as treatment outcome in malignant mesothelioma patients treated with cisplatin-based chemotherapy. However, larger studies are needed to confirm these associations.

P404. Comparison of functional CYP2A6 polymorphisms among three Asian populations

Yin-Jin Jang1, Woo-Young Kim2, Jae-Gook Shin3 and Sang Seop Lee2

1Pharmacology, Pharmacogenomics Research Center, Busan, South Korea, 2Pharmacogenomics Research Center, Busan, South Korea, 3Dept. of Pharmacology and Clinical Pharmacology, Pharmacogenomics Research Center, Inje University College of Medicine and Busan Paik Hospital, Busan, South Korea

The human cytochrome P450 2A6 (CYP2A6) enzyme metabolizes therapeutic drugs and appears to be the most impor-tant enzyme for nicotine metabolism. So the functional alteration of CYP2A6 activity may lead to altered drug responses such as nicotine addiction and clinical outcome of pharmacotherapy with anti-cancer drugs, antiepileptic drugs, etc. It is well known that the genetic polymorphism in CYP2A6 gene contributes to the functional variation and shows inter-individual and inter-ethnic difference in genotype-distribution. So far, more than eighty different allelic variants of CYP2A6 gene have been reported in CYP nomenclature committee. Previously, we sequenced CYP2A6 gene of 100 Korean subjects and found ten functional CYP2A6 alleles including CYP2A6*4, *7, *9A, *10, *11, *18A, *29,*30 and

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567C>T. Based on literature reviews, these alleles are also relevant in other Asian populations. In the present study, we screened those ten functional CYP2A6 alleles in 100 Korean, 98 Vietnamese and 100 Chinese subjects using multiplex minisequencing method and directly compared genotype distribution. All allelic variants were in Hardy-Weinburg equi-librium. CYP2A6*4 allele was most frequent in all of the three populations. The frequency of CYP2A6*4 in Korean (16%) was significantly higher than Chinese (7.5%) and Vietnamese (7.1%). CYP2A6*7 and *9 also showed ethnic-specific difference. Phenotype prediction based on known functionality of each genotype suggests that the frequency of poor metabolizers shows significant ethnic difference among three populations in order of Korean>Chinese>Vietnamese. Our results suggest that the variation of CYP2A6 genotype distribution among three Asian populations may contribute the difference of CYP2A6-related drug responses among these populations.

P405. Confounding Effects of C57BL/6 Substrains in Toxicology

Mohammed Bourdi1, John S. Davies1, Nalini Raghavachari2, Delong N. Liu3, Khalid Sendide1, Peter J. Munson3 and Lance R. Pohl1

1Molecular and Cellular Toxicology, Laboratory of Molecular Immunology, National Heart,Lung and Blood Institute, National Institutes of Health, DHHS, Bethesda, MD, USA, 2Genomics Core Facility, NHLBI, Bethesda, USA, 3Center for Information Technology, NIH

C57BL/6 mice are widely used in biomedical research for genetically engineered mice (GEM) and their background wild type (WT) controls. It is generally not known that substrains of C57BL/6 mice can differ genetically and phenotypically. We report here that this lack of information can lead to inaccurate findings as a result of improper pairing of GEM and WT C57BL/6 substrains. The Jackson Laboratory is the largest source of GEM on a C57BL/6 background and recom-mends in many cases for the C57BL/6J substrain to be used as a background control. This has led us to genotype 79 of these C57BL/6 GEM for the functional deletion of an important gene specific to the C57BL/6J substrain. Twenty GEM (∼25%) were found with the intact gene indicating that C57BL/6J is not the background. The mismatches of these mice with C57BL/6J controls may be responsible for conflicting findings in the literature. This possibility led us to determine whether C57BL/6 substrains differ in susceptibility to xenobiotic-induced liver injury. When a hepatotoxic dose of acetaminophen (APAP) was administered to substrains of C57BL/6 mice from four different vendors (Taconic Farms, Charles River, Harlan and The Jackson Laboratory), significant differences were found in their susceptibility to liver injury and survival. Further study revealed that C57BL/6J mice (The Jackson Laboratory), the least susceptible substrain tested, had lower mitochondrial APAP protein-adducts and glutathione levels than C57BL/6NTac mice (Taconic Farms), which was one of the most susceptible substrains to APAP-induced liver injury (AILI). Finally, GeneChip exon array analysis showed large changes in genes involved in cell death, inflammation, immune cell trafficking, cell proliferation and lipid metabolism in livers of C57BL/6NTac mice when compared to C57BL/6J mice. C57BL/6 substrains also differ in susceptibility to Concanavalin A-induced liver injury. These findings stresses the importance of carefully choosing the appropriate C57BL/6 mice substrain for control when using GEM on a C57BL/6 background, not only for toxicological research, but also for other biomedical studies.

P406. CYP2D6 Genotype and Phenotype Determination in the Hausa, Ibo and Yoruba Ethnic Populations of Nigeria

Benjamin U. Ebeshi1, Oluseye O. Bolaji2, Alice Matimba3 and Collen M. Masimirembwa4

1Pharmaceutical & Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island, Nigeria, 2Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria, 3Division of Human Genetics, University of Cape Town, South Africa, 4African Institute of Biomedical Science & Technology, Harare, Zimbabwe

In spite of the importance of the cytochrome P450 2D6 (CYP2D6) genetic polymorphism in understanding the interindi-vidual and interethnic variability in drug response of about 20 to 25% of clinically useful drugs [1-2], very few pharmacoge-netic data have been generated in African populations, including Nigerians. It is for this reason that we set out to determine CYP2D6 genotype and phenotype in the three major ethnic populations of Nigeria, which comprise of the Hausa, Ibo and

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Yoruba in a bid to ensuring rational use of drugs. Genotyping of five CYP2D6 mutant alleles by polymerase chain reaction (PCR) and sequencing techniques was performed in 298 Nigerians comprising of (Hausa=97), (Ibo=101) and (Yoruba=101) [3, 4]. Eighty six subjects were also phenotyped using dextromethorphan as the probe drug [5]. The frequency of the defec-tive CYP2D6*4 allele, which in homozygous state or in combination with other non-functional alleles of CYP2D6 predicts poor metabolism was found at 2% in Hausa, 8% in Ibo, and 3% in Yoruba subjects. The most commonly found alleles in these populations are the CYP2D6*17 and CYP2D6*29, which accounts for reduce enzyme activity. The frequency of CYP2D6*17 was not significantly different in the three population (P>0.05), which occurs at 18%, 14% and 22% in the Hausa, Ibo and Yoruba respectively. Also, the frequency of CYP2D6*29 was found to be 10%, 20% and 10% in the Hausa, Ibo and Yoruba respectively. CYP2D6*3 and *9 were absent in these populations, while CYP2D6*5 was inconclusive. Among the 86 phenotyped subjects, 3 (3.5%) were identified as poor metabolizers (PM), 68 (79%) were extensive metabolizers (EM) and 15 (17.4%) were intermediate metabolizers (IM). The mean and median log10 dextromethorphan/dextrophan metabolic ratios (MR) were 0.72 and 0.79 (SD=0.26) for PMs, -1.81 and -1.83 (SD=0.4) for EMs, and -0.51 and -0.62 (SD=0.37) for IMs respectively. The mean PM frequency of CYP2D6 in the population studied was 4%, which follows a similar trend with other African populations. These data show a trend toward a larger mean log MR (reduced enzyme activity) as the number of active alleles decreases.

References

1. Ingelman–Sundberg M. Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity. Pharmacogenomics J 5: 6–13, 2005.

2. Gaedigk, A., Coetsee, C. The CYP2D6 gene locus in South African Coloureds: unique allele distributions, novel alleles and gene arrangements. Eur J Clin Pharmacol 64(5): 465-475, 2008.

3. Masimirembwa, C., Persson, I., Bertilsson, L., Hasler, J., Ingelman–Sundberg, M. A. A novel mutant variant of the CYP2D6 gene (CYP2D6*17) common in a black African population: association with diminished debrisoquine hydroxylase activity. Br J Clin Pharmacol 42: 713–719, 1996.

4. Wennerholm, A., Johansson, I., Hidestrand, M., Bertilsson, L., Gustafsson, L. L., Ingelman-Sundberg, M. Characterization of the CYP2D6*29 allele commonly present in a black Tanzanian population causing reduced catalytic activity. Pharmacogenetics, 11: 417-427, 2001.

5. Minoo, A., Mohammad-Reza, R., Mohsen, A. Simple chromatography method for simultaneous determination of dextromethorphan and its main metabolites in human plasma with fluorimetric detection. Journal of Chromatography B 802: 317–322, 2004.

P407. Development of Real-Time PCR VKORC1 and CYP2C9 Genotyping Method for Dose Adjustment of Anticoagulant Warfarin

Woochang Lee1, Hee-Jung Chung1, Sollip Kim1, Eun Ju Cho1, Hye Won Lee1, Hae-Joon Park2, Sail Chun1 and Won-Ki Min1

1Laboratory Medicine, Asan Medical Center, Seoul, South Korea, 2Bioneer Corporation, Daejeon, South Korea

Background: Genetic polymorphisms in VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are the most important genetic factors related to dose adjustment of oral anticoagulant warfarin. U.S. FDA and NACB recommend those genetic testing along with the administration of warfarin. Fast and reliable test method is essential for prompt dose adjustment of warfarin to prevent serious hemorrhagic or thrombotic complications. Authors developed a real-time PCR-based method for VKORC1 and CYP2C9 genotyping.

Methods: Single nucleotide polymorphism (SNP) genotyping methods were developed under the real-time PCR platform using allele specific primers and 5′ nuclease probes. Four well-known SNPs for VKORC1 gene (g.3673G>A, g.6484C>T, g.6853G>C, and g.9041G>A) and two SNPs for CYP2C9 gene (c.430C>T and c.1075A>C) were genotyped based on the differences in Ct (cycle threshold) in the real-time PCR system. Each reaction was conformed by PCR-direct sequenc-ing method. Also multiplexing reaction was applied to reduce the number of wells and internal positive control (IPC) reaction was added to all the wells to guarantee successful reaction in each well.

Results: The validity of amplification reactions was assessed based on the difference in Ct (DCt) for the reactions tar-geting two different alleles at each SNP site. For each SNP site, homozygote specimens showed DCt greater than 3.0 and heterozygote specimens showed DCt smaller than 1.0. Also no interference was observed in the multiplexed PCR environment and all the IPC reactions were successfully completed. All these results were concordant with those con-firmed by PCR-direct sequencing method. Overall turnaround time was around 2 hours and 12 genomic DNA specimens could be analyzed simultaneously.

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Conclusion: We developed a genotyping kit for warfarin dosing using real-time PCR based allele-specific primers and probes, which has rapid turnaround time and consistent results with those by direct sequencing method. This method is expected to be practically applied in the clinical practice for the warfarin dose adjustment.

P408. EFFECT OF SILDENAFIL CITRATE (VIAGRA®) ON TRACE ELEMENT CONCENTRATION IN SERUM AND BRAIN OF RATS

Abdel-Hasseb A. Fayed and Shreen B. Gad

Department of Physiology, Faculty of Veterinary Medicine, Alexandria University, Egypt

As a vasodilator with good hemodynamic effects, sildenafil has been successfully used in the treatment of patients with pulmonary hypertension and cardiovascular diseases. By selectively inhibiting phosphodiestrase type 5 (PDE-5) and thus effectively reducing the breakdown of c GMP, sildinafil administration can markedly improve the erectile dysfunc-tion. Sildinafil also elevates localized cerebral blood flow in rat brain. The objective of the present study was to investigate the effect of sildenafil on the level of trace elements concentration (Zinc (Zn), copper (Cu), iron (Fe), selenium (Se), cobalt (Co), and chromium (Cr)) in blood and brain of rats. Sixteen male albino rats weighing 220-250 g were divided into two groups (8 rats/group). Sildenafil (Viagra, Pfizer Inc.) was dissolved in saline and administered at a dose of 10 mg/Kg i.p. (0.5 ml volume) to rats in the treated group every 72 hours for 12 injections. Rats in the control group were administered the same volume of saline as in treated group. All rats were sacrificed 24 hours after the last injection. Blood samples were collected and serum was separated and stored at frozen. Brains were dissected and stored frozen until analysis. Trace elements concentration were determined by flame emission atomic absorption spectrophotometer. Results showed that sildenafil injection significantly (P<0.05) increased serum Se concentration ( 4.97 vs 4.28 mg/L in the control) and Cu ( 0.34 vs 0.24 mg/L in the control). The brain Se, Cu, and Cr concentration significantly increased ( 3.14, 0.52, and 0.45 mg/Kg vs 2.55, 0.37, and 0.24 mg/Kg, in the control, respectively).It was concluded that sildenafil induced alterations in trace elements concentration in the blood and brain of rats which could be considered during treatment with this drug.

P409. Effects of Polymorphic ABCG2 and SLC2A9 on the serum Uric Acid Levels in Korean Healthy Subjects

Kyoung-Ah Kim1, Hyun-Jin Joo2 and Ji-Young Park1

1Dept. of Clinical Pharmacology & Toxicology, Anam Hospital, Korea University College of Medicine, Seoul, South Korea, 2Anam Hospital, Korea University College of Medicine, Seoul, South Korea

Serum uric acid (UA) is a highly variable trait among humans. Recently, it has been revealed that in addition to SLC2A9 (glucose transporter 9 [GLUT9]) ABCG2 (breast cancer resistance protein [BCRP]) transporter involves in the modulation serum UA levels. In this study, we assessed whether genetic polymorphisms of ABCG2 and SLC2A9 genes affect the serum UA levels in humans. We enrolled 250 healthy Korean subjects. We assessed their serum UA levels and analyzed genetic polymorphisms of ABCG2 (V12M, Q141K, and Q126X), and and SLC2A9 (V253I and R265H) using pyrosequenc-ing methods. The allele frequencies of ABCG2 V12M, Q141K, and Q126K were 0.196, 0.284, 0.012, respectively, and those of SLC2A9 V253I and R265H were 0.012 and 0.310. When we assessed the effect of these genetic polymorphisms on the serum UA levels, ABCG2 Q141K and R265H affected serum UA levels. Subjects with either polymorphic ABCG2 Q141K or SLC2A9 R265H exhibited significantly higher serum UA levels. In conclusion, both genetic polymorphisms of ABCG2 and SLC2A9 individually influence the serum UA levels in humans.

P410. EPHX1 GENE TYR113HIS VARIANT ALONE, or in COMBINATION with XRCC1 ARG399GLN POLYMORPHISM SIGNIFICANTLY and CONSIDERABLY INCREASED THE RISK of CHILDHOOD ACUTE LYMPHOBLASTIC LUKEMIA

Tugba Boyunegmez Tumer1, Duygu Yilmaz2, Cihan Tanrikut2, Gulen Ulusoy2 and Emel Arinç2

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1Vocational School of Health Services, Çanakkale Onsekiz Mart University, Çanakkale, Turkey, 2Biochemistry Graduate Programme and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

Today it is well known that the interindividual variation in the activity of xenobiotic metabolizing enzymes and DNA repair genes could modify an individual′s risk for several types of cancer. The observed effect for the risk of cancer devel-opment is more prominent when more than one polymorphism, especially those affecting xenobiotic metabolizing and as well DNA repair capacity are combined. We recently showed that DNA repair gene XRCC1 Arg399Gln polymorphism alone significantly increased the risk of childhood ALL 2.0-fold (OR: 2.0; p=0.04). Furthermore, its combination with CYP2E1 polymorphisms has stronger effect and increased the risk of childhood ALL 3.7 fold (OR: 3.7; p=0.049) (Tumer et al., 2010). In the current study, we investigated the possible association of microsomal epoxide hydrolase (EPHX1) exon 3 and exon 4 variants with the risk of incidence of childhood ALL in Turkish population comprised of 190 healthy controls and 167 ALL patients. EPHX1 is a phase I drug metabolizing enzyme which plays major role in the detoxification of highly reactive, pre-carcinogen epoxide intermediates. Exon 3 variant of EPHX1 gene is characterized by the substitu-tion of Tyr113His of the encoded protein, which is associated with the 50% decrease in the enzyme activity, whereas EPHX1 exon 4 variant comprises the replacement of His139Arg, enhancing the enzyme activity by 25%. In this study, for exon 3 polymorphism, His/His genotypes were significantly more common in ALL patients (31%) than controls (21%) and represent a considerably high risk factor for the childhood ALL (OR: 2.3, p=0.01). In case of exon 4 polymorphism, no significant associations have been found with the risk of childhood ALL. When the genotype distributions of both variants were combined and categorized according to predicted enzyme activities, it has been found that “the very slow” predicted enzyme activities significantly increased the risk of childhood ALL to 2.4 fold (p= 0.009). This result was in accordance with the single locus analyses and emphasizes the importance of exon 3 variant of EPHX1 for the risk of childhood ALL. In the scope of current work, we have also performed haplotype analysis for EPHX1 exon3 and exon 4 polymorphisms together with XRCC1 Arg399Gln variant. Accordingly, the co-presence of exon 3 polymorphism of EPHX1 and Arg399Gln variant of XRCC1 in the same individuals (either homozygous or heterozygous forms for both variants) significantly increased the risk of childhood ALL up to 2.1 fold (OR=2.1, p= 0.03). The more prominent effect has been observed when homozygous mutant genotype has been considered for both gene which significantly and considerably increased the risk of childhood ALL 8.9 fold (OR: 8.9, p= 0.02). Available literature suggest that this is the first study correlating exon 3 variant of EPHX1 either alone, or in combination with XRCC1 Arg399Gln polymorphism with the risk of development of childhood ALL.

References

Tumer TB, et al., DNA repair XRCC1 Arg399Gln polymorphism alone, and in combination with CYP2E1 polymorphisms significantly contribute to the risk of development of childhood acute lymphoblastic leukemia. Leuk Res. 2010, doi:10.1016/j.leukres. 2010.02.035.

This study was carried out in METU.

P411. Frequencies of three CYP2D6 nonfunctional alleles (CYP2D6*3, *4, and *6), and the metabolic capacity of CYP2D6 in Northern Iranian Population (Mazandaran)

Mohammad R. Shiran1, Mohammad B. Hashemi Soteh2, Fatemeh Merat3, Fatemeh Sarzare3 and Ebrahim Salehifar3

1Department of Physiology and Pharmacology,, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, 2Genetics, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, 3Pharmacotherapy, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran

Aim: To determine the frequency of three CYP2D6 nonfunctional alleles (CYP2D6*3, *4, and *6) and the metabolic capacity of CYP2D6 in Mazandarani ethnic group among Iranian Population. Methods: Blood samples from 100 subjects were genotyped by a polymerase chain reaction-based method. 71 subjects were also phenotyped using dextrometh-orphan as probe drug. Results: The frequencies for CYP2D6 alleles *3, *4, and *6 were 0.5%, 9%, and 0.5%, respectively. Homozygous or compound heterozygous genotypes that predict poor metabolizer phenotype i.e. *4/*4 or *4/*6 were not found in this study. A 560-fold interindividual variation in dextromethorphan MRs was observed in this study.

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Considering the antimode of 2 (0.3 in log scale), 7.04% (5/71) volunteers were identified as PMs. Conclusions: In con-clusion, the result of present study showed that the frequencies of CYP2D6 PM Phenotypes in Mazandarani ethnic group (7.04%) were comparable with those found in Caucasians (3–10%) and Turkish (6.7-9.4 %). The three inactive alleles, CYP2D6*3, CYP2D6*4, CYP2D6*6, accounted for 10% of CYP2D6 alleles in our sample versus 16-18% reported in European populations. Despite these finding, we propose further processing of a broad spectrum of CYP2D6 alleles to provide a wider image and to get more valuable information upon pharmacogenetic basis for individual therapy and personalized medicine.

P412. Genetic Polymorphism of Metabolizing Enzymes: Influence on DNA Damage, Paraoxonase and AChE Activity in Pesticide Exposed Subjects

Satyender Singh1, Vivek Kumar2, Grover S S1, Rawat D S1, Pasha S T1, Jain S K3 and B. D. Banerjee2

1Biochemistry & Biotechnology, National Centre for Disease Control, Delhi, India, 2Department of Biochemistry, UCMS & GTB Hospital, Delhi, India, 3Centre for Epidemiology and Parasitic Disease, National Centre for Disease Control, Delhi, IndiaPesticides pose a clearly identifiable risk to those who are occupationally exposed to this carcinogen causing acute to chronic health impacts including various neoplastic diseases and congenital malformations. Studies have indicated that pesticides are metabolized by various phase I and phase II metabolizing enzymes which provide critical defence against carcinogen. Their polymorphism has been found to be associated with various forms of cancer. Therefore, it is important to identify the potential genetic susceptibility factors affecting individual responses to pesticides exposure. Hence, the present study was designed to evaluate genetic polymorphism in CYP1A1 (m1, m2 and m4), CYP3A5 (A44G), CYP2C9 (Arg144Cys and Ile359Leu), CYP2D6 (*3 and *4) and PON1 (Gln192Arg and Leu55Met), GSTM1, GSTT1, GSTP1 and NAT2 and its correlation with DNA damage, AChE and paraoxonase activity in pesticides exposed workers. Using the comet assay and PCR-RFLP, the extent of DNA damage and genotype frequencies were evaluated in the lymphocytes of 115 pesticides exposed workers and equal number of age, ethnicity and gender matched control subjects. Exposed workers showed significantly higher levels of DNA damage compared to controls as measured through various comet parameters. The AChE and Paraoxonase activity was also found suppressed. The prevalence of m1, m2, m4 CYP1A1, A44G CYP3A5, CYP2C9, CYP2D6, PON1, GSTM1, GSTT1, GSTP1 and NAT2 genotype frequencies did not differ significantly in pesticide exposed and control subjects. CYP2C9 (Cys144Cys), PON1 (Arg-Gln, Gln-Gln, and Met-Met) and GSTP1 (Ile-Ile) individuals had higher comet assay parameters as compared to other forms of these enzymes in pesticide exposed subjects. Influence on paraoxonase activity was also observed due to genetic polymorphism of PON1. The prevalence of slow acetylation was found significantly higher in smokers as compared to non-smokers in pesticide exposed subjects which was not seen in control subjects who were smokers. In conclusion, the genetic polymorphism in CYP1A1, CYP3A5, CYP2D6, GSTM1 and GSTT1 has no influence on DNA damage while that of CYP2C9, PON1, and GSTP1 increases the risk of DNA damage. This study is helpful towards identifying individual markers of susceptibility and toxicity toward pesticides.

P413. HLA-B*1502 Allele in Chinese mainland patients with carbamazepine induced Stevens-Johnson syndrome and toxic epidermal necrolysis

Yan Zhang, Wei Peng and L.Y. Miao

Clinical Pharmacology Research Lab, The First Affiliated Hospital of Soochow University, Soochow, China

Purpose: Previous studies found a strong association between HLA-B*1502 and carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis (CBZ-induced SJS/TEN) in Han Chinese and in a Thai population, but not in Caucasian populations or in Japanese. This study is to clarify the involvement of HLA-B*1502 allele with CBZ-induced SJS/TEN in the Chinese mainland population.

Methods: HLA-B*1502 genotyping was performed on 14 CBZ-induced SJS/TEN patients, matched with 21 CBZ-tolerant controls, and with 185 healthy controls during past years from 2008 to 2009.

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Results: 71.4 % (10/14) HLA-B*1502 allele was presented in CBZ-SJS/TEN patients, but 9.5% (2/21) in CBZ-tolerant patients and 9.2% (17/185) in healthy controls. In addition, 2 CBZ-SJS/TEN patients who did not have the HLA-B*1502 allele had HLA-B*1521 allele, which was not defected in CBZ-Tolerant and healthy controls.

Conclusions: We also found a significant association between HLA-B*1502 and CBZ-induced SJS/TEN in Chinese mainland population. Furthermore, HLA-B*1521 allele might also have some association with SJS/TEN.

P414. Implication of genetic polymorphisms in CYP2C9 and CYP2C19 on drug metabolism in iso-lated cryopreserved human hepatocytes

Roberto Tolondo1, Caitlin Brown2 and Scott Heyward2

1Sales and Marketing, Celsis In Vitro Technologies, Baltimore, MD, USA, 2Research and Development, Celsis In Vitro Technologies, Baltimore, MD, USA

The cytochrome P450 enzymes 2C9 and 2C19 in the IIC cluster on chromosome 10 are responsible for the metabolism of a number of clinically relevant drugs. Single Nucleotide Polymorphisms (SNPs) have been delineated in both genes which affect the enzyme activity or expression. The importance of certain polymorphisms is highlighted by the FDA recommendation for utilizing warfarin based on CYP2C9 genotype information and clopidogrel labeling based on the genotype of CYP2C19. Cryopreserved human hepatocytes have gained importance as a model for in vitro testing of drug metabolism and interactions in the modern drug discovery process. The ability of researchers to have access to reagents which are representative of the population at large is of critical importance. Here we explore the population representation and the correlation to activity of SNPs in 2C9 and 2C19 in our cryopreserved hepatocyte repository. Genomic DNA was isolated from cryopreserved human hepatoctye samples taken from 91 individual donors. Genotypes were generated utilizing Taqman PCR SNP assays for 2C9*2, *3 and 2C19*2, *3, *4, *5, *17. A metabolism assay was performed with tolbutamide and mephenytoin for these individuals generating a clearance rate based on formation of 4′-methylhydroxytolubamide and 4′-hydroxymephenytoin respectively. The activity data, genotype, and race data were compiled and analyzed for basic statistical components and significance. Polymorphism frequency for 2C9*2, *3 and 2C19*2, *3, *4, *5, *17, closely match the frequency data from the HapMap survey. Sample number did not allow for analysis of the distribution by race. The mean rate of tolbutamide metabolism for CYP2C9*1/*2, *1/*3 and *2/*3, was significantly lower than that of *1/*1. 2C19*3, *4 and *5 were not present in our sample which correlates with their low population frequency. 2C19 *1/*2 heterozygous, wild type *1/*1 and heterozygous 2C19 *1/*17 showed a rank order relationship in mean rate of metabolite formation with mephenytoin corresponding with their Poor Metabolizer, Extensive Metabolizer, and Ultra Rapid Metabolizer phenotypes. The differences between these groups were not statisti-cally significant and there was overlap in the range of values. These results confirm the utility of isolated cryopreserved human hepatocytes in in vitro drug development studies. The presence and proper phenotype of polymorphic alleles in isolated hepatocyte repositories allows for researchers to perform studies which require donors with specific genetic backgrounds earlier in development.

P415. In Vivo and In Vitro Pharmacokinetic Characterization of Calycosin

Jianqing Ruan1, Yitao Wang2 and Ru Yan3

1Institute of Chinese Medical Sciences, University of Macau, Macau, China, 2Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China, 3Institute of Chinese Medical Sciences, University of Macau, Macau SAR, Macau

Background Calycosin is one of the main bioactive and characteristic constituents in Radix Astragali (Chinese name Huangqi), a Chinese medicinal herb noted for its immune promotion function. The beneficial actions of calycosin have been evidenced by in vivo and in vitro studies. However, the in vivo fates and hence the bioactive forms of calycosin remain unknown. The present study characterized, for the first time, the in vivo forms of calycosin in the rat plasma and delineated its absorption and metabolic properties on in vitro mod-els. Methods Calycosin (50 mg/kg) was orally administrated to male Sprague-Dawley rats and blood samples

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collected at appropriate time intervals. The intestinal permeability of calycosin was measured on a Caco-2 cell model. Hepatic metabolic stability of calycosin was examined in pooled human liver microsomes (HLMs) or S9 (HLS) fractions. Reaction phenotyping was conducted in HLMs and 12 recombinant UGTs. Structural identification was performed using HPLC-MS/MS and quantitative analysis completed using HPLC-UV. The major metabolite G2 was biosynthesized and its structure unambiguously identified by HPLC-MS/MS and NMR spectrometry. Results After oral administration, calycosin was absorbed rapidly then eliminated quickly from the rat plasma. Two metabolites of calycosin, 1 mono-sulfated and 1 mono-glucuronidated, were detected in the plasma. The mono-glucuronidated metabolite was the main drug-related component in the plasma. Bidirectional transport study on Caco-2 monolayer revealed a high permeability of calycosin (Papp values ∼3 × 10 −5 cm/s in both absorptive and secretory directions). Calycosin underwent glucuronidation, sulfa-tion and oxidation in human hepatic subcellular fractions with the average elimination rate of calycosin via glucuronidation >100 times those via other two pathways. Totally six metabolites, 2 mono-oxidated (O1, O2), 2 mono-glucuronidated (G1, G2) and 2 mono-sulfated (S1, S2) of calycosin, were tentatively identified. G2 was generated from a large-scale incubation, purified and unambiguously identified as calycosin-7-O-glucuronide. G2 was proved to be the glucuronidated metabolite observed in rat plasma. Kinetic studies showed that G2 formation by HLMs followed substrate inhibition kinetics (Km 13.58 uM; Vmax 5.65 nmol/min/mg protein). The recombinant human UGT1A9 exhibited the highest affinity to G2 (Km 4.39 uM; Vmax 9.11 nmol/min/mg protein). UGT1A7, 1A8, 1A1 and 1A3 also contributed but to a less extent. Propofol, the selective inhibitor of UGT1A9 could abolish G2 formation in a dose-dependent manner. Conclusions When taken orally, calycosin is absorbed rapidly, then undergoes extensive first-pass hepatic metabolism. Its glucuronidated metabolite formed in the liver is the main drug-related component in rat plasma. UGT 1A9 was the major isoform catalyz-ing calycosin glucuronidation in HLMs.

P416. Investigation of association between paraoxonase-1 192/55 polymorphisms and serum paraoxonase activity in lung cancer

Pinar Aksoy1, Bedia Agachan2, Turgay Isbir3, Ahmet Kizir4, Erkan Topuz4 and Hakan Berkkan5

1Biochemistry, Faculty of Pharmacy, İstanbul University, Istanbul, Turkey, 2Molecular Medicine, Istanbul University, Istanbul,3Medical Biology, Medical Faculty, Yeditepe University, Istanbul, Turkey, 4Institute of Oncology, Istanbul University, Istanbul, Turkey, 5Istanbul Bilim University, Istanbul, Turkey

Paraoxonase 1 (PON1) is a serum enzyme closely associated with high density lipoprotein (HDL). Studies suggest that PON1 is a primary determinant of the antioxidant and anti-inflammatory capacities of HDLs. Serum PON1 eliminates carcinogenic lipid-soluble radicals. Therefore, varies on PON1 gene might be associated with increased risks of cancer. In our study, we investigated the association between PON1 192/55 polymorphisms and lung cancer and, the effect of these polymorphisms on serum PON1 activity in Turkish population. We conducted a study on 223 lung cancer patients and 234 controls. We showed that PON1 192 and 55 genotypes had a major effect on serum PON1 activity. This association was evident only among controls. Among cases, PON1 192 and 55 genotypes were found to be no association with serum paraoxonase activity. We found that frequency PON1 192 R allele was significantly different in cases (58.3%) as compare to controls (48.7%). This difference was also observed among nonsmokers (p<0.05). However, distribution of PON1 55 genotypes were similar in cases and controls. Compare to smokers cases, MM genotype frequency was found to be higher among nonsmokers cases but not statistically significant (p>0.05). In addition, frequency of PON192 R allele was found to be higher in light smoker cases (<50pack/year) (64.5%) than that of the light smoker controls (48.3%) and same association was also observed in heavy smoker controls (>50 pack/year) (76.5%) as compare to light smoker controls (48.3%) (p<0.05). Frequency L allele in late stage (98.2) was significantly different as compare to early stage (86.5%) and frequency R allele was in small cell and epidermoid lung cancer found to be higher in comparison to controls. Among individuals who carrying LL genotype, R allele in cases in comparison to controls was found to be higher and also statistically significant (p<0.001). The results suggest that the PON1 192 R allele may play an important role as a risk factor for lung cancer in Turkish population and R and L alleles may be associated with malign prognosis. The interaction between PON1 192 polymorphism and smoking status was statistically significant.

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P417. Pharmacogenomic factors of tamoxifen efficacy in women with early breast cancer

Ondrej Slanar1, Sona Argalácsová2, L. Petruželka3, B. Konopásek3, M. Drazdakova4, A. Cerna3 and T. Zima4

1Institute of Pharmacology, 1st Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Prague, Czech Republic, 2Department of Oncology, 1st Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Prague, Czech Republic, 3Department of Oncology, 1st Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Prague, Czech Republic, 4Department of Clinical Biochemistry and Diagnostics, 1st Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Prague, Czech Republic

Introduction Tamoxifen (TMX) is the first line of adjuvant hormonal therapy in premenopausal patients (pts) and had been the first choice of therapy in hormonal-dependent postmenopausal women with breast cancer till lately. Metabolism of TMX – its conversion into active endoxifen is mainly dependent on CYP2D6. The process of TMX utilisa-tion could be affected by external factor - e.g. concomitant medication with inhibitors of CYP 2D6 or internal factors – polymorphisms of genes for CYP2D6 or MDR-1.

Methods: premenopausal and postamenopausal women with hormonal - dependent early breast cancer with TMX in the first line of adjuvant hormonal therapy were treated in the Department of Oncology of the General Teaching Hospital in Prague. Genotype of CYP2D6 was established by AmpliChip (Roche) and MDR-1 polymor-phisms C3435T and G2677T/A were detected by PCR- RFLP. The co-medication with CYP2D6 inhibitors was also noted.

Results: In the first phase of our study 81 postmenopausal pts (median age 62,4 yrs) and 26 premenopausal pts (median age 46 yrs) were examined. Median tamoxifen therapy duration was in UrM, EM, IM and PM genotype groups 34.5, 33.6, 36.2, and 30.2 months (m.), respectively. MDR1 polymorphism had no significant effect on duration of TMX therapy, while in premenopausal women with variant hymozygotes of C3435T and G2677T/A had prolonged treatment duration (54.3 m. vs. 28.4 m.). Comedication with inhibitors CYP2D6 decreased treatment duration from 39.3 m. to18.4 m. The relaps on TMX therapy was seen in 14 postmenopausal pts with DFS 30.7 m. and in 7 premenopausal pts with DFS 46.8 m. Significantly longer DFS was seen in homozygous carriers of 3435TT allele (39.6 m. vs. 16.6 m.) in wild-type homozygotes and heterozygotes.

Conclusion: Our preliminary results tentatively support hypothesis, that the genetic variants of CYP2D6 and MDR-1 and the concomitant medication influenced the effecacy of TMX therapy. We suggest that these factors could help clinicians in determining the therapeutic strategy.

P418. Protective Role of DNA Repair XRCC3 Thr241Met Polymorphism on Development of Childhood Acute Lymphoblastic Leukemia

Cihan Tanrikut1, Tugba Boyunegmez Tumer2 and Emel Arinç3

1Biochemistry Graduate Programme and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey, 2Vocational School of Health Services, Canakkale Onsekiz Mart University, Canakkale, Turkey, 3Graduate Programme in Biochemistry and Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

For transmission of genetic information with high fidelity, cells have evolved DNA repair mechanisms which also play vital role for genome stability. However, recent epidemiological studies well have shown that genetic polymor-phisms impairing the DNA repair capacity can disrupt the genomic integrity and potentially modulate individual′s susceptibility to various cancers. A recent publication from our laboratory indicated that mutant allele of X-ray repair cross complementing group 1 (XRCC1) gene which is involved in base excision repair mechanism, significantly increased the risk of development of childhood acute lymphoblastic leukemia (ALL) (Tumer et al., 2010). In this study, we investigated the role of two DNA repair genes, X-ray repair cross complementing group 3 (XRCC3) Thr241Met

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polymorphism and Rad51 G135C variant in the risk of development of childhood ALL in Turkish population among 193 healthy controls and 184 ALL patients, by using PCR-RFLP technique. The Rad51 and XRCC3 genes are involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Although XRCC3 and Rad51 have been studied extensively in relation to risk of development of various cancer types, there is no study about the effect of these two polymorphisms on the risk of development of childhood ALL. In the current study, for XRCC3 Thr241Met polymorphism, the frequencies of both heterozygous and homozygous mutant genotypes were found to be higher in the controls compared to ALL patients (OR: 0.59, p = 0.02; OR: 0.48, p = 0.02, respectively). In addition, either het-erozygous (Thr/Met) or homozygous mutant (Met/Met) genotypes were significantly more common in the controls than the ALL patients (OR: 0.55, p =0.005). In case of Rad51 G135C polymorphism, no significant associations have been found with the risk of childhood ALL. Combination of XRCC3 heterozygote genotype and Rad51 heterozygote genotype increased the protective effect for risk of childhood ALL. (OR=0.35; p =0.02). Moreover, combination of homozygote mutant genotype of XRCC3 with homozygote wild type genotype of Rad51 gave a highly statistically proved protective effect for the development of disease. (OR= 0.36; p= 0.004). To our knowledge, this is the first study showing the protective role of XRCC3 Thr241Met polymorphism either alone or in combination with Rad51 G135C variant on the risk of development of childhood ALL.

References

1. Tumer TB, et al. DNA repair XRCC1 Arg399Gln polymorphism alone, and in combination with CYP2E1 polymorphisms significantly contribute to the risk of development of childhood acute lymphoblastic leukemia. Leuk Res (2010), doi:10.1016/j.leukres.2010.02.035.

This study was carried out in METU.

P419. The Alteration of the Genetic Variants of CYP2A13 on Courmarin 7-hydroxylation and Frequency Distribution of the Genetic Variants of CYP2A13 in a Chinese Han Population

Ting Liu1, Jun-Yan Hong2, Su Zeng3 and Shuqing Chen1

1Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences Zhejiang University, Hangzhou, China, 2Environmental and Occupational Health, School of Public Health/EOHSI, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, American Samoa, 3Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

A study indicated that coumarin acts as a co-mutagen with aflatoxin B1 when incubated with human liver S9 (Goeger, et al., 1999). CYP2A13 is highly expressed in human lung (Su T, et al., 2000) and identified as an efficient catalyst for the metabolic activation of AFB1(He XY, et al., 2006) and 7-hydroxylation of coumarin (Su T, et al., 2000). CYP2A13 variants may have an altered coumarin 7-hydroxylation and AFB1 activation activity and may alter human susceptibility to co-mutagen coumarin with aflatoxin B1. In present study, the frequency distributions of eight SNPs of CYP2A13 (R257C, 133_134insT, R101Q, F453Y, R494C, R101X, D158E, V323L), were examined in a Chinese Han population. Five CYP2A13 variants, CYP2A13*2, CYP2A13*5, CYP2A13*6, CYP2A13*8, and CYP2A13*9, were expressed in sf9 and evaluated for coumarin 7-hydroxylation and AFB1 activation. Our results demonstrated that the coumarin 7-hydroxylation activities of two variants CYP2A13*5 and CYP2A13*6 were two-fold higher than that of wild-type CYP2A13 while CYP2A13*2, CYP2A13*8 and CYP2A13*9 were slightly higher. The frequency distributions of CYP2A13 alleles and SNPs in a Chinese Han population were statistically different from the reported value in Japanese (p < 0.05). CYP2A13 polymorphisims could lead to an increase in coumarin 7-hydroxylation and, combined with AFB1 activation results (data unfinished), may result in an increased susceptibility to co-mutagen coumarin with aflatoxin B1. Reference Goeger, D. E., A. W. Hsie, et al. Co-mutagenicity of coumarin (1,2-benzopyrone) with aflatoxin B1 and human liver S9 in mammalian cells. Food Chem Toxicol. 1999.37(6): 581-589. He XY, Tang L, Wang SL, Cai QS, Wang JS, Hong JY. Efficient activa-tion of aflatoxin B1 by cytochrome P450 2A13, an enzyme predominantly expressed in human respiratory tract. Int J Cancer. 2006.118(11):2665-71. Su T, Bao Z, Zhang QY, Smith TJ, Hong JY, Ding X. Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco-specific car-cinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 2000.60:5074-5079. Supported by the NSFC (30771825)

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P420. The effect of related metabolizing enzyme polymorphism and active metabolites on thiopurine treatment in inflammatory bowel disease

Min Huang, Liang Ding, Pin-jin Hu, Xue-ding Wang and Hui-chang Bi

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China

Inflammatory bowel disease (IBD), includes Crohn′s disease (CD) and ulcerative colitis (UC), is a chronic non-specificity bowel inflammation with unknown etiopathogenesis[1]. The thiopurine drugs, such as Azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are immunosuppressive agents widely used in the treatment of IBD and have been proven to be effective in both inducing and maintaining remission of CD and UC. Thiopurine drugs metabolizing enzymes include thiopurine methyltransferase (TPMT), xanthine oxidase (XO), and hypoxanthine guanine phosphoribosyltransferase (HPRT). 6-MP can be catalyzed into active 6-thioguanine nucleotides (6-TGNs) by HPRT. The TGN metabolites act as purine antagonists and induce cytotoxicity, and their accumulation may contribute to a more persistent neutropenia. The interindividual differences in therapeutic effect and toxicity of thiopurine drugs could be a result of genetic variation in these metabolic enzymes. Therefore, the aims of this study were to 1) detect the genotype of TPMT, the phenotype of TPMT, HPRT, XO and the concentration of 6-TGNs in Chinese IBD patients, observe the clinical efficacy and adverse effects, and investigate the relationship among related metabolizing enzymes, active metabolites and clinical outcomes; 2) evaluate and verify the risk therapeutic concentration threshold of 6-TGNs in Chinese IBD patients; 3) formulate the pharmacogenetic-based metabolites monitoring therapy to improve the clinical efficacy and safety in thiopurine therapy. The genotype of TPMT was performed by RFLP-PCR and AS-PCR method. The phenotypes of TPMT, HPRT and 6-TGNs levels were detected by HPLC method. The phenotype of XO was detected by UV-spectrophotometric method. The clinical efficacy and adverse effects were observed in all patients. The genetic variation of TPMT could predict the myelosuppression of IBD patients undergoing AZA/6-MP therapy. However, as the allele frequency of TPMT was very rare in Chinese, the clinical significance of TPMT polymorphism in Chinese IBD patients was limited. In the cohort of patients who developed myelosuppression within 3 months, TPMT activity was significantly lower than in the patients without myelosuppression. Moreover, HPRT activity was correlated with thiopurine-induced adverse effects, especially myelosuppression, while XO activity was probably correlated with thiopurine-induced adverse effects, especially hepatotoxicity, in IBD patients. The ROC curve was obtained to plot the sensitivities and specificities of various 6-TGNs levels to predict the development of myelotoxicity of AZA/6-MP treatment. A sensitivity of 56.3% and specificity of 84.4% were seen at the cut off level of 420 pmol/8x108 RBC with a negative predictive value of 95.9% on myelosuppression, and a positive predictive value of 21.2%. To describe the linear association among HPRT activity, TPMT activity and 6-TGNs concentration, a multiple linear regression model was developed by stepwise method. The regression model was: 6-TGNs concentration = antilog (2.324 + 0.09198x5-ASA +0.115xHRRT activity-0.008xTRMT activity). Therefore, the pharmacogenetic-based metabolites monitoring therapy was established to help identify patients at risk of associated hematological toxicity and can be served as a guide for dose individualization. (The work was supported by Science and Technology Ministry of China 2009ZX09304-003 and 2008ZX09312.)

P421. Interspecies allometric scaling of therapeutic monoclonal antibodies

Masataka Oitate, Noriko Masubuchi, Takashi Ito, Yoshiyuki Yabe, Tsuyoshi Karibe, Takanori Aoki, Nobuyuki Murayama, Atsushi Kurihara and Noriko Okudaira

Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan

In order to seek the fixed exponent in the allometry equation that can predict human clearance (CL) and volume of dis-tribution at steady state (Vss) of monoclonal antibody (mAb) from those of animal data, PK parameters of 11 therapeutic mAbs were collected and analyzed based on the targeted antigens (soluble or membrane bound). The fixed exponents for CL and Vss prediction were back-calculated to be 0.788 and 1.130 (for soluble), and 0.970 and 1.002 (for membrane bound), respectively. Using these exponent values, human CL and Vss of mAbs can be predicted from monkey data (at > 1 mg/kg) with reasonable accuracy.

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P422. Comparison of pharmacokinetics of dapsone in rats following blood sampling via retro-orbit-al, jugular vein and saphenous vein

Tausif Ahmed, Prashant N, Sudipta B, Rajanikanta S, Vijay J, Abdul B, Kaustubh T, Amol G, Siree-sha D, Swapnakumari T and Subrahmanyam V

DMPK & Toxicology, Sai Advantium Pharma Ltd, Pune, India

Pharmacokinetic (PK) studies are an important part of the drug discovery process. Typically rats are used for PK screening of new chemical entities (NCEs) during drug discovery and lead optimization process because they require small quanti-ties of compound and can yield multiple blood samples. In the present study the effect of three blood sampling techniques (retro-orbital, jugular vein and saphenous vein) on PK of dapsone was evaluated. Dapsone was administered both orally and intravenously at a dose of 12 mg/kg to a group of 4 or 5 male Sprague Dawley rats. Blood samples were collected up to 24 hours for the determination of dapsone concentrations in plasma. The PK parameters between group I (retro-orbital) and group II (jugular vein) were not statistically different (p> 0.05), while the PK parameters of both group I and II were statistically significant (p< 0.05) from group III (saphenous vein). The authors recommend saphenous vein sampling to be the most appropriate from scientific and ethical perspective for conducting short term PK studies in rat.

P423. Development of the Next Generation of On-Line Radiochromatography Detectors

Tom Deakin1, Lee F. Thompson1, John K. Clapham2 and Richard A. Brown2

1Physics and Astronomy, The University of Sheffield, Sheffield, United Kingdom, 2LabLogic Systems Ltd., Sheffield, United Kingdom

Since the 1980s, on-line radiochromatography measurements have formed an integral part of metabolite profiling stud-ies and are nowadays coupled with advanced HPLC, UPLC and MS analysis techniques. Currently, the commercially available on-line detectors are being pushed to their operational limits as demands for better sensitivity and higher resolution are ever-increasing, along with the added pressure from desirable shorter run-times. Part of this demand is being fuelled by the necessity to work with much lower levels of β-radioactivity and so a significant improvement in the signal to noise ratio will be of paramount importance in the near future. In this work, we describe and present some experimental results of an on-going research collaboration that aims to identify key areas for on-line radio-detector instrument development and improvement through appealing to both the fundamental physics of the measurement process and the latest technological advances. The collaboration is between the University of Sheffield′s Department of Physics and Astronomy and LabLogic Systems Ltd. and is funded by the UK Government′s Knowledge Transfer Partnership (KTP) initiative. The primary aim of the research is to reassess the status of the current instrument tech-nology via a combination of theoretical and experimental studies, which are being supported by a constant stream of communication with LabLogic′s end users. Use is being made of state-of-the-art measurement techniques normally applied to advanced experimental particle physics and also of major research facilities such as the Boulby Underground Laboratory in the UK which will enable experiments to be performed in a unique, ultra-low background environment. To date, a number of potential areas for the improvement of LabLogic′s current β-RAM® Model 5 instrument have been identified and recent focus has been on understanding the physical behaviour of current photomultiplier tube (PMT) technology, with a view to being able to give better differentiation between genuine counts and those from noise and other background sources during on-line radiochromatography measurements. The culmination of the work will see future generations of LabLogic′s β-RAM® on-line radiochromatography detector leading the way in terms of resolution and reproducibility, as well as providing a detailed insight into the fundamental scientific processes that are in play.

P424. Anti-Arthritis Effect of a Novel Bruton′s Tyrosine Kinase Inhibitor in Rat Collagen-Induced Arthritis and Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling: Relationships between Inhibition of BTK Phosphorylation and Efficacy

Lichuan Liu1, Julie Di Paolo2, Jim Barbosa2, Hong Rong2, Karin Reif3 and Harvey Wong4

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1Department of Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA, USA, 2CGI Pharmaceuticals, Branford, CT, USA, 3Genentech, South San Francisco, CA, USA, 4Drug Metabolism and Pharmacokinetics, Genentech, Inc, South San Francisco, CA, USA

Bruton′s tyrosine kinase (BTK) plays a critical role in the development, differentiation and proliferation of B-lineage cells, making it an attractive target for the treatment of rheumatoid arthritis (RA). GDC-0834 is a potent and selective small molecule inhibitor of BTK. The objective of this study was to demonstrate the anti-arthritis effect of GDC-0834 and characterize the relationship between inhibition of BTK autophosphorylation (pBTK) and efficacy using a mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model. In the rat collagen-induced arthritis (CIA) model, GDC-0834 directly inhibited pBTK with an IC

50 (drug concentration that

produces 50% of the maximum inhibition) of 3.3 μM and max

(maximum pBTK inhibition) of 81% in the rat blood. Furthermore, administration of GDC-0834 resulted in a dose-dependent reduction of ankle swelling (from 30 to 100 mg/kg). Finally, the alleviation of the morphologic pathology of ankles confirmed the anti-arthritis effect of GDC-0834. A disease progression PK/PD model was constructed by integrating an indirect response model of pBTK inhibition with a transit model used to describe time-dependent decreases in ankle swelling due to spontaneous disease remission. The base model (without BTK inhibition) described well the natural progres-sion of inflammatory arthritis in vehicle-treated animals with an estimated k

in (net ankle swelling increase rate

constant) of 0.019 hr−1 and a kout

(rate constant describing ankle swelling decrease due to spontaneous disease remission) that increased with time. The simultaneous fitting of data from vehicle and GDC-0834 treated groups showed that overall 77% inhibition of pBTK was needed to decrease the k

in by half. These findings suggest that

a high degree of pBTK inhibition is required for maximal activity of the pathway on reduction of ankle diameter in CIA rats.

P425. PK-PD Modeling of Combination Efficacy Effect from Administration of a MEK Inhibitor, GDC-0973 and PI3K Inhibitor, GDC-0941 in A2058 Xenografts

Edna F. Choo1, Marcia Belvin2, Klaus Hoeflich2, Mark Merchant2, Douglas Dan Otter2 and Laurent Salphati1

1Drug Metabolism and Pharmacokinetics, Genentech Inc, South San Francisco, CA, USA, 2Cancer Signaling and Translational Oncology, Genentech Inc, South San Francisco, CA, USA

Combining multiple targeted anti-cancer agents with the goal of providing effective therapy has been increasingly explored in oncology. Prior to clinical combination trials, initial proof of concept studies are usually conducted using mouse xenograft models. Here we describe PK/PD modeling of A2058 melanoma xenograft efficacy data after co-administration of two anti-cancer agents, a MEK inhibitor (GDC-0973) and a PI3K inhibitor (GDC-0941), both acting on parallel pathways. Using an indirect response model and an “additive model” (sum of effect from 2 single agents), the following PK/PD evaluations were conducted: 1) single agent model fit of efficacy data; 2) parameters from single agent fits were then used to simulate an “additive” efficacy effect from combined agents; 3) the “additive” model was verified by fitting it to efficacy data from combined agents. Similar parameters were obtained from fitting the “additive” model to the efficacy data compared to parameters obtained from single agent fits. The constant associated with tumor kill from fitting individual efficacy data for GDC-0973 and GDC-0941 were 0.0108 μM−1 hr−1 and 0.000484 μM−1 hr−1, respectively. The constant associated with tumor kill from the “additive” fit of the combination data was 0.00985 μM−1 hr−1 and 0.000559 μM−1 hr−1, for GDC-0973 and GDC-0941, respectively. The tumor net growth rate constants were also similar (0.00453-0.00487 hr−1). These findings suggest that parameters from single agent fits adequately described the combination efficacy effect. Although the selected model does not provide mechanistic insight into the biology, e.g., it does not provide biological evidence that effects are additive, it provides a means to simulate regimens and doses that have not been experimentally determined based on single agent data and provides the opportunity to simulate and predict efficacy based on human PK inputs.

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P426. Pharmacokinetics of Subcutaneous Single Doses of Pegylated Recombinant Human Growth Hormone(PEG-rhGH)in rhesus Monkeys

Yong M. Cai1, M. Li2, Zong P. Zhang2, Zheng M. Chen2 and Chang X. Liu2

1State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin University,Tianiin Institute of Pharmaceutical Research, Tianjin, China, 2State Key Laboratory of Pharmacokinetics and Pharmacodynamic, Tianiin Institute of Pharmaceutical Research, Tianjin, China

Background: Human growth hormone (GH) is a 191-amino-acid pituitary protein that stimulates the production and release of insulin-like growth factor-1 (IGF-1) into the systemic circulation. IGF-I is responsible for most anabolic and mitogenic GH actions and consequently for growth promotion and development of organs and tissues during all growth stages. GH is approved for the treatment of adult and paediatric GH deficiency.

Materials and Methods: PEG-rhGH, created by the covalent attachment of a 40 kDa polyethylene glycol (PEG) molecule to the N-terminal residute of rhGH, has an average molecular weight of approximately 60 kDa resulting in a longer half-life and sustained duration of action. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated for precision, accuracy, dilution, specificity and stability. The single-dose pharmacokinetics studies was conducted in six monkeys following subcutaneous administration of 125µg/kg PEG-rhGH using the ELISA, and compared with rhGH at the same dose. Pharmacokinetic parameters were calculated with 3P97 software.

Results: The concentration-time profile of PEG-rhGH in monkeys was described by the one-compartment model. The mean time to reach peak concentration (t

max) was 15.0 h. The elimination half-life (t

1/2) was (16.2 ± 1.5) h. The drug

clearance from the serum (CL) was (2.8 ± 0.9) ml/h/kg. The peak concentration (Cmax

) was (1065.3 ± 258.2) ng/ml. The area under the serum concentration time curve (AUC) was (53846 ± 17356) ng·h/ml. The monkey receiving s.c. dosing PEG-rhGH had a 5-fold longer terminal half-life and 110-fold slower clearnance than GH at the same dose of 125µg/kg. In addition, the time to reach peak concentration was achieved much later (approximately 13h) and the peak concentration was much larger (approximately 17 times) for PEG-rhGH than for GH.

Conclusions: This study showed that the PEG-rhGH offers a superior pharmacokinetics profile compared with that of GH when administered s.c. in the monkeys.

P427. Preclinical Profiling, Prediction of Human Pharmacokinetics and Pharmacokinetic-Pharmacodynamic Modeling of a Potent and Selective Met Kinase Inhibitor

Bianca M. Liederer1, Leonid M. Berezhkovskiy1, Brian Dean1, Vikki Dinkel2, John Gaudino2, Mark Merchant1, Emile Plise1, Dan Sutherlin1, Harvey Wong1 and Xingrong Liu1

1Genentech, Inc., South San Francisco, CA, USA, 2Array BioPharma, Inc., Boulder, CO, USA

GNE-A (N-(4-(3-((3S,4R)-1-ethyl-3-fluoropiperidine-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide) is a novel small molecule Met kinase inhibitor devel-oped as a potential orally available drug for the treatment of human cancers. The objective of this study was to assess the preclinical ADME characteristics of GNE-A and its potential as a human drug candidate. The pharmacokinetic (PK) properties of GNE-A were evaluated following single IV and PO dose administration to mouse, rat, dog and monkey. Plasma protein binding, blood-plasma partitioning and permeability/transporter characteristics were determined in vitro. The plasma clearance was low to moderate (7.9-66% of hepatic blood flow) and the

d ranged from 2.1 to 9 L/kg

in preclinical species. The mean terminal elimination t1/2

values ranged from 1.7 hr in rat to 16.3 hr in dog. The mean %F values in rats at 5 mg/kg, in mice at 10 mg/kg and in monkeys and dogs at 3 mg/kg were approximately 11, 88, 72 and 56%, respectively. Absorption was moderately rapid with T

max values ranging from 2.7 to 5 hours. All preclinical

species, and human, exhibited high plasma protein binding (96.7-99% bound). MDR1-transfected MDCK cells showed a marked efflux ratio of 42. P-gp inhibitor GF120198 inhibited this polarized efflux to 1.2, suggesting that GNE-A is a substrate for MDR1. In vitro blood-plasma ratios ranged from 0.78 to 1.46 in all species, indicating that GNE-A did not preferentially distribute into red blood cells. Human PK parameters were predicted using allometric scaling with four preclinical species. Results projected a low drug clearance and a high

d of approximately 4.8 L/kg in human. PK-PD

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modeling of tumor growth inhibition was performed to determine the relationship of GNE-A plasma concentrations to tumor growth inhibition in the MET amplified EBC-1 human non-small cell lung carcinoma tumor xenograft model grown in female nude (CRL nu/nu) mice. From these studies, estimates of human doses needed for 50% and 90% tumor growth inhibition were made. The data were fitted using a linear effects indirect response model and simulations were performed using projected human PK parameters from simple allometric scaling. Once daily PO doses of 5.6 mg/kg/day and 13 mg/kg/day GNE-A were estimated for 50% and 90% tumor growth inhibition, respectively. Overall, GNE-A exhibited favorable preclinical properties and projected human dose estimates thereby warranting further investigation for its therapeutic potential in human.

P428. Pharmacokinetics, Disposition and Metabolism of A Novel Peptidomimetic in the Rat

Bin Ma1, Chun Yin1, Dan Yang2 and Ge Lin3

1School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China, 2Department of Chemistry, The University of Hong Kong, Hong Kong, China, 3School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China

Alpha-Aminoxy acid peptides are a novel class of peptidomimetics. They are small molecules mimicking the functions of natural peptides and have been developed recently by our research team. These newly developed peptidomimetics showed a therapeutic potential for the treatment of Cl− channel dysfunctional diseases. The present study aimed to inves-tigate the pharmacokinetics of leucine-peptidomimetic (LeuP), a representative peptidomimetic, in Sprague-Dawley rats. The animals received single i.v. (5 mg/kg) or p.o. (100 mg/kg) dose of LeuP, and biological samples, including blood, major organs, urine and feces were collected in the designed time intervals. Stability of LeuP in gastrointestinal (GI) tract was determined by incubation with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively. All the samples were analyzed by HPLC-UV and/or HPLC-MS. After i.v. administration, LeuP was eliminated rapidly with t

1/2 of 0.30 ± 0.01 h and CL of 0.72 ± 0.02 l/h/kg. LeuP widely distributed to the most organs with about 60% of the dose

administered found in the liver and lung within 5 min. However, only negligible amount of LeuP was determined in the brain, suggesting that it could not permeate through blood-brain barrier. Two monohydroxylated metabolites and one dihydroxylated metabolite were detected in the liver, and the monohydroxylated metabolites were also detected in the urine, while no metabolites were found in the plasma. Oral bioavailability of LeuP was remarkably low (0.36%) partially due to its instability of in the stomach (41% degradation within 1 hr) and intestine (47% within 3 hr) and extensive hepatic first-pass metabolism (45% metabolism within 30 min) determined by our previous in vitro metabolic study. In addition, poor intestinal absorption of LeuP due to P-glycoprotein (P-gp)-mediated efflux transport revealed by our previous study also contributed to its low oral bioavailability. About 30% of the amount orally administered was detected in the feces within 24 hr, indicating that LeuP was poorly absorbed and/or significant amount of the intact LeuP underwent biliary excretion. In conclusion, LeuP exhibited rapid elimination and wide distribution with predominant amount in the liver and lung. LeuP had a very low oral bioavailability (<1%), which was attributed mainly to its instability in the GI tract, poor absorption, and extensive first-pass metabolism in the liver. Our findings suggested that other administration routes such as sublingual or intravenous injection, which can avoid GI tract degradation and hepatic first-pass metabolism, are recommended as more appropriate delivery methods for LeuP. [Supported by RGC Central Allocation fund (HKU 2/06C) and Chinese University of Hong Kong Direct Grant (2041542).]

P429. Changes in Expression and Function of Pept1 in Indomethacin-induced Acute Intestine Injury in Rats and Damaged Caco-2 Cells after Treatment of JBP485

Kexin Liu1, Wei Wang1, Qi Liu1, Changyuan Wang1, Qiang Meng1, Jian Zhang1, Xinjin Guo1 and Taiichi Kaku2

1Department of Clinical Pharmacology,College of Pharmacy, Dalian Medical University, Dalian, China, 2Bioproducts Industry Co. Ltd., Tokyo, Japan

Objectives: To investigate the changes in expression and function of the intestinal oligopeptide transporter, Pept1, in indomethacin-induced acute small intestine injury in rats and damaged Caco-2 cells after treatment of JBP485 (a dipeptide with anti-inflammatory action).

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Methods: The effects of JBP485 on indomethacin-induced intestinal histological changes, MDA levels and MPO activity, inflammatory mediators release and cell LDH-release were examined. Uptakes of Glysar were determined by in vivo oral administration, in situ intestinal perfusions, in vitro everted small intestinal sac preparations and RT-PCR and Western blot studies for determing Pept1 mRNA and protein were utilized.

Results: (1)JBP485 decreased in MDA concentration and MPO activity significantly, improved inflammatory charac-teristics in histochemistry and attenuated cell damage induced by indomethacin. (2)The uptake of Gly-Sar by Pept1 in indomethacin-induced rats were significantly decreased, whereas the Gly-Sar concentration in plasma in JBP485 treated rats were markedly higher than indomethacin-induced rats. (3)Indomethacin caused a significant decrease in the expression of pept1 mRNA and protein in the small intestine. When compared with indomethacin-induced rats and cells, levels of Pept1 mRNA and protein were increased after administration of JBP485.

Conclusions: JBP485 improved acute intestinal inflammation condition and cells damage induced by indomethacin. The mechanism is related to decrease the inflammatory mediators release and improve the expression and func-tion of the intestinal oligopeptide transporter (Pept1). JBP485 might be a new therapeutic agent for acute intestinal injury.

P430. Pharmacokinetic interactions between three major bioactive flavones in Radix Scutellariae

Chenrui Li1, Ge Lin2 and Zhong Zuo1

1School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China, 2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Objectives: As two major bioactive flavones in Radix Scutellariae, the intestinal absorption and disposition as well as their pharmacokinetic profiles of Wogonin (W) and Oroxylin A (OA) were investigated. Their potential pharmacokinetic interactions with Baicalein (B), the most abundant flavone in Radix Scutellariae were also studied.

Methods: Rat in situ single-pass intestinal perfusion has been used to investigate the intestinal absorption and disposi-tion of W and OA. In addition, the effect of co-existing components on the absorption and metabolism of each bioactive component in RS has been evaluated. Afterwards, the pharmacokinetic of W and OA as well as their interaction with B were studied in rat.

Results: The data obtained from rat in situ single-pass perfusion model demonstrated a satisfactory absorption of W and OA, however, with extensive first-pass metabolisms during absorption, mainly in the form of glucuronidation. Formed glucuronides could be effluxed to the lumen side and the blood side of intestine epithelium. After perfusing three flavones together, the parent drugs absorbed into mesenteric blood significantly increased whereas their cor-responding glucuronides effluxed to the lumen side significantly decreased. The further in vivo rat pharmacokinetics studies showed that W and OA could be readily absorbed and extensively metabolized with no parent compound detectable in blood after oral administrations of W and OA. The plasma concentration versus time profiles for detect-able glucruonides of baicalin (BG), wogonoside (WG) and oroxylin A-7β-D-glucuronide (OAG) were similar with a fast metabolite formation followed by a long elimination phase. Comparisons of the oral absorptions of each component in rat after oral administration of single component form versus their mixture form found that only the absorption of OA increased significantly after administration of the mixture form.

Conclusion: Similar to B, W and OA could be well absorbed followed by extensive first-pass metabolism mainly in the form of glucuronidation in rat. During the absorption process, the formed WG and OAG were effluxed to both the lumen and blood side of the intestine. Interaction among B, W and OA would lead to the absorption enhancement of parent drug by in situ model, however increased absorption in vivo was observed only for OA. (CUHK 478607 from the Research Grants Council of the Hong Kong SAR, China)

P431. Blood Sampling Method Influences Pharmacokinetics in the Rat

Natasha Nikolaidis1, Candace Rohde-Johnson1, Nick Suttles2, R. Tyler DeGraw1, Nathanael Lichti3, Rachel Sun1 and David Hopper2

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1BASi, West Lafayette, IN, USA, 2BASi, Evansville, IN, USA, 3Purdue University, West Lafayette, IN, USA

In early drug discovery, various sampling techniques are employed to collect blood samples for pharmacokinetic stud-ies. Blood collection methods involving restraint are known to elevate stress hormones, especially when compared to automated sampling methods. Physiologic responses to stress include increased heart rate and blood flow, as well as diversion of blood away from the liver and the gut. Stress induced changes in hepatic blood flow may impact PK param-eters, especially for compounds with a high hepatic extraction ratio, where kinetics are highly dependent on blood flow. In this study we examined the effect of sampling method on the pharmacokinetics of three compounds with varying hepatic extraction ratios (carbamazepine, codeine, and propranolol). Three groups of rats were sampled manually and with a Culex Automated Sampler. Each rat was dosed with one compound into the jugular vein, and then blood samples were collected either manually from the tail vein or automatically via the Culex sampler from an implanted femoral vein catheter. After a 48 hour washout period, the groups were crossed over with dosing repeated as previously, and sampling accomplished by the alternate method. There was a significant interaction between compound and sampling method on pharmacokinetic parameters. AUC was lower and clearance was higher in rats sampled via Culex, and the difference between manual and automated groups was positively correlated with hepatic extraction ratio. The results suggest that sampling method can influence pharmacokinetics, and that certain drugs may be more susceptible to this effect.

P432. Pharmacokinetics and NONMEM analysis of curcumin and its metabolite THC

Habibur SM Rahman1 and Ramanathan M2

1Pharmaceutics, PSG College of Pharmacy, Coimbatore, India, 2Pharmacology, PSG College of Pharmacy, Coimbatore, India

Hypothesis: Curcumin (diferuloylmethane) is a potent phytomolecule possess low bioavailability profile due to its rapid metabolism. To understand the mechanism behind the low bioavailability and interplay of metabolism, it is necessary to study the pharmacokinetics of curcumin along with its metabolites and simulating the PK data by additive model and covariance model may help in identifying the various factors which affect the bioavailability of curcumin. The present work is proposed to study the pharmacokinetics of curcumin along with its metabolite tetrahydrocurcumin (THC) and simulation of PK data using Non Linear Mixed Effects Modeling (NONMEM).

Methods: Simple HPLC method was developed for estimation of curcumin and THC in plasma. The method involves extracting the curcumin from 0.1ml sample volume of plasma in PBS buffer along with β glucuronidase for hydrolyzing the curcumin and THC conjugates at 370C for 1h. Ethyl acetate was used as extraction solvent. Salbutamol was used as internal standard. The method had LOQ of 1μg/ml in rat plasma. The precision, accuracy, recovery and applicability were found to be adequate for pharmacokinetic studies. Analysis utilizes a reversed-phase phenominex C

18 column

and PDA detection at 280 and 430 nm for THC and Curcumin respectively. Curcumin was administered orally to rats in two doses 25 and 50 mg/kg. Blood samples were collected in the time intervals of 0.25, 0.5, 1, 2, 3, 4, 6 and 8h from the time of administration. Pharmacokinetic parameters like Tmax, Cmax, AUC, t

1/2 were evaluated by the developed

HPLC method. The treatment was continued for seven days and on the seventh day pharmacokinetic parameters were evaluated. Curcumin treated and control animals were administered with phenobarbitone (40mg/kg) and parameters like onset and duration of sleep was noted.

Results and Conclusion: PK parameters suggest that curcumin is undergoing first order kinetics with high elimination half life. Further PK results are subjected to various modeling process and simulation of results by additive model and covariate models were using NONMEM programme. HPLC results and sleeping behavior studies suggests that curcumin shows dose dependent affect on CYP enzyme activity.

P433. Pharmacokinetics (PK) and pharmacodynamics (PD) of RO4929097 (RO), a novel gamma secretase inhibitor, in a phase I study in patients with advanced solid tumors

Karen Wang

Clinical Pharmacology, Hoffman-La Roche, Nutley, NJ, USA

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Pharmacokinetics (PK) and pharmacodynamics (PD) of RO4929097 (RO), a novel gamma secretase inhibitor, in a phase I study in patients with advanced solid tumors Ka Wang, Zhi-Xin Xu, Mark DeMario, Stanislaw Mikulski, John Boylan, Steven Middleton Hoffmann- La Roche, Inc., Nutley, NJ

Background: RO is a novel oral gamma secretase inhibitor developed to treat patients with advanced solid tumors. Preclinical studies have demonstrated potent activity in a range of human tumor xenografts models at 10 and 30 mg/kg (daily or intermittent dosing regimens) with efficacy observed up to 91% TGI. RO was identified as a P450 3A4 (3A4) substrate (microsomal incubation in the presence of chemical inhibitors) and as a potential inducer of 3A4 in strength comparable to known clinical inducer rifampicine. (human hepatocytes incubation).

Methods: This is an open label, repeat dose escalating study to evaluate safety/tolerability, PK, PD/biomarker and tumor responses to two RO dosing regimens (3 weeks per cycle, Sch-A: 3-day-on-4-day-off, for two weeks up to 270 mg; Sch-B: daily dosing for 1 week up to 135 mg). Drug-drug interaction (DDI) liability with RO was investigated in an end-study cohort expansion (“tail”). The PK profile of RO was determined after the 1st dose and after the last doses in the first cycle in all the patients. The PK of MDZ and its metabolite was determined at baseline and after the last doses of RO in the first cycle, in “tail” only.

Results: A total of 89 patients were evaluated in dose escalation. RO was well tolerated in all patients. Serum Ab40 showed response to dose in a nonlinear pattern as a potential biomarker for notch signaling modulation via gamma secretase inhibition. The plasma exposures of RO (Cmax and AUC

0-24, respectively) increased approximately propor-

tionally with escalating single dose up to 180 mg and appeared to plateau at the highest dose (270 mg). The mean RO exposure in the 180 mg dose cohort was 43400 ng*hr/mL for AUC

0-24h and 2800 ng/mL for Cmax. With large variation, the

median t1/2

of RO after single dose was 26 hrs. After repeated dosing, decreased exposures were observed consistently in higher doses (>/=80 mg in Sch-A and >/=60 mg in Sch-B). However, after “drug holidays” in both schedules, exposures nearly reverted to day-1 levels. This observation suggests autoinduction of 3A4 at higher doses; the effect appears to be time dependent and reversible. Importantly, RO exposures in patients demonstrating autoinduction after repeat dosing remained above the levels associated with TGI in preclinical studies mentioned above. In the “tail” portion, evaluation of the extent of potential RO-related autoinduction via 3A4 on drug exposures and clinical outcome is ongoing with lower doses given in different dose regimens.

Conclusion: Preclinical testing suggested 3A4 induction potential for RO. This finding supports the clinical obser-vation of decreasing drug exposure at higher doses after repeated administration. This warranted an early clinical assessment of RO autoinduction and DDI potential in a Phase 1 substudy, which has been successfully implemented in patients with advanced cancers.

P434. Pharmacokinetics of recombinant human apolipoprotein(a) kringle V in rats

Byoung Soo Kim1, Eun Jung Kim1, Jang Seong Kim2, Gi Jeong Cheon1, Ho Jeong Lee2 and Tae Hyun Choi1

1Radiopharmaceuticals Development Team, Korea Institute of Radiological and Medical Science, Seoul, South Korea, 2Mogam Biotechnology Research Institute, Yongin, South Korea

Purpose: Recombinant human apolipoprotein(a) kringle V (rhLK8), which consists of 86 amino acid residues, inhibits angiogenesis in vitro and in vivo. This study aimed to investigate the pharmacokinetics of rhLK8 in rats

Methods: Radiolabeled rhLK8 ([125I]iodo-rhLK8) was prepared by chloramine T method and purified using ultra-centrifugal filter device. Stability of radio-iodinated rhLK8 was evaluated by radio-TLC in rat serum and in rat liver microsome. [125I]iodo-rhLK8 was administered to S.D. rats by single i.v. bolus at 50 mg/kg dose. Blood samples were collected from cannulated femoral artery prior to dosing and at 5, 10, 15, 30 min and 1, 2, 4, 6, 9, 24, 48 and 72hr after drug administration. Drug levels were determined in plasma by gamma counter. The pharmacokinetic parameters calculated by WinNonlin version 2.1.

Results: After the radio-labeling and purification procedure, final recovery and radiochemical purity of [125I]iodo-rhLK8 were 69.6% and over 99.0%, respectively. [125I]iodo-rhLK8 was very stable in rat serum up to 6hr and in rat liver microsome up to 30min. After a single i.v. bolus dosing, plasma rhLK8 concentration decreased bi-exponentially. The C0 and AUCinf were 374?76.3 ug/mL and 1168?116 ug*hr/mL, respectively. The terminal half-life and clearance were 20.0?2.88 hr and 43.1?4.44 mL/hr.

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Conclusion: For the pharmacokinetic study, a stable radio-labeled rhLK8 was prepared successfully. Comparing to the pharmacokinetic profile of recombinant human endostatin (rhEndo) reported before, rhLK8 has longer half life and higher exposure value.

P435. Accurate prediction of variability in CLint and Fm via 3A4 is only obtained by assessing a series of individual cryopreserved human hepatocyte batches

Coraline Desbans1, Constanze Hilgendorf2, Lysiane Richert1, Mareike Lutz2 and Anna-Lena Ungell2

1KaLy-Cell, Illkirch, France, 2DMPK and Bioanalytical Chemistry, AstraZeneca R&D, Mölndal, Sweden

Multiple in-vitro and in-vivo methods are currently assessed and under discussion to predict human clearance and pharmacokinetics from preclinical studies. A combination of high f

m with a high fraction metabolism via a single path-

way, e.g. via CYP3A4 (fmCYP3A4

), has been recognized as a high risk factor for Drug Drug Interactions (DDI) in the clinical setting[1],[2],[3],[4]. Thus, an early predictive tool to allow for appropriate modeling of this potential risk for DDI is highly warranted. Hepatocytes, capable of both phase I and phase II reactions, are an attractive system to study fraction metabolized (f

m) via a single pathway. In the present study, intrinsic clearance (CL

int) was determined in cryopreserved

human hepatocytes in suspension for a set of five compounds with known and variable fm

via CYP3A4 (amitriptyline, loratadine, methylprednisolone, midazolam, and tacrolimus) in the absence or presence of ketoconazole. In order to get an insight into the influence of inter-individual variability, twelve batches of cryopreserved human hepatocytes with either high, moderate or low CYP3A4-dependent activity towards midazolam (MDZ) were chosen. Cl

int values

were determined as substrate depletion under shaking conditions (900rpm) using an elliptic shaker as previously reported[5]. For all compounds, the mean CL

int for individual donors in absence of ketoconazole correlated very well

with literature data on the mean of individual donors1,2,3, and/or pools of donors5. Average fmCYP3A4

for midazolam was 83%, tacrolimus 64%, methylprednisolone 55%, amitriptyline 28%, and loratadine 19% are also well within the literature data2,3,4. Interestingly, the results obtained for a homogenous subpopulation regarding MDZ CL

int and percent inhibition

by ketoconazole, were not directly related to the ketoconazole sensitive CLint

for the other CYP3A4 substrates tested. The variability in CYP3A4 contribution for compounds having multiple metabolic pathways cannot be predicted by the fm

3A4 for MDZ. This suggests that an overall prediction of CL

int or fm via CYP3A4 for compounds partially metabolized

by this enzyme is not possible. Thus, the individual differences in CLint

for a given compound and fmCYPi can only be well covered by assessing a series of individual cryopreserved human hepatocyte batches.

References

[1]. Lu, C., Miwa, G. T., Prakash, S. R., Gan, L-S. and Balani, S. K. (2007), Drug Metabolism And Disposition, 35: 1, 79–85[2] Lu, C., Hatsis,P., Berg,C., Lee, F. W. and Balani, S. K. (2008), Drug Metabolism And Disposition, 36: 7, 1255–1260[3] Lu, C., Hatsis,P., Berg,C., Lee, F. W. and Balani, S. K. (2008), Drug Metabolism And Disposition, 36: 7, 1261–1266[4] Emoto, C., Murase, S. and Iwasaki, K.(2006), Xenobiotica, 36: 8, 671 — 683[5] Simon, S., Blanchard, N., Alexandre, E., Hewitt, N. J., Bachellier, P., Heyd, B., Coassolo, P., Schuler, F., Richert, L., (2009) ‘MV-HUF

Copenhagen′

P436. Single rat full PK-profiling to allow for better science and for the use of less testing animals

Mira Wenker, Michael Eman, Danielle Broekhuizen, Theo Noij and Harry Emmen

NOTOX B.V., Hertogenbosch, Netherlands

Obtaining pharmacokinetic data from plasma or blood is a very important aspect of the safety evaluation of new pharma-ceuticals. Rats are often used to evaluate the safety of new chemical entities and are therefore also the rodent species of choice to generate pharmacokinetic data. One important drawback in the use of these rodents is their relative small size and therefore the inability to draw large amounts of blood from these animals. Often composite plasma concentration-versus-time curves are prepared, whereby different animals are used at different time points. However, this means a higher use of animals and a more ambiguous interpretation on pharmacokinetic data. New analytical techniques like

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UPLC allow the analysis of very small sample volumes while still preserving the sensitivity of conventional HPLC-MSMS analysis. When a smaller volume of blood is needed, a complete PK profile from one rat can be drawn. In this study, we have compared the pharmacokinetics of two known compounds, Nimesulide and Moxifloxacine, obtained by composite and single-profile curves. Rats were orally dosed with either 50 mg/kg Moxifloxacine or 3 mg/kg Nimesulide and sampled at 7 time points after dosing by the retro-orbital punction, tail vein puncture or saphenous vein puncture. Blood samples of 100 microliter were drawn, and plasma samples of 10 microliter were analyzed by UPLC/MSMS. The calculated PK parameters (Cmax, AUC and t1/2) were quite similar between the different blood sampling methods. However, resulting plasma concentration time curves showed a double peak pattern for data obtained by the retro-orbital punction, as a result of the composite sampling. The variability observed in Cmax concentration was up to 60% for the orbita sampling while this was only 10-20% for the other blood sampling techniques. In summary, using a combination of small volume blood sampling and UPLC analysis, a single PK profile of one rat can be obtained, which leads to the use of less animals, less stress and discomfort to the animals and a better interpretation of pharmacokinetic data.

P437. Pre-clinical pharmacokinetic studies of hemoglobin-vesicle as an artificial oxygen carrier

Kazuaki Taguchi1, Toru Maruyama1, Hiroshi Watanabe1, Hiromi Sakai2, Eishun Tsuchida2 and Masaki Otagiri1

1Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan, 2Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan

Hemoglobin-vesicle (HbV) is an artificial oxygen carrier that encapsulates concentrated hemoglobin (Hb) in phos-pholipid bilayer membrane, and has considerable promise for use in clinical settings. In this study, we examined the pre-clinical pharmacokinetic studies of HbV. Using HbV in which the internal Hb was labeled with iodine-125 (125I-HbV) and cell-free 125I-Hb, it was found that encapsulation of Hb increased the half-life by 30 times, accompanied by a decreased distribution in both the liver and kidney. The uptake clearance for the liver and spleen were decreased with increasing dose of HbV, and the half-life of HbV was increased up to approximately 19 hours at a dose of 1400 mg Hb/kg. The pharmacokinetics of HbV components were also investigated using 125I-HbV and 3H-HbV (liposomal cholesterol was radiolabeled with 3H). As a result, similar plasma concentration curves of 125I-HbV were observed for 3H-HbV, and time course for the plasma iron concentration curve derived from HbV was consistent with the plasma concentration curves for both labeled-HbV. 125I-HbV and 3H-HbV were mainly distributed to the liver and spleen. In an in vitro study, the specific uptake and degradation of HbV in RAW 264.7 cells was found, but this was not the case for parenchymal and endothelial cells. After degrading HbV in liver and spleen, internal Hb disappeared from both the liver and spleen 5 days after injection, and the liposomal cholesterol disappeared at about 14 days. Internal Hb was excreted into the urine and cholesterol into feces via biliary excretion. Next, we investigated the pharmacokinetics of HbV during hemor-rhagic shock. The half-life was shorter in hemorrhagic shock rats as comparison with normal rats. Finally, the half-life of HbV in humans was estimated to be approximately 3-4 days using an allometric equation. We could realize the detail pharmacokinetics properties of HbV, and our results suggest that HbV could be used as an oxygen carrier from the view point of pharmacokinetic studies.

P438. Absorption, Distribution, Metabolism and Excretion (ADME) Study With a Potent Thiazolidinedione Insulin Sensitizer, Lobeglitazone

Se-Mi Kim, Nina Ha, Kyu-Tae Park, Hyun-Tak Oh, Hosung Yu, Young Min Kim, Sungsook Lee and Dal-Hyun Kim

Research Institute, Chong Kun Dang Pharm, Cheonan, South Korea

Absorption, Distribution, Metabolism and Excretion (ADME) Study of a Potent Thiazolidinedione Insulin Sensitizer, Lobeglitazone Se-Mi Kim, Ni-na Ha, Kyu-Tae Park, Hyun-Tak Oh, Ho-sung Yoo, Yong-Min Kim, Sungsook Lee, Dal-Hyun Kim CKD Research Institute, Cheonan, PO Box 74, South Korea Lobeglitazone, a potent peroxisome proliferator-activated receptor gamma agonist, is currently under investigation as an oral agent that decreases hyperglycemia by reducing insulin resistance in patients with type 2 diabetes mellitus. In vitro and in vivo studies were conducted to character-ize the pharmacokinetic profiles. The oral bioavailability of Lobeglitazone was ∼100% in rats and 56% in dogs and its

276

half-life was 1.5 hrs in rats and 8 hrs in dogs. In rats, the oral pharmacokinetic profiles increased over the dose range studied (2.5∼40 mg/kg) according to dose proportionally. Protein binding was high with an unbound fraction less than 2% in all species tested. In distribution studies in rats dosed orally, Lobeglitazone was mainly distributed in liver, lung and kidney, but its exposure in all tissues tested was less than that of plasma. Lobeglitazone was found to have comparable metabolic stability both in dog and human liver microsomes where there was no difference between male and female. However, the metabolism rate in rat liver microsomes was markedly different between male and female because of CYP2C11 and we confirmed it by in vivo study. Experiments using recombinant human CYP enzymes and human liver microsomes suggested that CYP2D6, CYP2C19 and CYP3A4 are likely to play a key role in the metabolic clearance of Lobeglitazone; in addition, sulfation of metabolites was also observed in plasma and bile of rats. The major metabolite of Lobeglitazone is M9 in rat liver microsome, but M7 in human and dog. In rats, the excretion of Lobeglitazone in urine, bile and feces amounted to 1.8%, 14.4% and 7.1%, respectively. Based on these results, clinical studies and the mass balance studies in monkey to understand fully Lobeglitazone pharmacokinetics are under the progress.

P439. Pharmacokinetics of tiapride and its principal metabolite in human blood plasma

Milan Nobilis1, Zuzana Vybíralová1, Barbora Szotáková2, Kveta Sládková1, Martin Kunes1 and Zbynek Svoboda1

1Bioanalytical department, Institute of Experimental Biopharmaceutics, Joint Research Centre of AV CR and PRO.MED.CS, Hradec Králové, Czech Republic, 2Dept. of Biochemical Sciences, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic

Tiapride, N-(2-diethylaminoethyl)-2-methoxy-5-methylsulfonylbenzamide hydrochloride, is an atypical neuroleptic (antipsychotic) agent, a selective adenylate cyclase-independent dopamine D

2-receptor antagonist which lacks affinity

for D1-receptors. It is employed for controlling severe agitation and agressiveness, in the treatment of dyskinesias and

other movement disorders, sleep disorders, anxiety and alcoholism. Tiapride is minimally metabolized in humans, 70 % of the drug is eliminated in unchanged form in the urine within 24 hours. Only low concentration of N-desethyl tiapride and tiapride N-oxide and no phase II metabolites were detected in former pharmacokinetic studies. For our disposition studies, a new bioanalytical HPLC method for the determination of tiapride and its metabolite(s) was needed. The principal problem to be solved was the possibility of unexpected coelution of drug metabolite(s) with internal standard and/or drug determined. In preliminary “in vitro” and “in vivo” xenobiochemical experiments with tiapride biotransformation in human and rat microsomes was found only one metabolic product in the extracts from microsomal biomatrices. Using a simple chemical modification of tiapride, we had synthesized expected principal metabolite N-desethyl tiapride. LC-MS experiments confirmed its chemical structure. The retention time and UV and FL spectral properties of N-desethyl tiapride standard and the metabolite present in microsomal extracts were identical. All three standards of tiapride, N-desethyl tiapride and sulpiride (I.S.) were used for the optimization of chromatographic and detection conditions. Chromatographic separation was performed on the Discovery HS F5 250 x 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel, mobile phase (acetonitrile – 0.01 M phosphate buffer pH3 in linear gradient) had flow rate of 1 ml.min−1. UV photodiode array and fluorescence detectors in tandem were used for the benzamide derivatives determination. Lower concentrations of tiapride and N-desethyl tiapride were monitored by selective and sensitive fluorescence detector (l(exc.) / l(emis.)=232nm/334nm), higher concentrations (500-6000 pmol/ml) were determined with linear response using UV PDA detector at 212 nm. Each HPLC run lasted 15 minutes. The sample preparation process involved a pH-dependent solid-phase extraction of analytes from blood plasma (SPE cartridges DSC-PH Supelco, U.S.A.). The developed method was validated in accordance to the official requirements for bioanalytical studies and was applied in xenobiochemical and pharmacokinetic studies in humans and animals.

P440. ABSTRACT WITHDRAWN

P441. Pharmacokinetics of recombinant human basic fibroblast growth factor following intravenous bolus plus infusion in rhesus monkey

Xiuwen Liu, Zhongming Tang, Haifeng Song and Baozhen Zhu

Department of Pharmacology, Beijing Institute of Radiation Medicine, Beijing, China

277

AIM: To study Concentration-time profiles and pharmacokinetics of recombinant human basic fibroblast growth factor (rhbFGF) following intravenous bolus injection plus infusion of the drug ( 20 %: 80 % ) at 10, 20, and 40 µg°¤kg−1 and compared with bolus injection in monkey.

METHOD: The specificity, sensitivity, precision and accuracy of determination by ELISA was satisfied. RESULT: The endogenous level of bFGF was 700 ± 490 pg°¤mL−1 in plasma. C

max of 32.7 ± 15.9, 55.9 ± 8, and 91.3 ± 28.2 ng°¤mL−1, respec-

tively, was observed immediately after injection. Levels maintained at 32.7 ± 15.9, 55.9 ± 8.0, and 91.3 ± 28.2 ng°¤mL−1, respectively during infusion. Concentration rapidly decrease after the end of administration, with an average terminal t

1/2 of 2.6∼2.9 h. AUC

(0-°Þ) increased proportional with dose, and systemic clearance CL

Swas closed among doses.

CONCLUSION: In dosage range studied pharmacokinetic

Foundation item: the project supported by National Natural Science Foundation of China (39930180) and The National High Technology Research and Development Program of China (2003AA2Z347B)

P442. An aqueous extract of Poncirus Fructus activates 5-HT receptor subtype 4 for its prokinetic effect without hERG interaction

Won-Sik Shim and Chang-Koo Shim

National Research Laboratory of Transporters Targeted Drug Design, College of Pharmacy, Seoul National University, Seoul, South Korea

Poncirus Fructus (PF) – also known as the dried, immature fruit of Poncirus trifoliata (L.) Raf. (Rutaceae) – is a natural substance that has long been used for various gastrointestinal disorders in eastern Asia. An aqueous extract of PF (PF-W) has particularly potent gastroprokinetic effects, but its molecular mechanism was not well understood. Identification of the underlying prokinetic mechanism of PF-W was pursued in the present study. Changes in in vitro cAMP levels and in vivo intestinal transit rate (ITR) caused by PF-W were measured after pretreatment with GR123587, an antagonist for serotonin receptor subtype 4 (5-HT4R). An [3H] astemizole binding assay and electrophysiology experiments were performed to determine if PF-W has any interaction with the human ether-a-go-go related gene (hERG) potassium channel. PF-W induced an increase in intracellular cAMP in 5-HT4R-expressing HEK293T cells, indicating that PF-W does activate 5-HT4R. Moreover, pretreatment with GR125487 successfully blocked the increase, suggesting that the response was 5-HT4R-specific. More importantly, pretreatment of GR125487 in rats inhibited the elevation of ITR by PF-W, indicating that the prokinetic effect of PF-W was indeed exerted via 5-HT4R. On the other hand, both [3H]-astemizole binding assay and electrophysiological experiments revealed that PF-W did not interfere at all with the hERG channel. It was found that PF-W exerts its prokinetic activity through a 5-HT4R-mediated pathway, with no interaction with hERG channels. Therefore, PF-W is a good candidate that might be developed as a prokinetic agent with fewer expected cardiac side effects.

P443. FXR, PXR and CAR were Decreased in the Liver but not in the Jejunum after Intestinal Ischemia-reperfusion

Ogura Jiro, Terada Yusuke, Masaki Kobayashi, Shirou Itagaki, Hiroaki Yamaguchi and Ken Iseki

Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Intestinal ischemia-reperfusion (I/R) causes gut dysfunction and promotes multi-organ failure (MOF). The liver and kidney can be affected by MOF after intestinal I/R. We previously reported that MRP2 expression was decreased in the jejunum and liver after intestinal I/R (Ogura J. et al. Alteration of Mrp2 and P-gp expressions, including expression in remote organs, after intestinal ischemia-reperfusion. Life Sci. 2008, 82, 1242-1248). Farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have known as transcriptional factors which regulate MRP2 expression. We hypothesized that FXR, PXR and CAR were affected by intestinal I/R injury. We used the intestinal I/R model rats. Surgical procedure was briefly described as below. Male Wistar rats were subjected to 30 min of ischemia by ligating small anastomosing vessels and occluding the superior mesenteric artery (SMA). Reperfusion was induced by removing the clamp. Caco-2 cells were used for a model of the intestinal epithelium and HepG2 cells were used for a model of the liver. Messenger RNA levels were assessed by real-time RT-PCR. Protein expression was

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investigated by Western blot analysis. It was found that FXR, PXR and CAR mRNA levels were decreased at 6 h in the liver but not in the jejunum after intestinal I/R. FXR, PXR and CAR protein were also decreased at 6 h in the liver but not in the jejunum after intestinal I/R. FXR, PXR and CAR have known to activate transcription of target gene in the nuclei. Therefore, we investigated these receptors levels in the nuclei after intestinal I/R. FXR, PXR and CAR levels in the nuclei were decreased at 6 h in the liver after intestinal I/R. Reactive oxygen spices and inflammatory cytokines play a major role in intestinal I/R injury. Subsequently, we investigated effects of hydrogen peroxide and IL-6 on MRP2, FXR, PXR and CAR expressions. Hydrogen peroxide decreased MRP2 expression but not FXR, PXR and CAR expressions. IL-6 decreased MRP2, FXR, PXR and CAR expressions. IL-6 also decreased FXR, PXR and CAR levels in the nuclei. These results indicated that the decreases in FXR, PXR and CAR expression in the liver after intestinal I/R were caused by IL-6. In conclusion, FXR, PXR and CAR expressions were decreased in the liver after intestinal I/R. Decreases in these receptors were caused by IL-6. In the jejunum, FXR, PXR and CAR expressions were not altered after intestinal I/R, although MRP2 expression was decreased.

P444. Intracellular Mechanism Involved In Downregulation of Hepatic Cytochrome P450 by Chronic Renal Failure and Parathyroid Hormone

Josée Michaud1, Judith Naud1, Stephanie Beauchemin2, Francois A. Leblond2 and Vincent Pichette2

1Departement de Pharmacologie, Centre de recherche de l′Hopital Maisonneuve-Rosemont, Montreal, QC, Canada, 2Departement de pharmacologie, Centre de recherche de l′Hopital Maisonneuve-Rosemont, Montreal, QC, Canada

Chronic renal failure (CRF) is associated with a decrease in the metabolism of many drugs, due to a down-regulation of hepatic cytochrome P450 (P450). Previous studies indicated that CRF modifies activity, protein and mRNA expression of different isoforms of P450 in vivo and in vitro. We have demonstrated that the down-regulation of P450 in CRF is caused by the presence of circulating mediators. One of them has been identified as parathyroid hormone (PTH). CRF and PTH cause an inhibition of P450 although the mechanism remains to be defined. The aim of this study was to evaluate the contribution of different signaling factors like; pregnane X receptor (PXR), nuclear factor kappa B (NF-kB), vitamin D receptor (VDR), protein kinase A (PKA) and protein kinase C (PKC). Four groups of rats were obtained CTL, CRF, CRF with parathyroidectomy (CRF-PTX) and CTL-PTX. Livers and hepatocytes PXR mRNA expression was measured by qPCR and PXR protein expression was measured by Western blots. Cultured hepatocytes were incubated with CTL, CRF, CRF-PTX and CTL-PTX sera, or with or without PTH and nuclear extracts were obtained. Nuclear extracts were used for NF-kB ELISA, flux cytometry and Western blots (p50 and p65). We observed a down-regulation of 43% of protein and 40% mRNA expression of PXR in the liver of CRF rats, and PTX prevents the down-regulation of PXR in CRF rats. Furthermore, blocking NF-kB with inhibitors (such as PDTC and Andrographolide) counteracts the effect of CRF and PTH on P450 expression. In conclusion, PXR and NF-kB could be implicated in the down-regulation of hepatic P450 by CRF and PTH.

P445. Nuclear receptor CAR transactivates the expression of DHCR24 gene encoding a cholestero-genic enzyme, 24-dehydrocholesterol reductase, in human hepatocytes

Kouichi Yoshinari, Hitoshi Ohno and Yasushi Yamazoe

Div. of Drug Metab. & Mol. Toxicol., Tohoku University, Graduate School of Pharmaceutical Sciences, Sendai, Japan

Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, is a key transcription factor for the xenobiotic-induced expression of numbers of genes involved in the drug metabolism and transport. The receptor is also suggested to associate with the physiology and pathophysiology of the liver, such as lipid and glu-cose metabolisms, hepatocarcinogenesis and liver/hepatocyte hypertrophy. In addition, it has long been reported that the treatment with phenobarbital, a representative CAR activator, increases serum cholesterol levels in human and rodents. These facts suggest the association of CAR with the cholesterol homeostasis although the molecular mechanism remains unclear. In the present study, we have investigated a possible involvement of CAR in the tran-scriptional regulation of cholesterogenic genes. In mice, treatment with a potent mouse CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene increased the hepatic content of total cholesterol and hepatic mRNA levels of several

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cholesterogenic genes, including those encoding HMG-CoA synthase 1, HMG-CoA reductase, squalene epoxidase and 24-dehydrochoresterol reductase (DHCR24). In this study, we have focused on DHCR24, which catalyzes the last step of cholesterol biosynthesis. In reporter assays with HepG2 cells, reporter gene constructs containing ∼4 kb of the 5′-flanking region of either human DHCR24 or mouse Dhcr24 was transactivated by human CAR (hCAR) and mouse CAR, respectively. Detailed assays with various deletion constructs in combination with hCAR-expressing plasmid identified the region from -1532 to -1445 of human DHCR24 as a CAR-responsive region. Because the region was found to contain 3 putative binding motifs (half-sites) for nuclear receptors, reporter assays were performed with the construct containing a mutation in each half-site. The results demonstrated that one of the half-sites (termed g half-site in this study) was necessary for the CAR-mediated activation of human DHCR24. In electrophoretic mobility shift assays with radiolabeled oligonucleotides and in vitro synthesized nuclear receptors, hCAR, as a heterodimer with human retinoid X receptor a, bound to the oligonucleotide containing the DR-4-type motif (corresponding to the region from -1464 to -1449) with the g half-site. In cultured human hepatocytes, the infection with hCAR-expressing adenovirus and treatment with 6-(4-chlorophenyl)-imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, an hCAR activator, increased DHCR24 mRNA levels. In conclusion, these results indicate that activated CAR enhances the transcription of human DHCR24 through binding to the DR-4-type motif located at around -1.5 kb of the gene in human livers. The present results thus suggest a possible involvement of CAR in the hepatic cholesterol biosynthesis.

P446. Nuclear receptors as controlling factors in chemical metabolism: Determination of regulatory signal network crucial for co-ordinating cellular response to chemicals

Alexey Kolodkin1, Anna Phillips2, Steve R. Hood3, Hans V. Westerhoff4, Frank Bruggeman5 and Nick J. Plant6

1Regulatory Networks Group, Netherlands Institute for Systems Biology, Amsterdam, Netherlands, 2University of Surrey, Guildford, United Kingdom, 3Dmpk-MET, GlaxoSmithKline R&D, Ware, United Kingdom, 4Manchester Centre for Integrative Systems Biology, University of Manchester, Manchester, United Kingdom, 5Netherlands Institute for Systems Biology, Amsterdam, Netherlands, 6Centre for Toxicology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom

The body is exposed to a wide range of external chemicals, both deliberately (e.g. medicines) and accidently (e.g. envi-ronmental contaminants). In addition, the body contains a large number of endogenous chemicals whose levels alter around a physiological mean: This may be as a result of circadian rhythms, normo- or pathophysiological processes. The body responds to changes in these chemical levels by altering flux through metabolic pathways, ensuring healthy physiology. Proteins involved in this biological response include active transport pumps, plus Phase I and Phase II metabolic enzymes, which together act to reduce the level of stimulating chemical through active efflux and metabo-lism respectively. The expression of many drug metabolising enzymes and drug transport proteins are under control of members of the nuclear receptor superfamily 1. These ligand-activated transcription factors include members who predominantly regulate endogenous functions, including the glucocorticoid receptor (GR), the progesterone receptor (PR) and the androgen receptor (AR), and those that act as sensors for external chemical challenge, such as the pregnane X-receptor (PXR) and the constitutive androstane receptor (CAR). However, it is becoming increasingly clear that these nuclear receptors do not function in isolation, but contribute towards a complex regulatory signal network, which allows the control of overlapping target gene sets and the refinement of biological response(s) to individual chemical challenges. Such interactions are required to meet the challenge of balancing endogenous and exogenous, producing a sensitive response to challenge by external chemicals, yet maintaining robustness within endogenous metabolic processes. We have developed a deterministic model of the nuclear receptor interaction network that regulates expres-sion of target proteins involved in the response to both external chemical challenge and internal steroid metabolism based upon in vitro and in vivo derived data. Examining the known interactions between PXR and GR2,3, we initially developed a model that demonstrates the value within the network of feedforward and feedback loops in refining both the magnitude and duration of response to stress challenges. GR autoregulation is a requirement for the attenuation of biological response to stress signals, and to increase the rate of activation of the metabolic response, which is medi-ated via PXR. Finally, GR-mediated activation of PXR gene expression is important for controlling the duration of the physiological response to stress signals. In addition, we hypothesis that this response network forms part of a larger,

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canonical, regulatory signal network between PXR, GR, PR, PPARα, AR and FXR nuclear receptors, and present a more complex model demonstrating how such a network would function. Taken together these networks allow the rapid response to external chemical challenge whilst maintaining normophysiology.

References

Plant, N. and S. Aouabdi. Nuclear receptors: the controlling force in drug metabolism of the liver? Xenobiotica 2009; 39(8): 597-605Pascussi, J.-M. et al. Dexamethasone induces pregnane X receptor and retinoid X receptor-a expression in human hepatocytes: Synergistic increase of

CYP3A4 induction by pregnane X receptor. Mol. Pharm. 2000; 58: 361-372Oakley, R. H. and J. A. Cidlowski. Homologous down regulation of the glucocorticoid receptor: The molecular machinery. Crit. Rev. Euk. Gene Exp.1993;

3(2): 63-88.

P447. Assessment of Sodium Dodecyl Sulfate-Induced Irritation on a Preclinical Model of Pig Ear Skin Organ Culture

Daniel Bacqueville, Valérie Ortis, Anne-Sophie Jimenez, Marie Boisson and Hélène Duplan

Laboratoire de Pharmacocinétique Cutanée, Centre de Recherche et Développement Pierre Fabre Dermo-Cosmétique, Vigoulet-Auzil, France

The assessment of skin irritancy potential of xenobiotics is a major issue to ensure the consumer safety exposed to a wide range of substances designed for industrial, pharmaceutical or cosmetic use. Over the last few years, many different human skin models have been reconstructed in vitro and validated by the European regulatory agencies to replace animal experimentation in irritation testing. However, these alternative models have a limited barrier function that might overestimate the toxicological risk of a potential irritant. Since porcine skin is highly related to human and recommended by percutaneous absorption guidelines, the aim of this study was to investigate Sodium Dodecyl Sulfate (SDS)-induced irritation in a short-term ex vivo skin organ culture obtained from domestic pig ears, and to perform a multiple endpoint analysis to identify relevant irritation biomarkers in this preclinical skin model. The porcine skin organ culture was initiated by seeding explants in gelatin-coated polycarbonate Transwell® culture inserts in 12-well plate, and feeding them using Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with pyruvate, glutamine, fungizone and antibiotics. Thereafter, the explants were topically irritated with a single dose of SDS from 0.5 to 2% up to 24 hours before biochemical analysis. Skin viability was assessed using the classical tetrazolium salt reduction (MTT) assay, but also by measuring remaining glucose, lactate production and lactate dehydrogenase enzyme (LDH) leakage into the culture medium. In addition, the releasing of the pro-inflammatory cytokine interleukin 6 (IL-6) in response to SDS exposure was quantified by specific sandwich ELISA. All assays were validated from fresh and frozen skin samples cultured for 24 hours. Topical application of 2% SDS during 24 hours greatly increased LDH activity and IL-6 production in culture medium whereas the glucose consumption slightly decreased. These cytotoxic effects were time and dose-dependent, and were confirmed by histological analysis after hematoxylin eosin staining. In contrast, both MTT and lactate production tests were not enough sensitive to detect significant variations in pig skin viability following irritation. Altogether, these data showed that LDH and IL-6 assays might be relevant endpoint biomarkers to assess irritation in ex vivo porcine skin organ culture, and suggest that this preclinical model could be used as a surrogate to human skin for the risk assessment of skin irritancy potential of xenobiotics.

P448. Covalent Binding of Drug Candidates to Proteins as a Reactive Metabolite Risk Assessment Tool

Emre M. Isin1, Jesper Nilsson1, Johanna Midlöv1, Göran N. Nilsson2, Carina Leandersson1 and Richard A. Thompson1

1Biotransformation, Discovery DMPK, AstraZeneca R&D Mölndal, Mölndal, Sweden, 2Isotope Chemisty, Medicinal Chemistry, AstraZeneca R&D Mölndal, Mölndal, Sweden

Formation of reactive metabolites and their covalent binding to biological macromolecules, in particular proteins, have been proposed to be involved in adverse drug reactions (ADRs). Whereas predictable Type A toxicity is likely to be

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readily assessed in preclinical toxicology studies, Type B or idiosyncratic ADRs (IADRs) are difficult if not impossible to detect early on in the drug development process mainly due to the lack of appropriate animal models. In many cases, the rare events of IADRs are observed only after the launch of the drug when it reaches millions of patients which is an observation consistent with the proposed host-specific nature of these ADRs. Therefore, in the lack of any animal models to assess IADRs, and in an attempt to eliminate any potentially problematic drug candidates early on, pharmaceutical industry′s efforts in the recent years have focused on using the degree of covalent binding of drugs to proteins as a tool to progress safer drugs into development.[1] Although this measure can be used as a guideline for candidate selection, recent studies demonstrated that covalent binding by itself is not sufficient to predict IADRs in man.[2] In this study, we have assessed the degree of covalent binding in human hepatocytes for a set of radiolabelled marketed drugs covering a range of safety outcomes in patients. Our results, in agreement with recent studies,[3] showed that covalent binding can be used as a useful tool in risk assessment of drug candidates when viewed as one of the metabolic pathways in the context of the overall absorption, distribution, metabolism and elimination of the drug given at a certain daily dose. The analyses of the covalent binding results using different approaches to estimate the percentage of a given daily dose resulting in covalent binding (estimated “body burden”) will be presented. For a selected sub-set of compounds of interest, the mechanistic studies to understand better the pathways leading to covalent binding will also be discussed.

References

[1] Evans DC, Watt AP, Nicoll-Griffith DA, and Baillie TA. (2004) Drug-protein adducts: an industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem Res Toxicol 17:3-16.

[2] Bauman JN, Kelly JM, Tripathy S, Zhao SX, Lam WW, Kalgutkar AS, and Obach RS. (2009) Can in vitro metabolism-dependent covalent bind-ing data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction. Chem Res Toxicol 22:332-340.

[3] Nakayama S, Atsumi R, Takakusa H, Kobayashi Y, Kurihara A, Nagai Y, Nakai D, and Okazaki O. (2009) A zone classification system for risk assessment of idiosyncratic drug toxicity using daily dose and covalent binding. Drug Metab Dispos 37:1970-1977.

P449. Gene Profiling of Drug-metabolizing Enzymes in Normal Human Bronchial Epithelial Cells Exposed to Single-walled Carbon Nanotubes

Kotaro Hitoshi1, Miki Katoh1, Tomoko Suzuki2, Yoshinori Ando2 and Masayuki Nadai1

1Faculty of Pharmacy, Meijo University, Nagoya, Japan, 2Faculty of Science and Engineering, Meijo University, Nagoya, Japan

Currently, single-walled carbon nanotubes (SWCNTs) are attracting significant attention as innovative nanomaterials with a wide range of applications, including serving as drug-delivery carriers. However, research on the use of SWCNTs in the medical field is scarce. Further, the effects of SWCNTs on the human body are yet to be elucidated before they can be used in the medical field. Because the efficacy and toxicity of a drug are closely related to drug metabolism, we focused on investigating the effect of SWCNTs on the expression of drug-metabolizing enzymes. To elucidate the response of the human body to inhaled SWCNTs, we conducted gene profiling of drug-metabolizing enzymes in normal human bronchial epithelial (NHBE) cells. SWCNTs were manufactured by the arc electrical discharge method using Ni and Y as catalysts. SWCNTs were dispersed in cell culture medium with an ultrasonic homogenizer. NHBE cells were exposed to SWCNTs at the concentration of 0.1 mg/ml for 24 h. Expression of 84 genes including phase I and phase II drug-metabolizing enzymes were assessed using drug metabolism RT2 profiler PCR arrays (SA Biosciences). In addition, real-time PCR was performed to validate the results of the PCR Arrays. Exposure of NHBE cells to SWCNTs resulted in up-regulation of 4 genes by more than 2-fold as determined by PCR arrays. On the other hand, 15 genes were down-regulated by less than one half. We picked up 3 genes that were up-regulated (aldehyde dehydrogenase 1A1, glutathione S-transferase (GST) A4, hydroxysteroid (17-beta) dehydrogenase) and 5 genes that were down-regulated (carboxylesterase 2, cytochrome P450 (CYP) 1A1, CYP19A1, glutathione reductase, GSTM3) for real-time PCR analysis. CYP1A1 was the most affected enzyme, which was down-regulated 500-fold relative to unexposed cells. A strong correla-tion was observed between results of PCR array and real-time PCR. Additionally, real-time PCR analysis revealed that CYP1B1 expression was down-regulated 1,000-fold relative to unexposed cells. Among major CYPs that are expressed in the lung, expression of CYP3A5 and CYP4B1 were unchanged. Alterations in expression of drug-metabolizing enzymes after exposure to SWCNTs may affect the drug-metabolizing capacity of the lung, which may lead to the changes in

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pharmacokinetics of the co-administered drug. Moreover, we examined the differences in responses between normal cells and cells of the lung carcinoma cell line A549 against SWCNTs.

P450. Investigation into the incidence of control sample contamination in toxicokinetic samples generated during regulatory toxicology studies

Robert G. Turcan and Sarah Cahill

Programme Management, Covance Labs Inc, Harrogate, United Kingdom

In 2005 the EMEA issued regulatory guidelines on the evaluation of control toxicokinetic samples in non clinical safety studies. Significant contamination of toxicokinetic samples with test material may lead to the invalidation of a study and the guidelines require that the source of such contamination is investigated and if possible identified. This presentation will review the incidence of control sample contamination in non clinical studies conducted at Covance and elsewhere with particular focus on both its significance to overall study integrity and on the success of different control measures that can be used to try and minimise or avoid this problem.

P451. Methylation markers of arsenic associated urothelial carcinoma in peripheral blood leukocyte

Tse-Yen Yang1, Ling-I Hsu2 and Chien-Jen Chen3

1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, 2Genomic Research Center, Academia Sinica, Taipei, Taiwan, 3Genomics Research Center, Academia Sinica, Taipei, Taiwan

Epigenetics, the control of gene packaging and expression independent of alterations in the DNA sequence, is providing new directions linking genetics and environmental factors. Several epigenetic mechanisms, including DNA methyla-tion, histone modifications, and micro-RNA expression, can change gene function under exogenous influence. Current evidence suggests several environmental chemicals, such as arsenite, hexachlorobenzene, and nickel compounds, interfere normal gene function by alterations of DNA methylation. Recent findings show that aberrant DNA methylation participates in carcinogenesis and specific methylation profile can be a molecular hallmark of neoplastic cells. When consumed arsenic (As)-contaminated water or food, inorganic arsenic is enzymatically methylated and S-adenosyl methionine (SAM) is used as methyl group donor. DNA methylation catalyzed by DNA methyltransferases (DNMTs) also require SAM and the competition of SAM by DNA methylation and arsenic biotransformation may suggest a role of DNA methylation in arsenic carcinogenesis. To test the hypothesis that early life arsenic exposure may alter DNA methylation profile and be associated with increased risk of urothelial carcinoma, DNA methylation pattern between arsenic-associated urothelial transitional cell carcinoma and non-arsenic-associated tumors was compared by genome wide approach. Materials and Methods: Commercialized genome-wide method Illumina Infinium Methylation27 BeadChip, which includes 27,578 methylation sites, was used in the study. The study subjects were recruited from Chi-Mei hospital, which is located near arseniasis-endemic area. The specimens from the participants who lived in non-arseniasis-endemic areas or never consumed deep-well water were defined as “ non-As-associated (non-As) “ and the ones from the subjects with high exposure (cumulative arsenic exposure, CAE>5 mg/L*years) were defined as “As-associated (As) “. Three specimens of each subject including tumor tissue, adjacent normal tissue and peripheral blood cell were included for the study. Each pair of As-associated and non-As-associated tumors were matched on age, gender, smoking status and the tumor stage. Bisulfite conversion of specimens was performed by using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA), according to the manufacturer′s recommended protocol. DNA methylation status was assessed using Illumina Infinium Methylation27 beadsarray from Illumina (La Jolla, CA). Results: The correlation of DNA methylation pattern between tumor and adjacent normal and the correlation between tumor and peripheral blood is consistently higher among four “As-associated” subjects than that among “non-As-associated” ones The correlation between adjacent normal tissue and peripheral blood was quite close. Mean β value, the index of DNA methylation of each site, was used to screen for the sites showing striking differences between As-associated and non-As-associated specimens. Our preliminary results suggested five sites showing the difference of mean β value greater than 0.15 between As-associated and non-As-associated samples and the differences were consistently observed in three types of specimens. Although the sample size was limited, our findings suggested that chronic arsenic exposure

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may induce systemic DNA methylation in the organ and peripheral blood cells. Although the half life of peripheral blood leukocyte is quite short, some marked alterations of DNA methylation remains and can be used as the indicator of arsenic intoxification as well as detection markers of internal malignancy.

P452. Occurrence of ochratoxin A in various food products in Turkey

Buket Alpertunga, Sibel Ozden and Ayse Sibel Akdeniz

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey

Ochratoxin AspergillusPenicilliumwine, coffee, cacao, spices or dried fruits, resulting in chronic human exposure. The aim of our study was to investigate the presence of OTA in widely consumed food products such as cereals, cereal-derived products, cereal-based baby foods, dried grapes, grape juices, pekmez, boiled and concentrated grape juice and tarhana, a traditional fermented and dried yoghurt cereal food commercialized in Turkey. For this purpose, totally 227 samples were collected from different supermarkets and traditional bazaars in Istanbul during 2008–2010 years. The analytical methods used in our study involved the liquid/liquid extraction of OTA, immunoaffinity (IAC) and HPLC quantification with fluorescence detection. The detection limit of OTA was and OTA identity was confirmed by methyl ester formation. The Scientific Commission of the European Community have regulated the maximum allowable limits for unprocessed cereals (5 ppb), all products derived from cereals (3 ppb), processed cereal-based foods and baby foods for infant and young children (0.5 ppb), for grapes (10 ppb) and for grape juices (2 ppb). All cereal-derived products and grapes examined contained OTA at concentrations much lower below the limit recom-mended by European Commission Regulation. Only, 12 pekmez samples with OTA, with a higher concentration then the maximum allowable limits.

P453. Semi-quantitative assessment of reactive metabolite formation of 60 marketed drugs using the bi-functional trapping reagent dansyl-γGlu-Cys-Lys

Ralf Schmidt and Rosemarie Panetta

Dept. In Vitro Biology and DMPK, AstraZeneca R&D Montreal, St.Laurent, QC, Canada

The occurrence of certain types of drug-induced toxicities (DIT) is likely due to the existence of reactive metabolites (RM). In order to reduce the toxicological liabilities of new potential drug molecules in drug discovery, it is crucial to elucidate the structure of the reactive species and explore the metabolic pathways leading to their formation. The detection of reactive species alone is not sufficient for a meaningful RM risk assessment; quantitative information about the amount of reactive species formed is essential to guide medicinal chemistry efforts and to develop risk mitigation strategies. A simple, efficient and sensitive method to quantify RM is the use of glutathione (GSH) modified by addition of a fluorescence label. This approach was used recently to characterize highly prescribed drugs for their potential for drug induced toxicity (DIT) based on the extent of adduct formation (1). In the present study, the bi-functional trapping reagent dansyl-γ-glutamyl-cysteinyl-lysine (dECK, 2), able to react with soft and hard electrophiles such as reactive aldehydes, was used to characterize and quantify the adduct formation for 60 marketed drugs, including 23 drugs previously reported by Gan et al. (1). Incubations were performed at 10μM and/or 50μM drug concentration using human liver microsomes (1mg/mL) in the presence of NADPH (1mM) and dECK (2mM) for 30min at 37°C. In control experiments dECK was replaced by GSH to allow a comparison of adduct structure and trapping capabilities for both reagents. All samples were analyzed on an Acquity UPLC-QTof Premier system (Waters) allowing the simultaneous analysis by UV, quantification of the fluorescence signal and the structural characterization of the trapped adducts. Reactive metabolites were identified and quantified for 19 drugs using dECK, 15 of them were also confirmed in the GSH incubations, whereas for additional three drugs GSH adducts were only detected at 50μM. Interestingly, with the exception of pioglitazone all incubations at 10μM resulted in higher percentage of adducts trapped although the signal/noise ratio was better for incubations at 50μM. In summary, adducts for acetaminophen, amodiaquine, carbamazepine, clozapine, lansoprazole, losartan, mianserin, naltrexone, nefazodone, nimesulide, nomifensine, olanzepine, paroxetine, pioglitazone, raloxifene, risperidone, rosiglitazone, ticlopidine and troglitazone were quantified. The potential daily RM body burden was calculated by incorporating the amount adduct formed and the metabolic turnover obtained in the experiment, maximum recommended daily dose, fraction absorbed and fraction of oxidative metabolism from

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published data. The results indicate a separation of drugs considered to be safe from those with known DIT with a safe RM body burden around 1mg per day. In conclusion, dECK provides a valuable tool for the quantification of RM to guide compound optimization and combined with additional parameter to support the RM risk assessment for potential new drugs.

References

(1) Gan, J. et al. (2008) Chem. Res. Toxicol. 22, 690-698 (2) Schmidt, R. et al. (2008) Drug Metabolism Reviews 40, 221

P454. Human PPAR&alpha is regulated by miR-21

Katsuhiko Kida, Miki Nakajima, Takuya Mohri, Yuki Oda, Shingo Takagi, Tatsuki Fukami and Tsuyoshi Yokoi

Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

Peroxisome proliferator-activated receptor α (PPARα) belongs to the steroid hormone nuclear receptor superfamily and regulates genes encoding endo/xenobiotic and lipid metabolizing enzymes. In silico analysis raised many kinds of miRNA that may bind to 3′-untranslated region (UTR) of human PPARα. Among them, we focused on miR-21 as a potential regulator of PPARα, because it was commonly predicted by some algorisms including TargetScan and PicTar, and it showed the highest score and is one of the abundant miRNAs expressed in liver. When the precursor and antisense oligonucleotide for miR-21 were transfected into HuH7 cells, the endogenous PPARα protein level was significantly decreased and increased, respectively, but the PPARα mRNA level was not. The results suggest that the miR-21 negatively regulates the PPARα expression via translational repression. In the 3′-UTR of human PPARα mRNA, two potential recognition elements for miR-21, MRE21_1 (+2467 to +2495) and MRE21_2 (+3733 to +3757), were pre-dicted. Reporter plasmids containing each potential MRE were constructed and luciferase analysis was performed. The overexpression and inhibition of miR-21 in the cell lines transfected with the reporter plasmids revealed that only the MRE21_1 is functional. Finally, using a panel of 24 human livers, we found that the PPARα protein levels were not correlated with the PPARα mRNA levels, but were inversely correlated with the miR-21 levels. The result suggests that the miR-21-dependent regulation substantially impacts on the PPARα expression in human liver. In conclusion, we found that human PPARα is post-transcriptionally regulated by miR-21. We will report the effects of miR-21 on the expression of target genes of PPARα such as CYP4A and acyl-CoA synthetase. This study could provide new insights of miR-21 into the metabolism of lipid and xenobiotics, in addition to previous findings that miR-21 is associated with tumor progression, cell signaling, and aging.

P455. Participation of MicroRNAs in Phenobarbital-induced Changes in the Expression of Genes Encoding Drug-metabolizing Enzymes and Transporters in Mouse Liver

Satoshi Numazawa, Chiharu Murakami, Ryota Shizu and Takemi Yoshida

Department of Biochemical Toxicology, Showa University School of Pharmacy, Tokyo, Japan

MicroRNAs (miRNAs) play an integral role in the post-transcriptional control of gene expression. Although evidence, which proves involvement of miRNAs in the normal development and physiology as well as pathogenesis of diverse diseases such as cancer and disruption of immune system, is increasingly accumulated, its participation in the drug-induced change in gene expression is obscure. The present study demonstrates comparison of miRNA and gene expres-sion profiles induced by phenobalbital used as a model drug in the liver of mice. Total RNA extracted from liver of male C57BL/6 mice treated with phenobarbital for 12, 24 or 48 h was subjected to DNA and miRNA microarray technology (Agilent). Levels of mRNA and miRNA were determined by real-time RT-PCR using TaqMan probes. Reporter plasmids were constructed on pGL3 vector by inserting 3 tandem repeats of mir-29b and mir-30a target sequences in the 3′UTR of mrp3 and cyp2c29 mRNAs, respectively. These reporter vectors were transfected into mouse liver in vivo and reporter activity was evaluated 12 h later. DNA microarray analysis revealed that phenobarbital increased levels of mRNA for 10

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transporters and 43 enzymes involved in drug metabolism >2-fold any time point after treatment. miRNA microarray analysis revealed that phenobarbital increased 22 miRNAs and decreased 12 miRNAs >1.3-fold. miRNAs and mRNAs changed by the treatment were paired by using the target searching tools such as MicroCosm Targets and miRGen. In addition, time course changes in the levels of these miRNAs and mRNAs were precisely determined by real-time RT-PCR. Based on these observations, we focused on pairs of cyp2c29 and mir-30a and mrp3 and mir-29b because these genes possess the target sequence of respective miRNAs and these pairs of mRNA and miRNA levels changed reciprocally in terms of the time. Phenbarbital increased cyp2c29 mRNA and decreased mir-30a levels. In addition, mir-29b levels significantly increased 48 h after the treatment when increased mrp3 mRNA levels returned to the basal level. Transfection in vivo of the mir-30a and mir-29b target vectors indicated that phenobarbital significantly decreased the reporter activity to 10% and 62% of the control, respectively. Co-transfection of the mir-30a mimic decreased the reporter activity to 64%. These results indicate that phenbarbital induces changes in the level of diverse miRNAs in addition to mRNAs. It is suggested that gene expression of cyp2c29 is regulated by mir-30a and phenobarbital-mediated down-regulation of this miRNA would be involved in the cyp2c29 induction. Moreover, machinery controlled by mir-29b could be involved in the recovery phase of up-regulated mrp3 after treatment with phenobarbital.

P456. Comparison of aflatoxin B1-induced cytotoxicity and genotoxicity in hepatocyte-like cells and their progenitor mesenchymal stem cells isolated from human umbilical cord blood

Abdolamir Allameh1, Masoumeh Ghaderi1 and Masoud Soleimani2

1Biochemistry and Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran, 2Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

High rates of cell division and the proliferation nature of the stem cells are important factors responsible for enhanced susceptibility of these cells to toxigenic and mutagenic agents. Recently we showed that the hepatocyte-like cells dif-ferentiated from bone marrow mesenchymal stem cells (MSCs) express different classes of cytochrome-P450 (CYP) and glutathione S-transferase (GST) [1-3]. The susceptibility of different stem cells and their progenitor cells depends on various factors particularly the expression of xenobiotic enzymes. In the present study, for the first time hepatocyte-like cells and their progenitor MSCs isolated from human umbilical cord blood (UCB) were treated with a potent hepatotoxic agent, i.e., aflatoxin B1 (AFB1). After 24 h of exposure the cell viability (MTT assay) and DNA damage (Comet assay) were measured. The results showed that the Inhibitory Concentration (IC50) of AFB1 measured in hepatocytes and MSCs was approximately 45 ?M. The results obtained from Comet assay showed that the DNA damage expressed in terms of %DNA in tail is about two-fold higher in differentiated hepatocytes as compared to that measured in MSCs. AFB1 at concentrations of 0, 2.5, 10 and 20 uM caused ∼15, 55, 65 and 70% DNA in tail respectively. Higher rate of DNA damage measured in differentiated cells as compared to undifferentiated cells may suggest that the differentiated hepatocytes are more sensitive to AFB1-induced DNA damage. The differences in DNA damage induced by AFB1 in stem cells before and after differentiation can be assigned to the differences in the expression of CYP enzymes involved in AFB1 biotransformation. Currently the CYP profile and their inducibility by AFB1 as well as the DNA repair capacity of these cells are under investigation in our laboratory. References 1. Allameh, A., Esmaeli, S., Kazemnejad S., Soleimani M. (2009).Differential expression of glutathione S-transferases P1-1 and A1-1 at protein and mRNA levels in hepatocytes derived from human bone marrow mesenchymal stem cells. Toxicology In Vitro, 23: 674-679. 2. Kazemnejad S., Allameh A., Gharebaghian A, Soleimani M., Mohammadi Y., Amirizadeh N. and Jazayeri M. (2009). Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold. J Gastroenterol Hepatol. 24: 278-87. 3. Kazemnejad S., Allameh A., Gharebaghian A, Soleimani A., Amirizadeh N. (2008). Efficient replacing of fetal bovine serum with platelet lysate during propagation and differentiation of human bone marrow derived mesenchymal stem cells to functional hepatocytes-like cells. Vox Sanguinis (Journal of International Society of Transfusion Medicine), 95:149-58.

P457. Cytochrome P450 BM3 Mutants as NOVEL Biocatalytic TOOLS for Regioselective Hydroxylation of Steroids

Vanina Rea1, A.J. Kolkman2, E. Vottero1, M. Honing3, S.S. Wijmenga2, N.P.E Vermeulen1 and J.N.M. Commandeur1

286

1Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2Department of Biophysical Chemistry, Institute of Molecules and Materials (IMM), Nijmegen, Netherlands, 3MSD-Merck, Oss, Netherlands

Microbial transformations of steroids provide a potentially important method for obtaining new steroid deriva-tives with pharmaceutical activity. However, highly specific reactions are required to produce functionalized steroids with potential therapeutic use and commercial value. The complexity of steroid molecules renders the use of biocatalysts particularly interesting, due to the high regio- and stereo-selectivity of the reactions to be performed. Cytochome P450 BM3 (CYP102A1) from Bacillus megaterium is considered as one of the most promising monoxygenases for biotechnological applications. Several mutants of Cytochome P450 BM3 with high activity towards drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts for the stereoselective hydroxylation of steroids has been investigated. Using a rational site-directed mutagenesis approach, we introduced single mutations at position 82 into the substrate binding pocket of a known drug-metabolizing P450 BM3 mutants, which greatly changed the product pattern of Testosterone and Norethisterone hydroxylation. The single mutation in position 82 led to the stereoselective hydroxylation of the 16b-position of these steroids with high activity and high selectiv-ity (e.e. >90%). Nuclear Magnetic Resonance (NMR) was used for the metabolite identification. Spin relaxation NMR revealed information on the orientation of Testosterone in the active site of the mutants and rationalized the selectivity observed.

P458. Enantioselective Metabolism of Flurbiprofen by Human Cytochrome 2C9 and its Allelic Variants

Y. Q. Liu1 and S. Zeng2

1College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China, 2Department of Pharmaceutical Analysis and Drug Metabolism, Zhejiang University, Hangzhou, China

Flurbiprofen is a chiral nonsteroidal anti-inflammatory drug (NSAID) of the 2-arylpropionic acid class. The major oxidative pathway in flurbiprofen metabolism is to a 4¡ä-hydroxy metabolite and Cytochrome 2C9 is the only isoform of CYP450 involved in this process (Tracy et al, 1996). Flurbiprofen exhibits stereoselectivity in its pharmacokinetics (Davies, et al, 1995). Roles of CYP2C9 and genetic polymorphism in this stereoselectivity need to be investigated. In our study, the S- and R-flurbiprofen enantiomer were used as substrate in incubation with recombinant human CYP2C9 and its functional mutants *3,*13 and *16 which were expressed by Bac to Bac system. The formation of metabolite 4¡ä-hydroxyflurbiprofen was determined by Waters UPLC/MS/MS method developed in our lab. The S/R-flurbiprofen ratio of Vmax/Km is around 2 folds by the metabolism of CYP2C9 wild type and its mutants studied. The differences in the Vmax/Km values of S- and R-enantiomer between CYP2C9 wild type and its mutants vary from 6.0∼13.6 folds and 6.5∼11.6 folds respectively. For both S- and R- enantiomer, CYP2C9*3 exhibits the smallest Vmax, whereas the greatest reduction of Vmax/Km value comes from CYP2C9*13. CYP2C9*13 had higher Km than wild type and other two mutants for each enantiomer while CYP2C9*3 showed similar Km with wild type. In summary, these results indicated that CYP2C9 shows stereoselectivity in flurbiprofen metabolism, preferentially involved in the clearance of S-enantiomer. Genotype doesn¡¯t change this preference. The three functional variants exhibit reduced activity in the metabolism of flurbiprofen with the genotype dependent. Vmax and Km being differentially altered by different allelic variants suggests that the activity reduction of these mutants may be associated with different kinetic mechanism. Keywords: Cytochrome 2C9, Allelic Variants, Flurbiprofen, Enantioselective Metabolism References Davies NM (1995) Clinical pharmacokinetics of flurbiprofen and its enantiomers. Clin Pharmacokinet.28:100-114. Timothy S. Tracy, et al (1996) Studies of Flurbiprofen 4¡ä-Hydroxylation: Additional evidence suggesting the sole involvement of cytochrome P450 2C9. Biochem Pharmacol.52:1305-1309.

P459. Stereoselective Glucuronidation of Bufadienolide Epimers by Human Liver Microsomes: Effects of Structure Variants on Glucuronidation

Jing Ning1, Guang-Bo Ge2, Si-Cheng Liang3, Xiao-Chi Ma4 and Ling Yang3

287

1School of Pharmaceutical Sciences, Dalian Medical University, Dalian, China, 2Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 3Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China, 4School of Pharmaceutical Sciences, Dalian Medical University, Dalian, China

Bufadienolides, a class of natural products with a unique steroidal skeleton were separated and identified from tradi-tional Chinese medicine Chansu, has been reported with Na+-K+-ATPase inhibitory effects and outstanding antitumor activities [1,2]. In the current study, the glucuronidation of three pairs of bufadienolide epimers including Cinobufagin (CB), deacetylcinobufagin (DCB) and bufalin (BF), as well as their corresponding 3-epimers (ECB, EDCB and EBF), were investigated by using human liver microsomes (HLM). Interestingly, among these investigated compounds, only EDCB and EBF can form corresponding glucuronidated metabolite when the substrate was incubated with HLM in the presence of uridine-5′-diphosphoglucuronic acid (UDPGA), while no metabolites of CB, DCB, BF or ECB were detected under the same conditions. The EDCB mono-glucuronide and EBF mono-glucuronide were characterized by using LC-MS/MS, and these two glucuronidated metabolites were also confirmed by hydrolysis with β-glucuronidase. Further investigation on EDCB glucuronidation in human liver microsomes was carried out, due to the detection limitation for quantification of EBF glucuronide. A combination of assays with a panel of recombinant human UGT isoform(s) and chemical inhibition study were used to explore the involved isoform(s) in the EDCB glucuronidation, only UGT2B7 exhibited the catalytic activity toward EDCB glucuronidation, while the inhibitory effect of mefanamic acid (a potent inhibitor of UGT2B7) for EDCB glucuronidation in HLM was similar to its in recombinant UGT2B7. These results revealed that human UGT2B7 was the major isoform involved in the glucuronidation of EDCB. The formation of EDCB glucuronide in pooled human liver microsomes can be well fitted to a substrate inhibition model and the kinetic parameters of EDCB glucuronidation were as follows: K

m=48.4 ± 12.6 µM, K

si=342.2 ± 95.3 µM, and V

max=1.6 ± 0.3 nmol/

min/mg of protein. In summary, this study revealed a stereoselective glucuronidation pathway to bufadienolides, and our results indicated that the structure variants of these epimers including the C-16 ester and the configuration of C-3 hydroxyl may influence the glucuronidation of bufadienolides.

P460. OATP1B1-Mediated Uptake of Statins, Estrone 3-sulfate and Estradiol 17β-glucuronide in HEK293-OATP1B1 Cells and Inhibition by Rifamycin SV, Gemfibrozil and an Investigational Drug

Pradeep Sharma1, Caroline J. Butters2, Veronica Smith2, Robert Elsby2, Peter J. Lawrence2 and Dominic Surry2

1Clinical Pharmacology and DMPK, AstraZeneca R&D Charnwood, Loughborough, United Kingdom,2Clinical Pharmacology and DMPK, AstraZeneca R&D Charnwood, Loughborough, United Kingdom

OATP1B1 is an important hepatic uptake transporter shown to effect the therapeutic efficacy, drug-drug interaction potential, pharmacogenetics and toxicity of drugs [1]. Among the most commonly prescribed medications for older patients are lipid lowering HMG-CoA inhibitors (statins) which are substrates of OATP1B1, making this transporter important for close scrutiny during drug discovery and development. We have previously published an approach using the HEK293-OATP1B1 cell line for studying compounds as substrates and inhibitors of OATP1B1 [2]. Here we describe the use of this cell line to investigate kinetics of six statins (simvastatin, atorvastatin, pravastatin, rosuvastatin, pitavas-tatin, fluvastatin), estradiol 17β-glucuronide and estrone 3-sulfate and inhibition of their OATP1B1-mediated transport by investigational compound and known prototypical inhibitors (gemfibrozil and rifamycin SV). The study included determination of time linearity of substrates, apparent Michaelis-Menten constant (apparent K

m) and IC

50 values. The

results obtained demonstrated that Km

values of statins were in range of 0.6-29 μM, with atorvastatin exhibiting highest and pravastatin exhibiting the lowest affinity for OATP1B1. Estrone 3-sulfate exhibited biphasic transport kinetics com-prising both high affinity (K

m = 0.4 μM) and low affinity components (K

m = 42 μM). The Michaelis-Menten constant (K

m)

of simvastatin could not be determined due to its high passive permeability. The investigational drug inhibited OATP1B1 mediated transport of all statins and estradiol 17β-glucuronide and weakly inhibited estrone 3-sulfate. Rifamcycin SV significantly inhibited OATP1B1-mediated transport of all substrates while gemfibrozil exhibited weak inhibition for all statins, estradiol 17β-glucuronide and estrone 3-sulfate. Based on results obtained estradiol 17βglucuronide appears to be a good surrogate probe substrate to predict OATP1B1 based statin DDIs in vitro using this cell line. These studies have also helped shape the future clinical DDI program of the investigational compound.

288

References

(1) Kalliokoski A, Niemi M. Impact of OATP transporters on pharmacokinetics. Br. J. Pharmacol. (2009), 158: 693-705. (2) Sharma, P, Holmes, VE, Elsby, R, Lambert, C, Surry, D. Validation of cell-based OATP1B1 assays to assess drug transport and the potential for

drug-drug interaction to support regulatory submissions. Xenobiotica (2010), 40: 24-37.

P461. Role of Mrp3-mediated efflux in systemic exposure of SN-38, the active metabolite of irinotecan

Yoshiaki Kitamura1, Hiroyuki Kusuhara2 and Yuichi Sugiyama2

1Discovery Research Laboratories, Kyorin Pharmaceutical Co. Ltd., Tochigi, Japan, 2Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Irinotecan is an ester-type prodrug of SN-38, which is used for anticancer chemotherapy. Hydrolysis of irinotecan by carboxylesterase yields SN-38, which then undergoes biliary excretion or glucuronidation. Previously, the associa-tion of transporter function with adverse reaction to irinotecan, diarrhea and neutropenia, has been reported for OATP1B1 and MRP2, which account for hepatic uptake and canalicular efflux, respectively. This study was performed to examine the importance of an ABC transporter, Mrp3/Abcc3, in the disposition of SN-38 during irinotecan infusion using Mrp3(-/-) mice. Mrp3 is expressed in the sinusoidal membrane of hepatocytes and the basolateral membrane of enterocytes, and functions as an efflux pump from inside the cells to the blood circulation. When irinotecan was given to wild-type and Mrp3(-/-) mice by intravenous constant infusion, there were no differences in plasma concentrations of irinotecan, whereas those of SN-38 were significantly lower in Mrp3(-/-) mice (36%) compared with wild-type controls. The hydrolytic activity of irinotecan to SN-38 as well as the sequestration clearance (glucuronidation and canalicular efflux) of SN-38 in the liver were similar between wild-type and Mrp3(-/-) mice. When SN-38 was given intravenously, dysfunction of Mrp3 had no effect on the systemic clearance of SN-38. These results suggest that Mrp3 mediates the sinusoidal efflux of SN-38 in the liver. As the hepatic elimination of SN-38 was similar to the hepatic blood flow rate, whether the dysfunction of Mrp3 increased the hepatic elimination of SN-38 (vectorial transport of SN-38 from blood to bile) remains unclear. In addition to the liver, to clarify the role of Mrp3 in the intestine, irinotecan was added to the serosal side of the everted sacs prepared from the intestine of wild-type and Mrp3(-/-) mice. Mrp3 dysfunction markedly reduced the serosal efflux of SN-38 glucuronide, but not SN-38, in the jejunum. The permeability surface area product of SN-38 glucuronide across the serosal membrane in Mrp3(-/-) mice was reduced to 17% when compared to wild-type controls. In conclusion, Mrp3 mediated the sinusoidal efflux of SN-38 formed from irinotecan in the liver, playing a significant role in the systemic exposure of SN-38, but had a limited role in the intestine. Acknowledgement: We thank Dr. Piet Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for supplying Mrp3(-/-) mice.

P462. Involvement of Multidrug Resistance 1 (MDR1) for the Pharmacokinetics of YM155, a Novel Survivin Suppressant

Megumi Iwai1, Tsuyoshi Minematsu1, Qun Li2, Takafumi Iwatsubo1 and Takashi Usui1

1Drug Metabolism Research Laboratories, Astellas Pharma Inc., Osaka, Japan, 2Translational & Development Pharmacology,Applied Pharmacology Research Laboratories, Astellas Pharma Europe BV, Leiderdorp, Netherlands

Background: MDR1 (also known as P-gp) is an efflux transporter expressed in various tissues. The in vitro experiments for identifying whether a drug candidate is a MDR1 substrate and/or inhibitor are important in estimating its pharma-cokinetics and the possibility of drug-drug interactions.YM155 is a small molecule that suppresses survivin expression, which is a member of the inhibitor of apoptosis protein (IAP) family, and exhibits potent antitumor activities in multiple xenograft models including human hormone refractory prostate carcinoma cell lines. Several experiments have been performed with regard to an interaction between YM155 and MDR1. A bi-directional transporter assay using Caco-2 cells showed low permeability and no-vectorial transport of YM155, indicating that YM155 is not a substrate of MDR1. However, a growth inhibition assay using cancer cell lines suggested that MDR1 is involved in YM155 efflux from cancer

289

cells. Because of these apparently incompatible results, it was still an open question whether MDR1 transports YM155. Since YM155 has a hydrophilic property, its transporter-mediated uptake into the cell monolayer appears to be an important step in vectorial transport, and previous studies have suggested that organic cation transporter 1 (OCT1) is one of the transporters responsible for the hepatic uptake of YM155.

Method: In the present study, OCT1/MDR1-LLCPK1 cells, which express both basal uptake transporter OCT1 and apical efflux transporter MDR1, were established and used to examine vectorial transport of YM155. For the control experiments, OCT1-, MDR1-, and mock-LLCPK1 cells were also used.

Results and Conclusion: OCT1/MDR1-LLCPK1 cells showed vectorial transport of 14C-YM155 (1 μmol/L), and its flux ratio was 16.6 after 180-min incubation. In addition, the transport observed in OCT1/MDR1-LLCPK1 cells was inhibited by a MDR1 inhibitor (cyclosporin A) and an OCT1 inhibitor (1-methyl-4-phenylpyridinium). The vectorial transport was not observed in the other cell lines including MDR1-LLCPK1 in the control experiments, suggesting a necessity of the expression of both uptake (OCT1) and efflux (MDR1) transporters for the transcellular transport of YM155. It was therefore possible that insufficient expression of basal uptake transporter in Caco-2 cells might lead to an erroneous conclusion that YM155 is not a substrate of MDR1. These results of OCT1/MDR1-LLCPK1 cells indicate that YM155 is a substrate for both OCT1 and MDR1. MDR1 may play a role in the pharmacokinetics of YM155.

P463. Validation of BCRP and MRP2 vesicle transport assays for regulatory submissions

Veronica Smith1, Robert Elsby1, Lisa Fox2, David M. Stresser2, Caroline J. Butters1, Pradeep Sharma1 and Dominic Surry1

1Clinical Pharmacology and DMPK, AstraZeneca R&D Charnwood, Loughborough, United Kingdom,2BD Gentest Contract Research Services, BD Biosciences Discovery Labware, Woburn, MA, USA

Drug transporters have a major impact on the absorption, distribution, metabolism and excretion (ADME) of a diverse number of drugs. Breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 2 (MRP2, ABCC2) are adenosine 5′triphosphate (ATP)-binding cassette (ABC)-transporters that utilise ATP hydrolysis to provide energy for the efflux of drugs or xenobiotics from the cell. An understanding of the role that BCRP and MRP2 can play in the ADME of candidate drugs, and an assessment of how these processes might impact on toxicity and the potential for drug-drug interactions in the clinic, is required to support drug development and registration. Pivotal to any such assessment is the prior evaluation of a candidate drug as a substrate or inhibitor of these transporters using in vitro test systems that have been formally validated; a process we have previously demonstrated for P-gp (1) and OATP1B1 (2). Here we describe the validation of insect-cell derived and mammalian cell-derived membrane vesicle-based test systems to evaluate candidate drugs as substrates and inhibitors of BCRP and MRP2. The study included assessment of protein and time linearity for subsequent determination of apparent K

m (on three occasions) of the BCRP and MRP2

probe substrates estrone 3sulfate and estradiol 17βglucuronide, respectively. Assessment of other known BCRP sub-strates (methotrexate and rosuvastatin) and MRP2 substrates (pravastatin and carboxydichlorofluorescein) was also performed. Inhibition of transport was assessed on three occasions through the determination of IC

50 values against

the probe substrates above for the known BCRP and MRP2 inhibitors sulfasalazine, fumitremorgin C and novobiocin, and benzbromarone, MK-571 and terfenadine, respectively. The kinetic and inhibition data generated was comparable between the insect cell- and mammalian cell-derived test systems and has demonstrated the methodologies to be both robust and reproducible, thereby eliminating the need for assay repetition as currently proposed by the FDA. Subsequently using the validation data we have selected suitable positive control reference compounds and developed acceptance criteria to quality control future use of the BCRP/MRP2 substrate and inhibition assays in the routine evaluation of candidate drugs.

References

(1) Elsby R, Surry DD, Smith VN, Gray AJ. Validation and application of Caco2 assays for the in vitro evaluation of development candidate drugs as substrates or inhibitors of Pglycoprotein to support regulatory submissions. Xenobiotica 38(7/8): 1140-1164 (2008).

(2) Sharma P, Holmes VE, Elsby R, Lambert C, Surry D. Validation of cell based OATP1B1 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions. Xenobiotica 40(1): 24-37 (2010).

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P464. Charactrization of the In Vitro Uptake Mechanism and Kinetics of Sodium Fluorescein in Suspended Rat Hepatocytes

Sarinj Fattah, Tom De Bruyn, Patrick Augustijns and Pieter Annaert

Laboratory for Pharmacotechnology and Biopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium

Sodium fluorescein (NaFluo), a commonly available dye used for diagnostic purposes, has never been fully characterized as a probe substrate for active hepatic uptake (1). The cellular uptake mechanisms and kinetics of NaFluo were assessed in vitro using freshly-isolated rat hepatocytes in suspension. Incubations were performed at several concentrations (1 to 80 μM) at 37°C, and also at 4°C to correct for passive uptake. HIV protease inhibitors (PI) were used as model interacting drugs (2). The initial uptake rate of NaFluo (0-90 sec) was saturable and followed Michaelis-Menten kinetics: Km and Vmax were 18.7 ± 1.6 μM and 91.3 ± 3.8 pmol/million cells/min, respectively, yielding an active uptake clearance of 72.6 μl/million cells/min. Co-incubation with inhibitors of the sodium-independent transporter family Oatp (Organic anion transporting polypeptide) (3), resulted in a significant reduction of NaFluo uptake: rifampicin (10μM; 63% of control), bromosulphophthalein (10μM; 49% of control), quinine (10μM; 63% of control). A minor contribution of the sodium-dependent transporter Ntcp (sodium taurocholate cotransporting polypeptide) could not be excluded, while inhibitors of the Oat (Organic anion transporter) and Oct (Organic cation transporter) family had no significant effect. A concentration-dependent inhibition of active NaFluo uptake was observed by HIV PI, yielding the following Ki values: ritonavir (0.4 μM); darunavir (3.5 μM); atazanvir (4.7 μM); saquinavir (4.6 μM) and amprenavir (13 μM). In conclusion, our findings illustrate the utility of NaFluo as a commonly available probe substrate for studying Oatp- mediated hepatic uptake and associated interactions.

References

Gebhardt R, Jung W. Biliary excretion of sodium fluorescein in primary cultures of adult rat hepatocytes and its stimulation by nicotinamide. J of cell Sci. 56: 233-244 (1982).

Ye ZW, Augustijns P, Annaert P. Cellular accumulation of Choly-glycylamido- fluorescein in sandwich cultured rat hepatocyte: kinetic characterization, transport mechanisms and effect of HIV protease inhibitors. Drug Metab Disp. 36 (7): 1315-1321 (2008).

Shitara Y, Sugiyama D, Kusuhara H, Kato Y, Meier PJ, Itoh T, Sugiyama Y. Comparative inhibitory effects of different compounds on rat Oatp1 (Slc1a1)- and Oatp2 (Slc1a5)- mediated transport. Pharm Res. 19 (2): 147-153 (2002).

P465. Quantitative Visualization of Bile Canalicular Transporter Activity by Time-Lapse Imaging to Evaluate Drug-Drug Interaction in Sandwich-Cultured Hepatocytes

Ikumi Tamai, Yoko Fukuyama, Yuta Shibue, Yoshiyuki Shirasaka and Takeo Nakanishi

Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan

It is cumbersome to evaluate activity of transporter that functions as an efflux pump or is expressed in intracellular organelles. We explores a new imaging method to enable quantitative assay of the multidrug associate protein 2 (MRP2) transport activity by monitoring disposition of fluorescent substrates of MRP2 under fluorescent microscopy using sandwich-cultured hepatocytes (SCRHs) in rats. Carboxydichlorofluorescein (CDF) produced by de-esterification of CDF diacetate (CDFDA), a well-documented fluorescent substrate of MRP2 was used. Fluorescence from the canalicular space was visualized by live-cell time-lapse imaging under fluorescent microscope (BZ-9000, Keyence, Osaka, Japan). Function of rat Mrp2 was characterized by quantifying alteration in fluorescence from CDF accumulated in the canalicu-lar space in the presence of several types of MRP2 inhibitors, including MK571 selective to MRP2. Fluorescent intensity observed in the canalicular space was higher than that in cytoplasm or blank area within the visualized field under the fluorescent microscopy, and linearly increased for up to 5 min. In SCRHs from Eizai Hyper-bilirubinemnia Rat (EHBR) or in the presence of MK571, no significant increase in fluorescent intensity for 5 min was observed in the canalicular space, suggesting the accumulation of CDF is due to rat Mrp2 expressed in canalicular membranes in the SCRHs. Addition of Mrp2 inhibitors, e.g. cyclosporin A and rifampicin, decreased rate of CDF accumulation into the space in a concentration dependent manner. Based on the extracellular concentration of the MRP2 inhibitors, cyclosporin A and rifampicin, the concentrations to inhibit 50% of fluorescent intensity (IC50) in the canalicular space were estimated at

291

1.63 and 3.02 μM, respectively. These observations were consistent with those obtained by conventional BEI method, suggesting that current imaging method is useful for quantitative evaluation. Furthermore, these values were 3.1- and 6.7-fold lower, respectively, than those obtained by membrane vesicle study prepared from rMrp2-expressing Sf9 cells. However, IC50 value of rifampicin, which is transported via organic anion transporter polypeptides (OATPs) in rat hepatocytes, based on its intracellular concentration when applied at 3.02μM in extracellular medium, was estimated to be 8.9 μM, showing a value closer to that from the membrane vesicle study. Therefore, it was demonstrated that this real-time imaging method enables us to quantitatively evaluate bile canalicular transporter activity as well as to estimate effect of drugs on MRP2 activity. Currently, this method is being evaluated to quantify bile salt efflux protein (BSEP) that is a major efflux transporter of bile salts in to bile.

P466. Time Dependent Activity of the Uptake Transporter OATP1B1 in Plated Primary Human Hepatocytes

Maria Ulvestad1, Espen Molden2, Anders Åsberg2, Petter Björquist3 and Tommy B. Andersson1

1Clinical Pharmacology and DMPK, AstraZeneca R&D Mölndal, Mölndal, Sweden, 2School of Pharmacy, University of Oslo, Oslo, Norway, 3Cellartis AB, Göteborg, Sweden

Primary human hepatocytes are widely used as an in vitro model for evaluation of drug metabolism and transport, and for hepatotoxicity studies. However, it has been shown that the gene expression of many drug-metabolizing enzymes and transporters in primary hepatocytes may change in culture. The human organic anion transporter protein 1B1 (OATP1B1) is one of the major transport proteins involved in uptake of drugs into human hepatocytes. The aim of the present study was to evaluate the activity of OATP1B1 in plated primary human hepatocytes with increasing time in culture. Primary human hepatocytes were isolated from human liver resections obtained from 4 different donors using a collagenase perfusion method. The hepatocytes were plated on rat tail collagen, and cultured for 2, 6 and 24 hours. The kinetics of OATP1B1-mediated transport was evaluated by concentration dependent uptake studies of estradiol-17β-D-glucuronide (3H-E17βG) at each time point. CL

int values were calculated using V

max and K

m parameter estimates from

non-linear regression. Moreover, gene expression of OATP1B1 was determined by RT-PCR, while protein expression and localization of OATP1B1 was illustrated by confocal microscopy. Our results showed an active uptake of 3H-E17βG in plated primary human hepatocytes at 2 hours, with an average CL

int value of 20.8 ± 5.2 mL/min/mg protein. Compared

to the CLint

values at 2 hours, the median decrease in OATP1B1 activity was 15% after 6 hours in culture, but with a large variability range from a reduction of 81% to an increase of 18% (p=0.31). After 24 hours in culture, CL

int values could

only be estimated for 2 out of 4 individuals due to low absolute OATP1B1 activity in two of the samples. Compared to the average gene expression level of OATP1B1 at 2 hours, there was a 30% reduction in gene expression level after 6 hours in culture (p<0.05), and 85% reduction after 24 hours (p<0.05). Visual examination of OATP1B1 protein expres-sion by confocal microscopy showed localization to the membrane as expected and supported an extensive decrease in expression after 24 hours in culture compared to after 2 hours. In conclusion, our results show that plated primary human hepatocytes is a useful in vitro model for OATP1B1-mediated uptake studies, but only for a limited period of time (2 hours) after isolation. Longer culturing times induce an extensive variability that prohibits reliable results. Together with restricted tissue availability, this limits the potential of plated primary hepatocytes as an in vitro tool for evaluation of hepatic uptake, and alternative models should be explored for use in the future.

P467. Kinetic Characterization of Fluorescein Uptake In Human Cryopreserved Hepatocytes and in OATP1B1 and OATP1B3 Transfected CHO Cells

Tom De Bruyn1, Sarinj Fattah1, Bruno Stieger2, Patrick Augustijns1 and Pieter Annaert1

1Laboratory for Pharmacotechnology and Biopharmacy, Katholieke Universiteit Leuven, Leuven, Belgium, 2Department of Medicine, University Hospital, Zürich, Switzerland

Fluorescent bile salt derivates like Cholyl-Glycylamido-Fluorescein (CGamF) (1) and Cholyl-L-Lysyl-Fluorescein (CLF) (2) are being used as probe substrates to study hepatic transporter-mediated drug-drug interactions; CLF has also been proposed for the evaluation of in vivo liver function. Hepatic transport characteristics of both fluorescent

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substrates have been elucidated (1-3). However, the hepatic uptake mechanism of the fluorescein moiety as such has never been determined. The aim of the present study was to thoroughly characterize the in vitro hepatic uptake properties of sodium fluorescein. Uptake experiments were conducted using a pool of suspended cryopreserved human hepatocytes and in Chinese Hamster Ovary Cells (CHO) transfected with the human transporters OATP1B1, OATP1B3 and OATP2B1 (Organic Anion Transporting Polypeptide). Transporter inhibition experiments in hepato-cytes were conducted with the OATP inhibitor rifampicin and in sodium-free buffer. In CHO cells, different concen-trations of rifampicin and bromosulfophthalein (BSP) were used to inhibit OATP activity. Concentration-dependent uptake of fluorescein was shown to be saturable in hepatocytes as well as in CHO cells transfected with OATP1B1 and OATP1B3. In hepatocytes, fluorescein uptake at 37°C was corrected for passive diffusion by subtracting the uptake at 4°C. Kinetic parameters for net transporter-mediated uptake were 14.1 µM and 5.8 pmol/million cells/min for Km and Vmax, respectively. In CHO cells, uptake of fluorescein in wild type cells was used to obtain net uptake. Km values of 13.5 and 33.2 µM and Vmax values of 30.9 and 195.6 pmol/mg protein/min were obtained for OATP1B1 and OATP1B3-transfected cells, respectively. No increased uptake could be measured in OATP2B1 cells, compared to wild-type CHO cells. The uptake of fluorescein (10µM) in hepatocytes was reduced by 35% and 22% when fluorescein was co-incubated with rifampicin (25µM) and in the absence of extracellular sodium, respectively. IC50 values for inhibition of fluorescein uptake in OATP1B1 and 1B3- transfected CHO cells were: 0.57µM/0.24µM for rifampicin and 0.066µM/2.09µM for BSP. These data support a major role for OATP1B1 and OATP1B3 in mediating hepatic uptake of fluorescein in human liver, while a contribution of NTCP cannot be excluded. We conclude that fluorescein can be used as a commonly available probe substrate to assess OATP activity and related interactions.

References

1. Ye, ZW. et al. (2008) Cellular Accumulation of Cholyl-Glycylamido-Fluorescein in sandwich-cultured rat hepatocytes: Kinetic characterization, transport mechanisms, and effect of human immunodeficiency virus protease inhibitors. Drug Metab Dispos 36(7): 1315-1321.

2. De Waart, D. et al. (2010) Hepatic transport mechanisms of Cholyl-L-Lysyl-Fluorescein. J Pharmacol Exp Ther. (in press). 3. Annaert, P. et al. (2010) Interactions of HIV PI with OATP1B1, 1B3 and 2B1. Xenobiotica 40(3): 163-176.

P468. Impact of PEPT2 on the disposition of glycylsarcosine and cefadroxil in cerebrospinal fluid, choroid plexus, and brain parenchyma following intracerebroventricular administration to wild-type and Pept2 null mice

David E. Smith1, Yongjun Hu1, Hong Shen1, Tavarekere N. Nagaraja2, Joseph D. Fenstermacher2 and Richard F. Keep3

1Pharmaceutical Sciences, University of Michigan College of Pharmacy, Ann Arbor, MI, USA, 2Anesthesiology Research, Henry Ford Health System, Detroit, MI, USA, 3Neurosurgery and Physiology, University of Michigan Medical School, Ann Arbor, MI, USA

Little if anything is known regarding how specific transporters may influence the circulation and distribution of drugs or endogenous peptide/mimetic substrates in cerebrospinal fluid (CSF) and brain parenchyma. PEPT2 (SLC15A2), a high-affinity low-capacity carrier of the proton-coupled oligopeptide transporter family, is found in brain where it is expressed at the apical surface of choroid plexus epithelial cells (i.e., CSF-facing), as well as in astrocytes (newborn rats) and neuronal cells (newborn and adult rats).1 The purpose of this study was to define the CSF efflux kinetics, choroid plexus uptake, and parenchymal penetration of PEPT2 substrates in different regions of the brain following intracerebroventricular (icv) administration. To accomplish these objectives, biodistribution studies using [14C]glycylsarcosine (GlySar) and [3H]cefadroxil, along with quantitative autoradiography of [14C]GlySar, were performed using wild-type and Pept2 null mice. We found PEPT2 deletion markedly reduced choroid plexus uptake of GlySar and cefadroxil by 94% and 82%, respectively, at 60 min (p<0.001), and the substrates were cleared more slowly from CSF after icv injection (∼ 4-fold difference). Specifically, the CSF efflux half-life of [14C]GlySar increased from 23.6 min in wild-type mice to 84.7 min in Pept2 null mice and, for [3H]cefadroxil, from 46.3 min to 188 min in wild-type versus Pept2 null mice, respectively. Self-inhibition of radiolabeled GlySar by unlabeled GlySar in wild-type mice revealed a dose-dependent increase in [14C]GlySar in CSF (p<0.001), with a similar reduction in choroid plexus (p<0.001). In contrast, the [14C]GlySar in CSF and choroid plexus of Pept2 null mice did not change

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significantly with the addition of 20 mM GlySar. Autoradiography showed that GlySar concentrations in lateral, third, and fourth ventricle choroid plexuses were reduced in Pept2 null mice as compared to wild-type animals (p<0.01). Although PEPT2 had a major effect on GlySar disposition at the CSF-choroid plexus interface and ependymal region, changes in dipeptide distribution were smaller than expected in terms of coronal penetration into brain near the CSF system or along the rostro-caudal axis. By combining biodistribution and autoradiography methodologies with drug disposition studies in a mouse model of transporter gene deletion, these results demonstrate that PEPT2 knockout can significantly modify the spatial distribution of GlySar (and presumably other peptides/mimetics and peptide-like drugs) in brain.

References

1Shen H, Smith DE, Keep RF, Brosius III FC (2004) Immunolocalization of the proton-coupled oligopeptide transporter PEPT2 in developing rat brain. Mol Pharm 1:248-256.

P469. Characterization of Drug Transporters Involved in the Disposition of Dabigatran Etexilate and its active form, Dabigatran

Wataru Kishimoto1, Naoki Ishiguro1, Asami Saito1, Thomas Ebner2, Sebastian Härtter2 and Takashi Igarashi1

1Pharmacokinetics and Non-Clinical Safety, Nippon Boehringer Ingelheim Co., Ltd., Hyogo, Japan, 2Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

Dabigatran etexilate is a pro-drug of dabigatran, a reversible, competitive, direct thrombin inhibitor that has been shown to be efficacious and safe in the prevention of deep vein thrombosis in patients undergoing elective total hip or knee replacement, treatment of venous thrombosis and the prevention of stroke in patients with atrial fibril-lation. After oral administration, dabigatran etexilate is rapidly hydrolyzed to dabigatran, the active principle and major component in the systemic circulation, via two intermediate metabolites [Ref. 1]. Conversion of dabigatran etexilate to dabigatran is complete and only minute amounts of the prodrug and its intermediates can be observed in plasma [Ref. 2]. Beside hydrolysis of the ester pro-drug enzymatic processes such as cytochrome P450 catalyzed oxidations play only a negligible role in the disposition kinetics of dabigatran [Ref. 1]. The involvement of drug transporters in the absorption, disposition and excretion of dabigatran and dabigatran etexilate were investigated by using Caco-2 cells, organic anion-transporting polypeptide (OATP), organic cation transporter (OCT), or organic anion transporter (OAT) expressing HEK293 cells and P-glycoprotein (P-gp) expressing LLC-PK1 cells as well as multidrug resistance associated protein (MRP) 2 or breast cancer resistance protein (BCRP) expressing MDCKII cells. Dabigatran was no substrate of any drug transporter investigated. Among drug transporters expressed in the luminal membrane of the small intestine, P-gp appeared to be involved in the efflux of dabigatran etexilate. Efflux ratios of around 10 and 15 were observed in Caco-2 and P-gp-expressing cells, respectively, and the ratios approached unity in the presence of a P-gp inhibitor. Inhibitory potency of dabigatran and dabigatran etexilate was investigated using typical probe substrates. Dabigatran did not show any inhibition up to 50 µM, while dabigatran etexilate showed weak inhibition of OATP2B1 and OCT1 activities, which seems unlikely to be of any clinical importance. These findings indicate that drug drug interactions via drug transporters are unlikely to occur for the active principle dabigatran. The prodrug dabigatran etexilate exhibits only low systemic exposure. Therefore, the potential for clinically relevant interactions of dabigatran on other drugs transported by the investigated drug transporters is low. Only during the absorption phase, co-medications of P-gp inhibitors can result in increased dabigatran exposure.

References

1 Blech S, Ebner T, Ludwig-Schwellinger E, Stangier J, Roth W. The metabolism and disposition of the oral direct thrombin inhibitor, dabigatran, in humans. Drug Metab Dispos, 36(2): 386-99, 2008.

2 Stangier J. Clinical pharmacokinetics and pharmacodynamics of the oral direct thrombin inhibitor dabigatran etexilate. Clin Pharmacokinet, 47(5):285-95, 2008.

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P470. Effect of Structural Modification On Intestinal Absorption and Transport Mechanism of Alpha-Aminoxy Peptides

Bin Ma1, Ge Lin2 and Dan Yang3

1School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China, 2School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong, 3Department of Chemistry, The University of Hong Kong, Hong Kong, China

Clinical use of natural alpha-amino peptides is limited due to their conformational flexibility, rapid metabolism, and poor bioavailability. A series of alpha-aminoxy peptides based on the structural modifications of alpha-amino peptides with backbone-controlled secondary structures and improved stability have been developed recently by our research team. These newly developed alpha-aminoxy peptides showed a therapeutic potential for the treatment of Cl− channel dysfunctional diseases. The present study aimed to investigate the effect of structural modifications of five structurally related synthetic alpha-aminoxy peptides (P1 to P5) on their intestinal absorp-tion and transport mechanism. Three absorption models, including Caco-2 cell monolayer, ex vivo intestinal model and in situ rat single-pass intestinal perfusion (SPIP) model, were used, and all the samples were ana-lyzed by HPLC-UV and/or HPLC-MS. The potential interactions of P1 and P2 with P-glycoprotein (P-gp) were further studied by Western blot and P-gp ATPase assay. P1 showed significantly low intestinal permeability due to the efflux transporter P-gp-mediated mechanism, and its permeability was enhanced in the presence of P-gp inhibitors. The other peptides are the analogues of P1 with structural modifications to improve their hydrophilic solubility by replacing the two leucine side chains in P1 with two glutamic acids for P2, two lysines for P3, two alanines for P4 and two serines for P5. Interestingly, structural modifications resulted in remarkable alternations of transport mechanisms from P-gp-mediation (P1, P2) to multidrug resistance-associated protein (MRP) media-tion (P3), breast cancer resistance protein (BCRP)-mediation (P4) active transport, or even passive paracellular diffusion (P5) without the involvement of any efflux transporters. Absorption of the peptides was also improved significantly except for P2. Comparing with P1, the absorbable permeability in Caco-2 monolayer increased to about 4-fold (P4), 7-fold (P3) and 11-fold (P5), respectively, and the absorbed amount through intestine in SPIP model significantly increased to about 42-fold (P3), 55-fold (P4) and 102-fold (P5), respectively. P1 also showed uncompetitive inhibitory effect on P-gp ATPase without significant alternation of P-gp expression, while the other P-gp substrate P2 showed no significant inhibition on P-gp even at the high concentration of 150 µM. In conclusion, the intestinal absorbability and transport mechanisms of the alpha-aminoxy peptides tested varied significantly with different structures, and intestinal absorption of the alpha-aminoxy peptides can be significantly improved by structural modifications, which allow us to further design and synthesize better alpha-aminoxy peptide candidates with appropriate pharmacokinetic fates including intestinal absorption for potential clinical use. [The study was supported by RGC Central Allocation fund (HKU 2/06C) and Chinese University of Hong Kong Direct Grant (2041542).]

P471. Human transporters that function at ice-cold temperature - Important notice for transporter research -

Mikihisa Takano, Satoshi Suzuki, Hiroaki Imaoka, Junya Nagai and Ryoko Yumoto

Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

Purpose: Recently, transporter research is getting more and more important not only in basic research field but also in the field of drug discovery and development and of clinical pharmacotherapy. Generally, the optimum temperatures for functional proteins are near the temperatures surrounding these proteins. In human cells, the optimal temperatures for functional proteins including transporters are usually around human body temperature, and at low temperatures less than 4°C, it is assumed that the activity of transporter would disappear or be markedly suppressed. However, during our studies on nucleoside transport in membrane vesicles isolated from human erythrocytes, we obtained preliminary data indicating that most of uridine taken up by the vesicles was extensively effluxed during washing process with ice-cold buffer. Based on this finding, we have assumed that human equilibrative nucleoside transporter ENT1 may be function-ing even at ice-cold temperatures (ICT; measured temperature, 0.5 - 0.7°C). However, as far as we know, there has been no study aimed to clarify whether or not certain human transporters really function at extremely low temperatures near

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0°C. In the present study, we therefore attempted to clarify the functionality of ENT1 and other transporters in human erythrocyte membranes at ICT.

Methods: The uptake of a substrate by rightside-out membrane vesicles (ROVs) or by inside-out membrane vesicles (IOVs) was measured by a rapid filtration technique. [3H]Uridine, 5-(and 6)-carboxy-2′, 7′-dichlorofluorescein (CDCF), and [3H]D-glucose were used as substrates for ENT1, multidrug resistance-associated protein 5 (MRP5), and facilitative glucose transporter GLUT1, respectively. S-(4-nitrobenzyl)-6-thioinosine (NBMPR) and phloretin were used as specific inhibitors for ENT1 and GLUT1, respectively.

Results: The uptake of uridine showed saturation kinetics and was inhibited by NBMPR at both 23°C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabelled uridine or ribavirin, another sub-strate for ENT1, at ICT. In addition, even the overshoot phenomenon was observed at ICT. Trans-stimulated uptake of [3H]uridine at ICT was inhibited by NBMPR in a concentration-dependent manner. Like uridine, [3H]D-glucose uptake by ROVs mediated by GLUT1 was trans-stimulated by unlabelled D-glucose at ICT with overshoot phenomenon, and the uptake was inhibited by phloretin. On the other hand, ATP-dependent transport of CDCF via MRP5, a member of ABC transporter family, in IOVs disappeared completely at ICT.

Conclusion: These results clearly indicate that human erythrocyte transporters like ENT1 function even at very low temperatures near 0°C. It is important to recognize that there are such human transporters which function at ICT, in order to carry out transport study properly and to avoid misunderstanding of the data obtained. This work was sup-ported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

P472. IDENTIFICATION OF PHYTOCHEMICAL MODULATORS OF MULTI-Drug RESISTANCE PROTEIN 5 (MRP5)

Yan Li1, Jezrael Revalde1, Glen Reid2 and James W. Paxton1

1Department of Pharmacology and Clinical Pharmacology, The University of Auckland, Auckland, New Zealand, 2Asbestos Diseases Research Institute, Bernie Banton Centre, The University of Sydney, Sydney, Australia

Chemotherapy of pancreatic cancer often fails due to the development of intrinsic and acquired resistance during drug treatment. Recent studies have suggested that MRP5 conferred resistance to first-line drugs 5-fluorouracil and gemcitabine by active efflux from the cell (Oguri, Achiwa et al. 2006; Hagmann, Jesnowski et al. 2009). Our aim was to evaluate whether several phytochemicals (curcumin, biochanin A, indo-3-carbinol and diindolemethane) could reverse this multi-drug resistance by inhibition of MRP5 efflux. Cell proliferation and drug accumulation studies were undertaken in mock and MRP5 over-expressing HEK293 cells, Panc-1 and MiaPaCa-2 pancreatic cancer cells. The cellular accumulation of a specific MRP5 fluorescent substrate 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was measured by flow cytometry and the cell proliferation determined by a 72-h CyQuant assay. MRP5 mRNA level was determined by real-time PCR and MRP5 protein detected by immuno-cytochemistry using a monoclonal antibody. In the presence of curcumin (5 and 10 µM) and a MRP5 inhibitor MK571 (25 µM), the cellular accumulation of BCECF in HEK293-MRP5 cells increased 68 ± 9% (p<0.01), 215 ± 12% (p<0.01) and 397 ± 17% (p<0.01), respectively. Curcumin and MK571 had no effects on cellular accumulation of BCECF in mock HEK293 cells. Biochanin A, indo-3-carbinol and diindolemethane have no effects on BCECF accumulation in mock and MRP5 over-expressing HEK293 cells. In the proliferation assays, curcumin caused a concentration-dependant increase in the sensitivity to the cytotoxic drug 6-thioguanine and 5-fluorouracil in HEK-MRP5 cells, Panc-1 and MiaPaCa-2 pancreatic cancer cells, but not in HEK293 mock cells. Our results suggest that curcumin is an inhibitor of MRP5 and may be useful in the reversal of multi-drug resistance in pancreatic cancer chemotherapy.

References

Hagmann, W., R. Jesnowski, et al. (2009). “ATP-binding cassette C transporters in human pancreatic carcinoma cell lines. Upregulation in 5-fluor-ouracil-resistant cells.” Pancreatology 9(1-2): 136-44.

Oguri, T., H. Achiwa, et al. (2006). “The determinants of sensitivity and acquired resistance to gemcitabine differ in non-small cell lung cancer: a role of ABCC5 in gemcitabine sensitivity.” Mol Cancer Ther 5(7): 1800-6.

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P473. Inside-Out Vesicle Model as Straightforward Assay to Determine Inhibitory Potential of Drugs Against Human, Rat, Mouse and Dog MRP2

Maria Rosa1, Céline Jacques-Hespel2, Steven SMITH1 and Hugues Chanteux1

1NCD, UCB, Braine-l′Alleud, Belgium, 2Cardio3, Mont-Saint-Guibert, Belgium

MRP2 belongs to the important family of ABC transporters. By its localization in the canalicular membrane of hepa-tocytes, it plays a significant role in the efflux of anionic compounds, glutathione and glucuronide conjugates into the bile. In addition, its expression at the intestinal and kidney level can also impact the absorption and urinary excretion of compounds. Human, rat, mouse and dog MRP2 inside out vesicles were prepared from Sf9 insect cells expressing the corresponding transporters. They were supplied by Genomembrane. MRP2-mediated and ATP-dependent uptake of CDCF (5(and6)carboxy2′, 7′dichlorofluorescein), a well-known substrate of MRP2, was conducted using a rapid filtration system in the absence or presence of MgATP. First, kinetic parameters for CDCF uptake were determined in all species. Km values were 76.1, 203, 46.5 and 83.6 µM and V

max values were 1009, 571, 586 and 304 pmol/min/mg,

for human, rat, mouse and dog, respectively. Then, inhibitory effects of three well-known MRP2 inhibitors, MK571, benzbromarone and bromosulfalein were investigated. They caused strong inhibition of MRP2 from all species tested. Bromosulfalein was the most potent inhibitor with IC50 values from ca 9 µM to ca 18 µM. Inhibition observed with MK571 and benzbromarone depended on species tested with IC50 values from ca 11 µM to ca 49 µM and from ca 21 µM to ca 66 µM, respectively. Finally, we screened several drugs known as MRP2 substrates (ceftriaxone, pravastatin, probenecid, ritonavir, rifampicin, vinblastine, diclofenac, phenytoin, methotrexate, furosemide and cyclosporine A) for their inhibitory effect on MRP2, at 100 µM. Cyclosporine A showed strong inhibition of human, rat, mouse and dog MRP2. Rifampicin had significant inhibition on human, rat and dog MRP2 while exhibiting no inhibitory effects on mouse MRP2. Vinblastine caused inhibition of human, rat and mouse MRP2 but had no effect on dog MRP2. Ceftriaxone, diclofenac, methotrexate and furosemide caused significant inhibition of human MRP2 only. No significant inhibition was observed with pravastatin, probenecid, ritonavir and phenytoin. Our results demonstrated that inside out vesicles are a useful and straightforward model to examine the interactions between MRP2 and drugs. Furthermore, this model allows also a direct inter-species comparison that could help to explain species difference observed in vivo since we showed that a same drug could have different affinity for human, rat, mouse and dog MRP2.

P474. Abstract Withdrawn.

P475. Development of Vesicular Transport Assay Method for MDR1 (ABCB1) Using The Fluorescent Probe

Kenji Tanaka, Masa Yasunaga, Miyako Maeda, Shigeki Enomoto, Yoshiaki Hagiwara and Hirotugu Watabe

GenoMembrane, Yokohama, Japan

Multi drug resistance protein 1 (MDR1, P-gp) is the most famous transporter, which plays key roles in pharmacokinetic barriers at brush border membrane of small intestine, blood-brain barrier, proximal renal tubule, and canalicular membrane of hepatocyte. Investigation of the interactions between drugs and MDR1 in vitro provides valuable informa-tion in order to predict intestinal absorption, penetration into brain, and biliary/urinary excretion of candidate drugs before the clinical tests. Vesicular transport assay is the simple and powerful tool and enables us to obtain kinetic parameters of the interaction between ABC transporter and the candidate drugs. However it is difficult to perform vesicular transport assay for MDR1 because many substrates against MDR1 are lipophilic and are not detected clearly due to their background. The purpose of this report is to look for a standard compound as a substrate to analyze MDR1 activity by vesicular transport assay. Quinidine is known as a substrate for MDR1, and its derivative, N-Methylquinidine (NMQ), is a hydrophilic compound and has fluorescence caused by quinidine backbone. We tested the characteristics of NMQ whether it is the suitable substrate for vesicular transport assay for MDR1 or not. For the analysis, we prepared Sf9 membrane vesicles expressing human MDR1 (hMDR1 vesicles) using baculovirus system. The uptake of NMQ into

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hMDR1 vesicles in the presence of ATP was significantly higher than the uptake in the absence of ATP. This uptake was increased linearly for at least 1 min, while on the other hand, an ATP-dependent increase was not observed with the mock-membrane vesicles. In addition, mouse Mdr1a vesicles also transported NMQ in ATP-dependent manner. The uptake of NMQ with hMDR1 was significantly inhibited by digoxin, verapamil, and cyclosporine, which are reported as substrates or inhibitors for MDR1. In conclusion, NMQ enables us to figure out the interactions between drugs of interest and MDR1 prior to the cell based assay such as Caco-2 cell systems, and this information will minimize the labor for time-consuming cell based assay. Thus, the screening system using NMQ and membrane vesicles expressing MDR1 is useful assay system for the drug discovery in order to predict the effects of MDR1 in human and other experimental animals.

P476. Searching for a Fluorescent Probe to Construct High-Throughput Screening System for BCRP

Kenji Tanaka, Miyako Maeda, Masa Yasunaga, Shigeki Enomoto, Yoshiaki Hagiwara and Hirotugu Watabe

GenoMembrane, Yokohama, Japan

The ATP-Binding Cassette (ABC) transporter superfamily is found in various organisms, through fungal to humans. In human, 49 kinds of ABC transporters have found in genome, and about 10 kinds of them are known as efflux pump of drugs from inside to out of cells by ATP hydrolysis energy. It means ABC transporter is important for ADME as well as toxicity of many drugs. BCRP (Breast Cancer Resistance Protein, ABCG2) is one of the ABC transporters associated with multidrug resistance, which carries topotecan, methotrexate, and pitavastatin. It is highly expressed in tumor such as lung cancer, and also in normal cell such as small intestinal epithelial cell, hepatic parenchymal cell, and cerebrovascular endothelial cell, as an important determinant for the drug disposition through its roles in absorption at the small intestine, excretion into bile, and transfer across to the blood-brain barrier. In order to analyze the interactions between BCRP and drugs of interest, vesicular transport assay using membrane vesicles expressing BCRP and RI-labeled substrate has been performed widely. However, the cost of RI-compound is high and strict regulation is required to use it. The fluorescent substrate instead of RI-labeled one is possible to solve these problems. This study reports a useful fluorescent substrate for BCRP, Lucifer-yellow (LFY) among some fluo-rescent compounds. For uptake study, we prepared membrane vesicles expressing human BCRP (hBCRP vesicles) by baculovirus systems and performed vesicular transport assay. Uptake of LFY was observed with hBCRP vesicles in ATP dependent manner, while it was not observed with the mock vesicles. The uptake of LFY was reduced by increasing sucrose concentration in the uptake medium, which indicates that hBCRP transports LYF into the inside of the vesicles, not binding to the vesicle surface. In addition, several compounds, which are reported as substrates or inhibitors of BCRP, inhibited the LYF uptake with hBCRP in concentration dependent manner. Furthermore, the membrane vesicles expressing rat Bcrp also transported LYF. From these results, LYF is a convenient and cost-effective fluorescent tool for analyzing the interactions between drugs of interest and BCRP, and vesicular transport assay using LFY provides valuable information for predicting BCRP functions in human and other experimental animals.

P477. The Uptake and Transepothelial Transport of JBP485 by PEPT1

Kexin Liu1, Zhihao Liu2, Changyuan Wang2, Qi Liu2, Qiang Meng2, Jian Cang2, Xinjin Guo2 and Taiichi Kaku3

1Department of Clinical Pharmacology,College of Pharmacy, Dalian Medical University, Dalian, China, 2Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, China, 3Bioproducts Industry Co. Ltd, Tokyo, Japan

Background: Cyclo-trans-4-L-hydroxyprolyl-L-serine(JBP485) is a dipeptide which exhibits obvious anti-hepatitis activity and has been synthesized by chemical means. Previous experiments showed that JBP485 was well absorbed by the intestine in rats after oral administration. The human peptide transporter (PEPT1) is expressed in intestine and recognizes the compounds such as dipeptides and tripeptides.

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Objective: The purposes of this study are to understand whether JBP485 is a substrate of intestinal peptide transportor PEPT1 and to investigate the characteristics of JBP485 uptake and transepothelial transport by PEPT1. We employed human intestinal epithelial cell line Caco-2 cell as model to observe the uptake and transepothelial transport statuses of JBP485.

Results: A rapid and selective liquid chromatographic/tandem mass spectrometric method has been developed to quantify JBP485 in samples. The uptake of JBP485 at pH 6.0 is higher than that at pH 7.4. Aslo,the uptake of JBP485 was significantly inhibited by Gly-Sar (a typical substrate for PEPT1 transporters), JBP923(a derivative of JBP485)and cephalexin (CEX, b-lactam antibiotics) with Ki values of 16.94mM, 0.77mM, and 0.26mM,respectively. Meanwhile, the uptake of Gly-Sar was also strongly inhibited by JBP485, JBP923 and CEX with Ki values of 36.09mM, 2.55mM, and 1.10mM. The uptake of JBP485 was inhibited by presence of Zn2+ and increased by verapamil. We found that the ratio value for transepothelial transport of JBP485 from apical-to-basolateral was 1.5 times higher than its basolateral-to-apical transepothelial transport, suggesting the transport is active transport. Also the transepothelial transport of JBP485 was obviously inhibited by Gly-Sar, JBP923 and CEX. At the same time, the mRNA level of PEPT1 was enhanced in the presence of JBP485.

Conclusions: JBP485 could be actively transported by intestine oligopeptide transporter PEPT1, which is one of the reasons that JBP485 was quickly absorbed by the gastrointestinal tract after oral administration.

P478. Biliary Excretion Mechanism of Cefditoren and the Effects of Cefditoren on Hepatic Transporters Expression in Rats

Qiang Meng1, Changyuan Wang1, Qi Liu1, Jian Zhang1, Zhihao Liu1, Taiichi Kaku2 and Kexin Liu1

1Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, China, 2Bioproducts Industry Co. Ltd., Tokyo, Japan

Objective: Recently, transport proteins tend to gain more and more attention together with liver drug enzymes. Mrp2 and Bcrp are the members of ATP-binding cassette (ABC) superfamily on canalicular membrane of hepatocyte. They transport a great deal of endogenous substances and xenobiotics, such as drugs which may regulate the expression levels of these transporters. Cefditoren, a fourth generation cephalosporin antibiotics, has been used in clinic extensively. Evidence has indicated that cefditoren is excreted into bile as unmetabolized form. Many drug-drug interactions are mediated via drug transporters, so elucidating correlations between drugs and drug transporting proteins is helpful for clarifying the mechanism of drug-drug interactions, which are important for clinical pharmacotherapy. Because of the increasing appreciation of the role played by transporters, data about alteration of transporter levels in response to xenobiotic exposure is also available, especially with respect to drug-drug interactions and drug adverse events.

Methods: Perfused rat livers were performed to investigate whether Mrp2°¢Bcrp and P-gp were involved in the bil-iary excretion of cefditoren. We used RT-PCR and Western blot to study the alteration of expression levels of relative hepatic transporters. (1) The hepatobiliary disposition of cefditoren (1µM) was examined with probenecid (20µM), novobiocin (20µM) and verapamil (20µM) as the inhibitor of Mrp2, Bcrp and P-gp respectively in the perfused rat liver. The concentration of cefditoren in perfusate and bile were determined by HPLC. Then compare the hepatic extraction ratio and cumulative biliary excretion rates in control and experimental group; (2) 16mg/kg, 50mg/kg, 100mg/kg of cefditoren and physiological saline as control were administered twice a day. After one week, the expression levels of Mrp2, Bcrp, P-gp, Oct1 and Oat2 mRNA were examined by RT-PCR; (3) expression of Mrp2 was investigated by Western blot to observe the dose-dependence.

Results: (1) The hepatic extraction ratio showed no statistically significant differences, whereas cumulative biliary excretion rates were significantly reduced to 43.78% and 79.52% over 25 min in the perfused probenecid and novobiocin group, respectively. The cumulative biliary excretion rates in perfused verapamil group had no change compared with control; (2) One week after oral administration of cefditoren, the expression levels of Mrp2, Bcrp and Oat2 mRNA were markedly up-regulated, while P-gp and Oct1 mRNA were down-regulated; (3) In accordance with RT-PCR results, the Mrp2 expression was increased by Western blot. After administration of 100 mg/kg, the Mrp2 levels were markedly up-regulated to 1.70 fold of the control, while the Mrp2 expression levels in 16 mg/kg group increased to 1.32 fold over the control.

Conclusions: Mrp2 is the major protein of efflux transport into bile, while Bcrp is also involved in the biliary excretion of cefditoren, and P-gp has no contribution to this process. Mrp2, Bcrp and Oat2 mRNA are up-regulated, and P-gp and

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Oct1 mRNA are down-regulated after administration of cefditoren and cefditoren increases expression levels of Mrp2. These results provide important data for drug-drug interactions.

P479. Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1/SLC22A4

Tomoko Sugiura1, Noritaka Nakamichi1, Yoshiyuki Kubo1, Sayaka Kato1, Tomoyoshi Soga2, Akira Tsuji1 and Yukio Kato1

1Div of Pharmaceutical Scis, Kanazawa Univ, Kanazawa, Japan, 2Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan

[Purpose] Solute carrier, OCTN1/SLC22A4 was first identified in Kanazawa University and accepts various types of the compounds as substrates in vitro, but its physiologically important substrates are still unidentified. The purpose of the present study was to identify the in vivo substrate of OCTN1 and clarify functional expression of OCTN1 in several organs with an aim to understand physiological roles of OCTN1. [Purpose]: We first constructed octn1 gene knockout (octn1−/-) mice. Metabolome analysis was then performed to identify substrates in vivo. Functional expression of OCTN1 was further examined using the substrate identified as a probe compound.

[Results and Discussion]: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine (ERGO) in octn1−/- mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ERGO, both of which were much reduced in octn1−/- mice. The OCTN1-mediated reabsorp-tion was demonstrated by the overshoot phenomenon in [3H]ERGO uptake by renal brush-border membrane vesicles in wild-type mice, which was absent in the vesicles prepared from octn1−/- mice. The octn1−/- mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model, proposing that OCTN1 may play a role for maintenance of intestinal exposure of ERGO which could be important for protective effect against intestinal tissue injuries. After oral administration of [3H]ERGO, the amount of [3H]ERGO remaining in the small intestinal lumen was much higher in octn1−/- mice, compared with wild-type mice. In addition, uptake of [3H]ERGO by HEK293 cells heterologously expressing OCTN1 gene product, and uptake of [3H]ERGO at the apical surface of intestinal everted sacs from wild-type mice were inhibited by OCTN1 substrates, tetraethylammonium and verapamil. Immunohistochemical analysis revealed that OCTN1 is localized on the apical surface of small intestine in mice and humans. These results suggest that OCTN1 is responsible for small-intestinal absorption of [3H]ERGO. Since ERGO is not synthesized in mam-mals, OCTN1 in the small intestine would be responsible for the absorption of ERGO from daily foods and subsequent systemic exposure of ERGO in the body. The plasma concentration of [3H]ERGO after oral administration was higher in octn1−/- mice than in wild-type mice, despite the lower absorption in octn1−/- mice. This was probably because of efficient hepatic uptake of [3H]ERGO, as revealed by integration plot analysis; the uptake clearance was close to the hepatic plasma flow rate. The uptake of [3H]ERGO by isolated hepatocytes was minimal, whereas [3H]ERGO uptake was observed in isolated non-parenchymal cells. This is consistent with immunostaining of OCTN1 in liver sinusoids. Thus, our results indicate that OCTN1 is functionally expressed in non-parenchymal liver cells.

[Conclusion]: OCTN1 accepts ERGO as a physiological substrate in vivo. OCTN1 is functionally expressed in vari-ous organs, especially on apical membranes of renal proximal tubules and small intestine, and liver sinusoidal non-parenchymal cells.

P480. The CDK inhibitor roscovitine (Seliciclib) is a selective substrate of human P-glycoprotein (P-gp/ABCB1/MDR1)

Zsuzsanna Rajnai1, Dora Mehn1, Erzsébet Beéry1, Alper Okyar2, Márton Jani1, Ferenc Fulop3, Francis Lévi4 and Peter Krajcsi5

1R&D, SOLVO Biotechnology, Szeged, Hungary, 2Istanbul University Faculty of Pharmacy, Istanbul, Turkey, 3Inst of Pharmaceutical Chemistry, University of Szeged, Szeged, Hungary,

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4Hôpital Paul Brousse PV Couturier 14-16, INSERM, Villejuif, France, 5R&D, SOLVO Biotechnology, Szeged, Hungary

Introduction R-Roscovitine (Seliciclib), a CDK inhibitor in Phase-II clinical trials is a promising candidate to treat a variety of cancers. We tested the interaction of roscovitine with human ATP-binding cassette (ABC) transporters P-gp (MDR1, ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and BCRP (ABCG2) which may play a crucial role of drug pharma-cokinetics, multidrug resistance and toxicity.

Materials/Methods: Transporter interactions were studied by measuring ATPase activity of P-gp, MRP1, MRP2 and BCRP. Vectorial transport of roscovitine was evaluated in parental and MDR1-transfected MDCKII cells. Additionally, inhibition of fluorescent dye efflux Calcein AM and vesicular transport studies were carried out for P-gp. We have tested using HL60-MDR1, K562-MDR and MDCKII-MDR1 and control cells if P-gp overexpression may result in resistance to roscovitine.

Results and Conclusion: This study shows that roscovitine is a high affinity substrate of P-gp as it activates the ATPase activity of the transporter and shows vectorial transport in MDCKII-MDR1 cells. (AB/BA ratio: 3.1). Roscovitine inhibited sulfasalazine-stimulated BCRP ATPase activity. Therefore, roscovitine is a likely inhibitor of BCRP. No pharmacologically relevant interaction was detected with MRP1 and MRP2. The roscovitine – P-gp interaction may be behind the drug′s limited penetration of the blood-brain barrier. P-gp overexpression on the other hand does not confer resistance to the drug. These findings should be considered when designing treatment strategies utilizing roscovitine.

P481. Role of P-glycoprotein and Bcrp1 in the brain penetration and activity of the Novel PI3K inhibitor GDC-0941

Laurent Salphati, Leslie Lee, Jodie Pang, Emile G. Plise and Xiaolin Zhang

Drug Metabolism and Pharmacokinetics, Genentech Inc, South San Francisco, CA, USA

The phosphatidylinositol 3-kinase (PI3K) pathway is a major determinant of cell cycling and proliferation. Its deregula-tion, by activation or transforming mutations of the p110α subunit, is associated with the development of many cancers. In recent years, this pathway has emerged as a major target for the investigation of anticancer drugs. GDC-0941 is a novel small molecule inhibitor of PI3K currently being evaluated in the clinic as an anticancer agent. The objectives of these studies were to determine in vitro whether GDC-0941 was a substrate of P-glycoprotein and Bcrp1 and to investigate the impact of Pgp and Bcrp1 on the absorption, disposition and brain penetration of GDC-0941 in FVBn mice (wild type), Mdr1a/b(-/-), Bcrp1(-/-) and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. In vitro studies with MDCK cells transfected with Pgp or Bcrp1 established that this compound was a substrate of both transporters. Following intravenous (IV) and oral (PO) administrations, GDC-0941 brain-to-plasma ratios ranged from 0.02 to 0.06 in the wild type mice, were unchanged in the Bcrp1(-/-) and were 3- to 4-fold higher in the Mdr1a/b(-/-) knockout mice, ranging from 0.08 to 0.11. In contrast, the brain-to-plasma ratio of GDC-0941 in Mdr1a/b(-/-)/Bcrp1(-/-) was 30-fold higher than in the wild type mice, ranging from 0.5 to 1. The plasma clearance of GDC-0941 was similar in the four strains of mice, ranging from 15 to 25 mL/min/kg in the wild type mice and from 18 to 35 mL/min/kg in the knockout mice. Exposure following PO administration was also comparable in the wild type and all knockout mice. Following administration of GDC-0941 to Mdr1a/b(-/-)/Bcrp1(-/-) mice, the PI3K pathway was markedly inhibited in the brain for up to 6 hours post-dose, as evidenced by a 60% suppression of the pAkt signal, while no effect on the PI3K pathway was detected in the brain of wild type mice. These findings showing overlapping and concerted effects of Pgp and Bcrp1 in restricting GDC-0941 access and pathway modulation in mouse brain may have implications for the treatment of patients with brain tumors.

P482. Localization and Functional Analysis of Organic Cation Transporter 3 (OCT3) in Rat Placenta

Davoud Ahmadimoghaddam1, Jakub Hofman1, Eva Brcakova2, Martina Ceckova1, Stanislav Micuda2 and Frantisek Staud1

1Department of Pharmacology and Toxicology, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Hradec Kralove, Czech Republic, 2Department of Pharmacology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic

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The placenta is an organ that mediates the transfer of endo- and exogenous compounds between the mother and fetus. Understanding the role of drug transporters on the materno-fetal interface is essential to optimize pharma-cotherapy during pregnancy. While the role of several drug efflux transporters in the placenta has been studied by many research teams, functional analysis of solute carrier transporters (SLC) has not been described in detail. The exact function of Organic Cation transporter 3 (OCT3), the most abundantly expressed OCT in the placenta, is still not fully elucidated. The aim of this study was to investigate the expression, localization and function of OCT3 in the rat placenta. For quantification of mRNA expression and detection of protein, RT-PCR and western blotting were used, respectively. For the functional analysis of OCT3, dually perfused rat term placenta was employed to study the effect of OCT3 on the clearance of its substrate, 1-methyl-4-phenylpyridinum (MPP+). Transplacental passage of MPP+ was investigated in both directions, maternal-to-fetal and fetal-to-maternal. We observed striking asymmetry of MPP+ clear-ance between fetal-to-maternal and maternal-to-fetal directions at low substrate concentration; on the other hand, at high substrate concentrations this asymmetry was decreased. In addition, we observed concentration dependence of placental clearance of MPP+ in both directions suggesting saturability of the transporter. These findings indicate substantial role of OCT3 in transplacental passage of its substrates. Additional studies are needed to assess the exact mechanism and localization of OCT3 in the rat placenta. This work was supported by Grants GAUK 137010/C/2010 and SVV-2010-261-003.

P483. Differences in mRNA and Protein Expression of Organic Anion Transporting Polypeptides (OATPs) in Malignant and Non-malignant Human Liver Samples

Juliane Riha1, Katrin Wlcek1, Martin Svoboda2, Theresia Thalhammer2 and Walter Jaeger1

1Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna, Austria, 2Department of Pathophysiology and Allergology, Medical University of Vienna, Vienna, Austria

Organic anion transporting polypeptides (OATPs) are known to mediate the uptake of many endogenous compounds and xenobiotics and therefore play a critical role in their absorption, distribution and excre-tion. Although some of the eleven known human OATP family members have been detected in gastric, pancreatic, and colon carcinomas, the knowledge of their expression in liver cancer is still limited. Hence we investigated expression of OATPs and their localization in human malignant and adjacent non-malignant liver tissue samples using real-time PCR and immunofluorescence staining, respectively. For TaqMan real-time PCR analyses total RNA was isolated from tissue specimens of 22 patients suffering from hepato-cellular carcinoma (HCC), cholangiocellular carcinoma (CCC), or even liver metastases from the colon (MLT). Protein expression and localization of selected OATP family members were further investigated by immunofluorescence staining. TaqMan real-time PCR analyses revealed mRNA expression of all human OATPs, except OATP1C1 and 6A1, in most of the studied cancerous and non-cancerous tissues samples. Interestingly, significant differences in OATP expression pattern could be observed comparing malignant to non-malignant specimens. While in tumorous samples mRNA levels of OATP1A2, 1B1, 1B3, and 2B1 were decreased, OATP2A1, 3A1, and 5A1 showed clear up-regulation. Variations in OATP mRNA expression levels can also be seen among different tumor types occurring in the liver. Remarkably, OATP4A1 exhibits considerable higher expression in the MLT group whereas slightly reduced mRNA levels were detected in HCC and CCC samples. Additionally, OATP2A1 and 5A1 mRNA was increased in all three tumor types with highest expression levels in the MLT group. Up-regulated OATP2A1, 4A1 and 5A1 have been chosen for further investigations regarding their localization in malignant and adjacent non-malignant tissue sections using immunofluorescence staining. Confirming real-time PCR results, more intensive staining was seen in cryosections of tumorous samples with localization at the plasma membrane. To investigate whether these OATP family members may also be located in cells deriving from proliferating bile ducts, double staining with cytokeratin 19 (CK-19), known as a selective marker for biliary/progenitor cells, was performed. Clear co-localization with CK-19 was observed for OATP2A1 and 4A1. In conclusion, this study demonstrated significant differences in OATP presence between malignant and non-malignant human liver samples. Whether this distinct expression pattern of the OATP transporter family in liver cancer may influence uptake of anticancer drugs has to be evaluated.

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P484. Hepatic Uptake in the Dog: Comparison of Uptake in Hepatocytes and HEK cells Expressing Dog OatpC

Alison J. Wilby1, Paul F. Courtney1, Peter J.H. Webborn1, Rob J. Riley1, Kazuya Maeda2, Yasuyuki Debori2 and Yuichi Sugiyama2

1Discovery DMPK, AstraZeneca R&D Charnwood, Loughborough, United Kingdom, 2Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Introduction There is an abundance of publications with regard to transporters in human and also in rat, but relatively little is known regarding transporters expressed in other species. Dog is frequently used in pharmacological and safety studies but little is known about canine transporters expressed in hepatocytes. Only recently has organic anion trans-porting polypeptide C (OatpC), the canine orthologue of human OATP1B1/1B3 been cloned and characterized (Gui and Hagenbuch, 2010), so far little has been published on dog Oatps A and B. To assess the importance of dog OatpC in the hepatic uptake of a range of substrates, uptake in HEK cells expressing dog OatpC has been compared to that in freshly isolated dog hepatocytes.

Methods: Uptake of a range of acidic substrates including pitavastatin, pravastatin, bromosulphaphthalein, glibencla-mide, valsartan and olmesartan was measured at 1 µM in freshly isolated male dog hepatocytes at 1 x 106 cells/ml (n=4) using a spinning through oil approach (Petzinger and Fuckel, 1992). Uptake intrinsic clearance (CL

int,uptake) was estimated

by subtracting the initial rate of the time-dependent uptake at 4° from the initial rate at 37°. CLint,uptake

in OatpC-expressing HEK293 cells was obtained by subtracting the initial slope of the time-dependent uptake of compounds at 0.1 µM by that in vector-transfected control cells.

Results: All substrates studied were actively taken up into dog hepatocytes and into OatpC-expressing HEK cells. CLint,uptake

estimated in fresh dog hepatocytes ranged from <5 µl/min/1x106 cells for pravastatin to ∼130 µl/min/1x106 cells for glibenclamide. CL

int,uptake in the OatpC-expressing HEK cells ranged from ∼1 µl/min/mg protein for pravastatin to ∼40

µl/min/mg protein for glibenclamide. On comparison of CLint,uptake

estimated from dog hepatocytes with CLint,uptake

esti-mated from OatpC-expressing cells a strong correlation was found indicating that dog OatpC may be the predominant transporter in dog for these acidic compounds.

Conclusion: There is a strong correlation between dog hepatocyte CLint,uptake

and dog OatpC CLint,uptake

and, as such OatpC may be the major transporter associated with transport of acidic xenobiotics in dog. Measuring uptake in OatpC-expressing cells may be a good surrogate for dog hepatocyte CL

int,uptake and, using appropriate relative activity factors it

may be possible to estimate in vivo clearance from OatpC uptake data.

References

Gui, C. and Hagenbuch, B. (2010). Cloning/characterization of the canine organic anion transporting polypeptide 1b4 (Oatp1b4) and classification of the canine OATP/SLCO members. Comp. Biochem. Phys. C. 151:393-399.

Petzinger, E and Fuckel, D. (1992). Evidence for a saturable, energy dependent and carrier-mediated uptake of oral antidiabetics into rat hepato-cytes. Eur. J. Pharmacol. 213:381-391.

P485. Characterization of Expression of Cytochrome P450 and drug transport proteins in the intes-tine of NMRI wild type mice and nude mice

Anja Rayss and Andreas Reichel

Research Pharmacokinetics, Bayer Schering Pharma AG, Berlin, Germany

Intestinal Cyp P450s and drug transporters are crucial for oral absorption and oral bioavailability of many compounds. However, little information exists regarding their expression in gut, especially in terms of the spatial resolution along the whole length of the intestine and strain differences. Therefore, the aim of my work was to investigate the CYP P450 enzyme and drug transporter protein expression in the whole length of the intestine of NMRI wild type mice and nude mice. The analysis was done by complete segmentation of the entire length of the intestine. Protein expression was investigated by Western blotting followed by immunodetection. Furthermore, metabolic degradation and permeation behaviour of selected test compounds were investigated in a functional assay using everted sacs from different gut

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regions. The protein expression of the drug efflux pumps, Pgp and BCRP, and the CYP P450 enzymes CYP2C and CYP3A was analysed in the intestinal segments of both mice strains. Pgp and BCRP expression was continuously increased from proximal to distal small intestine. While Pgp was highest expressed in colon, BCRP was only detected in very low amounts. CYP3A and CYP2C expression were continuously decreased from proximal to distal small intestine. In the colon high expression was found for CYP2C, CYP3A was not detectable. Investigation of different enterocyte populations of the villus showed that the analysed CYP′s and transport proteins were mainly expressed in the mature enterocytes of the villus tips. Significant differences in the expression profile of investigated efflux transporters and CYP′s in the intestine from NMRI mice and nude mice could not be observed. The CYP P450 expression profile in the small intestine was also reflected by metabolic activity along the small intestine. No differences have been observed between the mouse strains investigated.

P486. In Vitro testing of Bilastine For Transporter Mediated Drug-Drug Interactions

Alvaro Ganza1, Ignacio Ortega1, Ana Gonzalo1, María Luisa Lucero1, Mirza Jahic2, Dallas Bednarc-zyk2, Szilvia Gedey3, Zoltan Nagy4, Viktoria Juhasz4 and Eniko Ioja5

1R+D+i, Faes Farma, Vizcaya, Spain, 2Optivia Biotechnology Inc., Menlo Park, CA, USA, 3SOLVO Biotechnology, Szeged, Hungary, 4R&D, SOLVO Biotechnology, Budaors, Hungary, 5Research and Development, SOLVO Biotechnology, Szeged, Hungary

Membrane transporters can be major determinants of the pharmacokinetics, safety and efficacy profile of drugs. Their role in drug disposition and toxicity has gained recognition over the last years, and their clini-cal and regulatory relevance has been recently acknowledged in the ITC White Paper. Due to the large list and widespread distribution of membrane transporters, they can affect many PK and toxicological issues, not only drug absorption and elimination, but also tissue distribution and the interplay with enzymes involved in biotransformation. The inhibition of transporter may also lead to accumulation of endogenous compounds or drug-drug interactions (DDI) with concomitant medication. DDI potential is an integral part of drug develop-ment, recognized as a key issue in the evaluation of new drug candidates. ITC White Paper has addressed seven membrane transporters for which clinically relevant drug-drug interactions have been demonstrated to play an important role in drug absorption, distribution, metabolism or elimination, acting alone or in concert with drug metabolizing enzymes. Bilastine (2-[4-(2-(4-(1-(2-ethoxyethyl) benzimidazol-2-ylpiperidin-l-yl)ethyl)phenyl]-2-methyl propanoic acid), is a novel antihistaminic intended for allergic rhinoconjuntivitis and urticaria, which is not metabolized and is mainly excreted in urine and faeces. It has previously demonstrated to be a P-glycoprotein substrate and co-administration with fruit juice (a P-gp and OATP inhibitor) leads to a significant decrease in plasmatic levels, indicating a likely interaction with other intestinal transporters. In order to establish possible drug-drug interactions, bilastine ability to inhibit twelve drug transporters was tested, including the seven recommended in the ITC White Paper and other five related to intestinal absorption and biliar or renal excretion. The study was divided in two steps. First, a standard inhibition assay at one or two concentrations was carried out against three ABC (MRP2, BCRP and BSEP) and eight SLC (OATP1B1, OATP1B3, OATP2B1, OCT1, OCT2, OAT1, OAT3 and NTCP) transporters. Bilastine only gave mild positive results for two uptake transporters, OATP2B1 and OCT1, mainly located in liver and intestine. In a second step, bilastine IC50 was determined for these two transporters and (based in previous knowledge), also for P-gp. The assay was carried out at seven bilastine concentrations ranging from 0.41 to 300 µM. Bilastine showed poor inhibition, as values of IC50 obtained for all the three transporters were ≥ 300 µM. Gut calculated concentration after a 20 mg therapeutic dose is 220µM, thus there is only a remote possibility of bilastine to inhibit OATP2B1 significantly. On the other hand, the Cmax obtained in clinical studies for this dose was < 0.65 µM, and thus no clinical relevant interactions may be expected after absorption. Taking this into account, clinically relevant drug inter-actions regarding transporters are not expected with bilastine as a perpetrator drug. Moreover, as bilastine has also been previously probed not to be a CYP inducer nor inhibitor, clinically relevant drug interactions related to metabolism are not expected either.

This work was supported in part by the Department of Industry, Commerce and Tourism of the Basque Government.

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P487. Contribution of active uptake to the hepatic clearance of seven OATP substrates in rat and human plated hepatocytes

Karelle Ménochet1, Kathryn E. Kenworthy2, Brian J. Houston1 and Aleksandra Galetin1

1School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom, 2DMPK, Mechanisms and Extrapalation Technologies, GlaxoSmithKline, Ware, United Kingdom

Physiologically-based pharmacokinetic models developed in the rat often provide a sound basis for predicting human hepatic disposition. Therefore, understanding of the interplay of active uptake, metabolism and binding in rat and human in vitro systems is crucial for mechanistic prediction of hepatic clearance in human. The passive (P

diff) and active uptake clearance (CL

active) of seven Organic Anion Transporting Protein (OATP) substrates were

assessed in both plated rat (n=3) and human (n=1) hepatocytes over a range of substrate concentrations (0.1-300 µM). Drug uptake at each substrate concentration was assessed at 4 time points (0.5-2 min) at 37 and 4°C to allow the delineation of passive and active processes. Rosuvastatin, pravastatin, pitavastatin, repaglinide, valsartan, tel-misartan and bosentan were selected as probe substrates as they exhibit differing extents of hepatic metabolism and active uptake. In addition, metabolic stability of bosentan, repaglinide and telmisartan was investigated in rat hepatocytes. Depletion of the parent compound was monitored by sampling both in the whole incubation and the media alone, in order to identify the rate-limiting process for these drugs. Finally, non-specific binding (fu

med) and

unbound cell-to-media ratio (Kpu

) were estimated from the uptake experiments for each drug. Generally, higher Km

and lower CL

active values were obtained in the human system, whereas P

diff was consistent between rat and human

hepatocytes. CLactive

showed a 6 and 34-fold range in rat and human hepatocytes, respectively. In both species, Pdiff

was positively correlated with LogD

7.4 while K

m and V

max did not appear to be related to physico-chemical descrip-

tors. Active uptake was identified as the driver of total uptake and subsequent metabolism at clinically relevant concentrations. In both rat and human cell systems, the contribution of active transport to the total uptake was greater than 80 % for all investigated drugs. Similarly, media-loss assay confirmed the importance of active uptake in the hepatic disposition of repaglinide, bosentan and telmisartan in rat. Non-specific binding was limited for all compounds in both species, with fu

med on average > 0.7. The K

pu were reasonably comparable between the two

species and >10 for all the drugs investigated with the exception of telmisartan and repaglinide. The current study highlights both similarities and differences in hepatic uptake observed in rat and human hepatocytes that must be taken into account when applying rat uptake data to predict human hepatic clearance.

P488. Interaction of 140 orally administered drugs with the liver-specific organic anion transporter OATP1B1 (SLCO1B1)

Maria Karlgren1, Gustav Ahlin2, Christel Bergström3, Johan Karlsson4 and Per Artursson2

1Department of Pharmacy, Uppsala University, Uppsala, Sweden, 2Department of Pharmacy, Uppsala University, Uppsala, Sweden, 3Department of Pharmacy, Uppsala University, Uppsala, Sweden, 4AstraZeneca R&D, Molndal, Sweden

Purpose: Active transport via OATP1B1, a highly expressed liver transport protein, is important for drug uptake from blood into the hepatocytes and several clinical drug-drug interactions with OATP1B1 has been observed. The aim of this study was to screen for drug interactions with OATP1B1 and to develop a computational model that discriminate OATP1B1 interacting drugs.

Methods: A large structurally diverse data set of 140 compounds was studied in HEK293 cells stably transfected with human wildtype OATP1B1. Compounds inhibiting the uptake of the model substrate estradiol-17β-glucuronide to 50% or more at 20 uM were defined as inhibitors. Based on the obtained experimental data, a computational model that discriminates OATP1B1-inhibitors was developed using multivariate data analysis. Finally, IC50-curves were generated for a selected subset of inhibitors and comparisons were made with clinical data, using the OATP1B1 specific substrate atorvastatin.

Results: 61 out of 140 studied compounds were defined as OATP1B1 inhibitors. Enrichment of inhibitors was e.g. seen for the groups of statins, protease inhibitors and bile acids. Characteristics of the identified OATP1B1 inhibitors were

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high polarity, high number of H-bonds as well as size. In addition, the new computational model correctly predicted 77% of the OATP1B1 inhibitors in a test set. Finally, inhibition of atorvastatin by strong and weak inhibitors was in good agreement with the corresponding clinical interaction studies.

Conclusions: In conclusion, by using the developed experimental assays it was possible to identify compounds inter-acting with OATP1B1 and to predict the effects in vivo of these compounds on OATP1B1 specific substrates such as atorvastatin.

P489. Molecular Cloning and Comparative Functional Characterization of Human Riboflavin Transporters RFTs

Atsushi Yonezawa1, Yoshiaki Yao1, Hiroki Yoshimatsu1, Satohiro Masuda1, Toshiya Katsura1 and Ken-ichi Inui2

1Department of Pharmacy, Kyoto University Hospital, Kyoto, Japan, 2Kyoto Pharmaceutical University, Kyoto, Japan

Riboflavin, a water-soluble vitamin B2, is essential for normal cellular function. Although riboflavin transporter should play an important role in the maintenance of riboflavin homeostasis, a mammalian riboflavin transporter has not been identified for a long time. We found a novel riboflavin transporter RFT1, using our rat kidney mRNA expression database. Moreover, its homologue RFT3 was also cloned. Human RFT1 shows 41% amino acid identity with human RFT2 and 87% with human RFT3. The tissue distribution and comparative functional characteristics of RFT1, RFT2 and RFT3 were examined. Real-time PCR analysis revealed that RFT1 was mainly expressed in the placenta and small intestine, RFT2 was expressed in the testis and small intestine, and RFT3 was expressed in the brain and several tissues. The uptake of [3H]riboflavin by human embryonic kidney 293 cells transiently transfected with the cDNA coding each RFT was significantly increased, compared with control cells. The apparent Michaelis-Menten constants of RFT1, RFT2 and RFT3 were 1.38 µM, 0.98 µM and 0.33 µM, respectively. RFT-mediated [3H]riboflavin uptake was independent of extracellular Na+ and Cl−. Transport of [3H]riboflavin by hRFT2 decreased when extracellular pH was altered from 5.4 to 8.4, although RFT1 and RFT3 were insensitive to a change of pH. RFT-mediated uptake of [3H]riboflavin was inhibited by some riboflavin analogs, but not D-ribose, cimetidine, probenecid, thiamine and folate. RFT1, RFT2 and RFT3 could play an important role in the riboflavin homeostasis.

P490. Specific and general inhibitors of the three hepatic organic anion transporters OATP1B1 (SLCO1B1), OATP1B3 (SLCO1B3) and OATP2B1 (SLCO2B1)

Maria Karlgren1, Gustav Ahlin2, Anna Vildhede1 and Per Artursson2

1Department of Pharmacy, Uppsala University, Uppsala, Sweden, 2Department of Pharmacy, Uppsala University, Uppsala, Sweden

Purpose: Knowledge about specific and general inhibitors of drug transporters is important for studying transport proc-esses in complex in vitro systems, like primary human hepatocytes, but also for drug interaction screening assays in e.g. drug development. The aim of this study was to screen for drug interactions with OATP1B1, OATP1B3 and OATP2B1 and to study and compare the inhibition patterns of these uptake transporters that all are expressed in the human liver.

Methods: A structurally diverse data set of 130 compounds was studied in HEK293 cells stably transfected with human OATP1B1*1a, OATP1B3*2 or OATP2B1*1. Compounds inhibiting the uptake of the model substrates to 50% or more at 20 uM were defined as inhibitors, and comparisons for all compounds tested were made between the three transporters.

Results: Approximately one third of the tested compounds were classified as inhibitor for two or three of the OATP transporters, whereas 10-15% of all compounds were specific inhibitors for one of the OATP transporters when using the 50% cut off classification.

Conclusions: In conclusion, this study show that the three OATP transporters, OATP1B1, OATP1B3 and OATP2B1, show a broad overlap with regard to interacting compounds, but also that some compounds are specific inhibitors for only

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one of the transporters. These inhibitors will be valuable for understanding hepatic drug transport mechanisms and can also serve as a starting point for development of computational models for prediction of specific and multi-specific OATP inhibitors.

P491. Role of H+/Organic Cation Antiporter MATE1/SLC47A1 in the Renal Tubular Secretion of Metformin and Cephalexin

Masahiro Tsuda1, Shingo Watanabe1, Atsushi Yonezawa1, Tomohiro Terada2, Satohiro Masuda1, Toshiya Katsura1 and Ken-ichi Inui3

1Department of Pharmacy, Kyoto University Hospital, Kyoto, Japan, 2Department of Pharmacy, Shiga University of Medical Science Hospital, Otsu, Japan, 3Kyoto Pharmaceutical University, Kyoto, Japan

Multidrug and toxin extrusions (MATE1/SLC47A1 and MATE2-K/SLC47A2) as well as organic cation transporter 2 (OCT2/SLC22A2) play important roles in the renal excretion of cationic drugs. Organic cation transporter OCT2 is expressed in the basolateral membrane of proximal tubules and mediates the uptake of cationic drugs from the circula-tion. MATE1 and MATE2-K are localized at the brush-border membrane of human kidney, which were characterized as H+/organic cation antiporters. In rodents, Mate1, but not Mate2, is expressed in the kidney. We have previously demonstrated the tissue distribution, membrane localization, functional characteristics and genetic variants of MATE1 and MATE2-K. In the present study, we examined the role of Mate1 in the renal excretion of cationic metformin and zwitterionic cephalexin, using Mate1 knockout mice. After a single intravenous administration of metformin (5 mg/kg), a 2-fold increase in the area under the blood concentration-time curve for 60 min of metformin in Mate1(-/-) mice was observed. Urinary excretion of metformin for 60 min after the intravenous administration was significantly decreased in Mate1(-/-) mice compared with Mate1(+/+) mice. The renal clearance and renal secretory clearance of metformin in Mate1(-/-) mice were approximately 18% and 14% of those in Mate1(+/+) mice, respectively. There were no significant difference in pharmacokinetics of metformin between Mate1(+/+) and Mate1(+/-) mice. Moreover, after a single intravenous administration of cephalexin (5 mg/kg), blood concentration and renal concentration were also significantly increased. The urinary excretion, renal clearance and secretory clearance of cephalexin were reduced in Mate1(-/-) mice. These results indicated that Mate1 plays predominant roles in the renal tubular secretion of metformin and cephalexin.

P492. Integrated cell models for prediction of drug transport and drug metabolism in the human hepatocyte

Maria Karlgren1, Richard Svensson2 and Per Artursson3

1Department of Pharmacy, Uppsala University, Uppsala, Sweden, 2Department of Pharmacy, Uppsala University, Uppsala, Sweden, 3Department of Pharmacy, Uppsala University, Uppsala, Sweden

Purpose: Recent research indicates an extensive interplay between drug transport and drug metabolism in the human liver. The aim of this study was to establish integrated cellular models were both drug transport and drug metabolism can be studied simultaneously. Here, we report results from the first of these models.

Methods: HEK293 cells were transfected with both the hepatic uptake transporter OATP1B1*1a and the drug metaboliz-ing enzyme CYP3A4. Expression and activity of the two proteins were validated using western blotting and with incuba-tions using model substrates. The interplay between the two expressed proteins was investigated using compounds that are substrates for OATP1B1 and CYP3A4, such as atorvastatin.

Results: Validation of the integrated cell model shows that both OATP1B1 and CYP3A4 are expressed and functional in the cell model. A time dependent interplay between OATP1B1 and CYP3A4 was observed.

Conclusions: In conclusion, by using the new integrated in vitro model we have proven that the interplay between transport and metabolism can be studied in simple cell systems such as HEK293 cells that lack significant background

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activity of drug transporting and metabolizing enzymes. In further work, we will incorporate efflux transporters of the ABC-transporter family into the models.

P493. Amino acid and peptide transport in primary rat enterocytes

Caroline MacLean1, Ulla Moenning2, Andreas Reichel2 and Gert Fricker3

1Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany, 2Research Pharmacokinetics, Bayer Schering Pharma AG, Berlin, Germany, 3Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany

The bioavailability of an orally administered drug is mainly influenced by its absorption across the intestinal epi-thelium. However, to be available many emerging drugs depend on an active uptake into enterocytes located in the intestinal epithelium, as current development suggests. Therefore, a useful cell-based tool for drug discovery should display relevant intestinal uptake transporters. For that purpose isolated rat enterocytes were evaluated regarding their potential for carrier-mediated uptake using different substrates of drug transporters. We have used glycyl-sarcosine as a substrate for the apical peptide transporter PepT1, which transports various drugs like ß-lactam antibiotics and peptidomimetics, and L-leucine as a model substrate of amino acid transporters like LAT2, which transport drugs like L-DOPA, alpha-methyldopa, melphalan, and gabapentin. As expected, PepT1 was active in these primary enterocytes. The time-dependent uptake was pH sensitive, and could be competitively inhibited by other peptidomimetics and dipeptides. The cells also showed a sodium-independent active uptake of L-leucine which could be inhibited by other neutral amino acids and BCH, which is a specific inhibitor for system L type amino acid transporters, but not by MeAIB. Taken together, these data indicate that several drug uptake transporters are expressed in isolated primary rat enterocytes and this cell model may be suitable for evaluating absorption behaviour of drug discovery compounds.

P494. Pharmacokinetics and Hepatic Uptake of Eltrombopag, A Novel Platelet Increasing Agent

Kazuya Takeuchi, Kazuki Matsubara, Saki Umeda, Tomoko Sugiura, Noritaka Nakamichi and Yukio Kato

Division of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan

Eltrombopag (ELT) is a novel small molecule, which is orally administered as a non-peptide trombopoietin receptor agonist for the treatment of immune thrombocytopenia. Limited information is available on the pharmacokinetic profile of ELT. ELT is thought to be mainly eliminated in the liver because of its minimal excretion into the urine. However, the mechanism of hepatic handling of ELT has not yet been understood. The aim of the present study is to investigate overall elimination mechanism for ELT. We conducted the pharmacokinetic studies of ELT after i.v. administration using bile duct cannulated rats, and the integration plot analysis for ELT uptake in the rat liver. Approximately 40% of unchanged ELT was excreted into the bile until 72 hours after i.v. administration, whereas ELT was not detected in the urine. The total clearance was much lower than the hepatic blood flow rate and very similar to hepatic uptake clearance obtained from integration plot analysis. Coadministration of rifampin reduced both total clearance and hepatic uptake clearance of ELT. These results suggest that hepatic uptake is a rate-limiting process in the overall elimination of ELT. Therefore, we further focused on hepatic uptake mechanism of ELT. Uptake of ELT in isolated mouse hepatocytes was evaluated in the absence and presence of various compounds. HEK293 cells transfected with various hepatic uptake transporters of human was used to identify the ELT transporter(s). Uptake of ELT in isolated mouse hepatocytes showed temperature- and incubation time-dependency, and saturated at higher ELT concentration. The ELT uptake was inhib-ited by both organic anion and cation compounds. These results suggest that carrier mediated transport is involved in hepatic ELT uptake. Uptake of ELT by HEK293 cells transfected with OATP1B1, OATP2B1 and OCT1 was higher than that found in mock-transfected cells. ELT showed inhibitory effect on OATP1B1- and OATP2B1-mediated uptake of [3H]estrone 3-sulfate, OATP1B3 mediated uptake of [3H]estradiol 17beta-glucuronide and OCT1 mediated uptake of [14C]tetraethylammonium. These results suggest that various transporters such as organic anion transporters and organic cation transporters could be involved in hepatic uptake of ELT.

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P495. Impact of chronic renal failure on the expression and activity of kidney drug transporters in rats

Judith Naud1, Josée Michaud1, Stephanie Beauchemin1, Marie-Josee Hebert2, Michel Roger2, Francois Madore3, Francois A. Leblond4 and Vincent Pichette1

1Departement de Pharmacologie, Centre de recherche de l′Hopital Maisonneuve-Rosemont, Montreal, QC, Canada, 2Centre de recherche de l′Hopital Notre-Dame, Montreal, QC, Canada, 3Centre de recherche de l′Hopital du Sacré-Coeur, Montreal, QC, Canada,4Département de Néphrologie et Métabolisme, Centre de recherche de l′Hopital Maisonneuve-Rosemont, Montréal, QC, Canada

Sirolimus is an immunosuppressive agent widely used to prevent kidney-graft rejection. Its use is privileged because, unlike calcineurin inhibitors (CNI) such as cyclosporin A and tacrolimus, it is believed to be less toxic to the kidneys although it can induce severe proteinuria. CNIs and sirolimus are known to be substrates of cytochrome P450 3A and of various drug transporters such as p-glycoprotein (P-gp) and organic-anion-transporting-polypeptides (Oatp) located in tubular cells. We have recently shown that the expression and activity of these transporters and enzymes are modified by chronic renal failure (CRF), affecting drug metabolism and elimination. Our hypothesis is that CRF (as observed in failing grafts) decreases kidney drug transporters. This could cause sirolimus or CNIs to accumulate in the kidneys of patients or animals with impaired renal function and cause toxicity to the kidneys. The goal of this study was to evalu-ate the expression of drug transporters in the kidney of rats with surgically-induced CRF and to evaluate the intrarenal accumulation of drugs including sirolimus in CRF rats kidneys compared to control. Two groups of rats were studied: control and CRF. CRF was induced in young male rats by 5/6 nephrectomy and remnant kidneys were collected for analysis 42 days post-surgery. Compared to control rats, we observed important variations in the protein expression of P-gp (56.1%, p<0.001), MRP3 (391.7%, p<0.001), MRP4 (278.4%, p<0.001), Oat3 (82.0%, p<0.05) and Oatp2 (79.9%, p<0.001) in CRF suffering rats with similar changes in gene expression. Furthermore, these changes could be reproduced by incubating a human proximal tubule cell-line (HK-2) with sera from CTL or CRF rats. Finally, the renal secretion of 3H-digoxin (a specific substrate of Oatp2 and P-gp, like CNIs), 3H-sirolimus and 14C-benzylpenicillin (a specific substrate of Oat3 and MRP4) were decreased in CRF rats, compared to control, as shown by a 2-9 times greater accumulation of these drugs in CRF rats′ kidney. Although MRP4′s expression is greatly upregulated in CRF, there is a 9 times greater accumulation of 14C-benzylpenicillin in the kidney of CRF-suffering rats. It is well known that many uremic toxins are also substrates of MRP4. Competition for MRP4 transport could thus explain this 14C-benzylpenicillin accumulation. Our results demonstrate that CRF affects the expression and activity of several kidney drug transporters leading to the intrarenal accumulation of drugs. This, and competition with uremic toxins, could explain the tubular toxicity of many drugs, including CNIs and sirolimus.

P496. Ubiquitination Is Associated With Degradation of ATP-binding Cassette Transporter A1 (ABCA1) On Cell Surface

Tadahaya Mizuno, Hisamitsu Hayashi and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to Apolipoprotein A-I (apoA-I), and thereby induces the formation of high density lipoprotein (HDL). Its physiological importance has been demonstrated by the presence of Tangier disease and familial HDL deficiency, both of which are attributed to a mutation in the ABCA1 gene and show a low level of HDL in the plasma, promoting atherosclerotic cardiovascular disease. Therefore, ABCA1 is believed to have a great potency as a therapeutic target. However, much less is known about the controlling mechanism of ABCA1 expression and function excluding the transcriptional regulation. In order to help the development of the therapy for atherosclerosis targeting ABCA1, we have explored the posttranslational sorting mechanism of ABCA1. Our current study specifically focuses on ubiquitination, a reversible post-translational modification, since ubiquitination is reported to be an important sorting signal for endocytosis and subsequent lyso-somal degradation of receptor and channel. Ubiquitinated ABCA1 was verified in human hepatoma cell line, Huh7 cells, and mouse plasma membrane fractions from liver, by co-immunoprecipitaiton. ABCA1 degradation on the cell surface

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was significantly inhibited by the overexpression of the dominant negative form of ubiquitin in Huh7 cells. Moreover, the disruption of the Endosomal Sorting Complex Required for Transport (ESCRT) pathway, a dominating machinery for endosomal-lysosomal degradation of ubiquitinated proteins, by siRNA-mediated knockdown of Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) in Huh7 cells, significantly delayed the degradation rate of ABCA1 from cell surface. This was accompanied by an increase in ABCA1 expression on the plasma membrane and apoA-I-mediated cholesterol efflux function. Together with the fact that lysosomal inhibitor treatments in Huh7 cells increase ABCA1 expression, all of these results suggest that the cell surface-resident ABCA1 is degraded by the ubiquitin-mediated endosomal-lysosomal degradation pathway. The modulation of ABCA1 ubiquitination can thus be a potential new therapeutic target for anti-atherogenic drugs.

P497. Influence of Roux-en-Y gastric bypass surgery on the pharmacokinetics of paracetamol, talinolol and amoxicillin in obese patients

Jette Peters1, Stefan Oswald1, Sierk Haenisch2, Kaja Ludwig3, J. Bernhardt3, Karen May4, Christiane Modess1, Ingolf Cascorbi2 and Werner Siegmund1

1Department of Clinical Pharmacology, University of Greifswald, Greifswald, Germany, 2Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Kiel, Germany, 3Klinik für Chirurgie, Klinikum Südstadt Rostock, Rostock, Germany, 4Department of Clinical Pharmacology, University of Greifswald, Greifswald, Germany

Background: Roux-en-Y gastric bypass, whereby the stomach, the duodenum and the jejunum are circumvented, is an accepted procedure in weight loss surgery which was shown to significantly reduce body weight and obesity-related co-morbidity. Despite its frequent application (United States: ∼150.000/year) and the well documented risk of nutritional deficiencies, only little is known about the pharmacokinetic consequences in these patients who are often subjected to multiple drug therapy. Therefore, this study aims to evaluate the influence of this bariatric surgery on pharmacokinet-ics of paracetamol (well absorbed along the gut), talinolol (ABCB1 substrate) and amoxicillin (PEPT1 substrate). We hypothesized that absorption of talinolol and amoxicillin is decreased after surgery because of the regional intestinal expression of ABCB1 and PEPT1.

Methods: Disposition of paracetamol (200 mg, po), talinolol (50 mg, po) and amoxicillin (250 mg, po) was studied in a three-period, cross-over study in 8 obese patients (7 females, 1 male, age 22-53 years, body mass index 44.3-61.9) before and after Roux-en-Y gastric bypass surgery. Serum concentrations of paracetamol, talinolol and amoxicillin were quantified using validated HPLC-UV and LC-MS/MS methods. Tissue specimens were taken from the duodenum and the jejunal anastomosis, respectively, before, during and one year after surgery.

Results: Bariatric surgery did not significantly affect Cmax

(2.63 ± 1.07 µg/ml vs. 2.00 ± 0.69 µg/ml), AUC0-24h

(6.11 ± 1.56 µg×h/ml vs. 5.94 ± 3.90 µg×h/ml) and half-life (4.35 ± 2.89 h vs. 3.97 ± 2.47 h) of paracetamol. C

max and AUC

0-24h of talinolol

(36.5 ± 17.3 ng/ml vs. 42.7 ± 17.2 ng/ml; 350 ± 115 ng×h/ml vs. 403 ± 155 ng×h/ml) and amoxicillin (2.57 ± 1.51 µg/ml vs. 3.84 ± 1.45 µg/ml, p=0.036; 7.47 ± 4.58 µg×h/ml vs. 8.23 ± 1.65 µg×h/ml) tended to be decreased. The differences in disposition were most prominent during the absorption period of talinolol (AUC

0-6h, 98.4 ± 23.8 ng×h/ml vs. 142 ± 57

ng×h/ml, p=0.075) and amoxicillin (AUC0-3h

, 5.02 ± 2.88 µg×h/ml vs. 6.14 ± 1.66 µg×h/ml).

Conclusions: Roux-en-Y gastric surgery may result in reduced oral absorption of talinolol and amoxicillin by bypassing the respective ABCB1- and PEPT1-mediated absorption windows for both compounds in the proximal intestine.

P498. Human Hepatic Ribavirin Transporter: Molecular Characterization and Expression Profile in Hepatocytes

Tomomi Furihata, Yukina Fukuchi, Minami Iikura, Misato Hashizume and Kan Chiba

Lab of Pharmacol & Toxicol, Grad Sch of Pharmaceut Sci, Chiba Univ, Chiba-Shi, Japan

Ribavirin is a nucleoside analog that is essential for the treatment of hepatitis C virus infection. It has been thought that equilibrative and concentrative nucleoside transporters (ENTs, SLC29 and CNTs, SLC28) are involved in membrane transport of ribavirin. Although ribavirin uptake by hepatocytes is thought to be a prerequisite step for ribavirin to

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exert its anti-viral activity, ribavirin uptake transporters in human hepatocytes remain uncharacterized. The purposes of the present study were to identify the ribavirin uptake transporter in human hepatocytes and to clarify the causal factors for different uptake levels among the human hepatocytes. When the uptake activities of ribavirin (10 µM) in three Caucasian hepatocyte lines (HH268, HH283 and HH291) were measured by an oil-filtration assay, all ribavirin uptake activities in HH268 and HH283 were Na+-independent. In HH291, Na+-independent uptake activity explained the largest fraction of the total carrier-mediated uptake (60%), and the rest of the uptake activity was Na+-dependent, of which molecular entity was unknown because Na+-dependent nucleoside transporter CNT2 and CNT3 mRNA were expressed at only trace levels in three hepatocyte lines. The Na+-independent uptake activities in three hepatocytes lines were abolished by the addition of a specific inhibitor of ENT1. In line with the result that ENT1 mRNA was abundantly expressed in three hepatocyte lines, our results suggest that ENT1 is a major ribavirin uptake transporter in human hepatocytes. In the above experiments, we found that the level of ENT1-mediated uptake in HH291 was higher than that in HH268 or HH283 and, consistently, that the level of ENT1 mRNA expression was higher in HH291 than that in HH268 or HH283. To characterize molecular mechanisms causing different expression levels among three hepatocyte lines, the 5′-side of the SLC29A1 gene structure was characterized by determination of transcription start sites and by reverse transcription-PCR. The results showed that the SLC29A1 gene has at least four alternative promoters and that several mRNA variants with the distinct 5′-untranslated region were expressed in human hepatocytes. Notably, the results also showed that the levels of four ENT1 mRNA isoforms (ENT1d1-d4) in HH291 were specifically higher than those in HH283 or HH268 (about 5-fold). No genetic polymorphism was detected in the coding region and the proximal promoter region of the SLC29A1 gene in three hepatocytes lines. In conclusion, our results suggest that ENT1 is a major ribavirin uptake transporter in human hepatocytes and that the different expression levels of specific isoforms of ENT1 mRNA cause different ribavirin uptake levels among the human hepatocyte lines.

P499. Characterization of the NOVEL TISSUE-SPECIFIC Promoter of the SLC29A1 GENE

Seiya Ishii1, Tomomi Furihata1, Minami Iikura1, Yukina Fukuchi1, Misato Hashizume1, Miki Nagai2 and Kan Chiba1

1Laboratory of Pharmacology and Toxicology,Graduate School of Pharmaceutical Sciences, Chiba University., Chiba-Shi, Japan, 2Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Human equilibrative nucleoside transporter 1 (hENT1, gene symbol SLC29A1) is mainly expressed at the plasma membrane in various cells and transports various endogenous nucleosides and anticancer or antiviral nucleoside analogs across the membrane. ENT1 functions as the regulator of adenosine signaling as well as pharmacological action of nucleoside analogs. Therefore, it is an important issue to clarify the mechanisms by which the SLC29A1 gene expression is regulated, which remains largely unknown. We have recently reported the results identifying the novel promoter regions located at approximately 3.5 kbp and 7.4 kbp upstream from the initially identified promoter region (P1) (1). More recently, we also revealed that the novel promoter region exists at approximately 10 kbp upstream from P1 promoter region, termed P4 promoter. In the present study, to clarify mechanisms controlling SLC29A1 gene expression, we characterized the P4 promoter region of the SLC29A1 gene. ENT1 mRNA isoform transcribed by P4 promoter, termed ENT1e1 mRNA, contained the 5′-untranslated region distinct from those of other ENT1 mRNAs. To analyze the tissue expression profile of hENT1e1 mRNA, we performed reverse transcriptase-polymerase chain reaction (RT-PCR). The result showed that ENT1e1 mRNA exhibited the pancreas-specific expression profile among seven tissues analyzed. When the P4 promoter region was characterized by deletion assay, it was found that the p4-238/+32 region was a minimal promoter region of the P4 promoter and that the GC-box and CCAAT-box, which were located close to the transcription start site (TSS), played critical roles in the promoter activity. In line with this result, the promoter activity of p4-75/+32 containing the GC-box was activated by exogenously expressed Sp1. To clarify a role of the GC box methylation in SLC29A1 gene expression, methylation status of CpG sequences within the GC box of P4 promoter region was examined by bisulfite sequencing. The results showed that the CpG sequences within the GC-box were mostly unmethylated in the pancreatic genomic DNA, whereas they were highly methylated in the kidney and lung genomic DNA. Taken together, the results of the present study showed that the P4 promoter is activated through the GC-box and CCAAT-box in a tissue-specific manner to generate ENT1e1 mRNA in the tissue. The tissue-specific epigenetic condition may be essential for the P4 promoter activation.

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Reference

(1) Fukuchi Y., Furihata T., Hashizume M., Iikura M., Chiba K. Characterization of ribavirin uptake systems in human hepatocytes. J Hepatol. 2010;52:486-92.

P500. Evaluation of In Vivo Hepatobiliary Transport Using Positron Emission Tomography (PET): Studies in Rats and Application to Microdosing Clinical Studies

Tadayuki Takashima1, Hiroko Nagata1, Hideki Ishii1, Ryosuke Ijuin1, Takahiro Nakae1, Masaaki Tanaka2, Yoshihito Shigihara2, Suzuka Ataka2, Hisashi Doi1, Yasuhiro Wada1, Masaaki Suzuki1, Kazuya Maeda3, Hiroyuki Kusuhara3, Yuichi Sugiyama3 and Yasuyoshi Watanabe1

1Center for Molecular Imaging Science, RIKEN, Hyogo, Japan, 2Graduate School of Medicine, Osaka City University, Osaka, Japan, 3Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

[Purpose]: It is now well accepted that drug transporters play important roles in tissue distribution and excretion of drugs and their metabolites. Clinical studies have shown that variation in drug transporter activity caused by genetic polymorphisms or drug-drug interactions can affect the variability in therapeutic efficacy and the incidence of adverse effects. Positron Emission Tomography (PET) is considered to be a useful tool for functional analysis of drug transporters in vivo since it enables real-time monitoring of tissue concentration of radiolabeled drugs. In this study, we developed a quantitative PET methodology with (15R)-16-m-[11C]tolyl-17,18,19,20-tetranorisocarbacyclin (15R-[11C]TIC-Me) for in vivo kinetic analysis of hepatobiliary transport in rats and also applied this approach to microdosing clinical PET study.

[Method]: 11C-labeled 15R-TIC-Me was synthesized from its demethylated derivative using a palladium-mediated rapid coupling reaction of [11C]methyl iodide. In rat study, serial abdominal PET scans was performed as well as continuous blood sampling after bolus injection of 15R-[11C]TIC-Me and tissue distribution of its radioactivity was analyzed. In microdosing clinical PET study, healthy male volunteers were enrolled after obtaining informed consent and PET images in abdominal regions were acquired for 90 minutes following intravenous bolus injection of 15R-[11C]TIC-Me.

[Results and discussion]: PET scans in normal rats revealed that the radioactivity was localized mainly in the liver and, to lesser degree, kidney within 5 min of injection, and then the radioactivity was observed mainly in bile at 90 min following the administration of 15R-[11C]TIC-Me. Introducing integration plot analysis to PET image results revealed the feasibility of simultaneous determination of tissue uptake and canalicular efflux in a single subject.Radiometabolite analysis in rats showed that 15R-[11C]TIC was converted to at least three metabolites (M1, M2, and M3), and that M3 is the major component in bile. The study in Mrp2-deficient rats showed the radioactivity was mainly excreted by renal excretion. These results show that the radioactivity derived from M3 is primarily excreted into bile,and Mrp2 is largely responsible for its excretion in normal rats. PET studies in healthy male volunteers using 15R-[11C]TIC-Me showed that the radioactivity was mainly excreted via hepatobiliary excretion, which was similar to the results in rat. Treatment of rifampicin (600 mg/body) significantly decreased both the hepatic uptake clearance and biliary excretion clearance of the radioactivity in human. These results indicate that the hepatobiliary excretion can be assessed quantitatively by PET image analysis and it was applicable to human study.

[Conclusion]: We demonstrated the utility of non-invasive PET imaging using 15R-[11C]TIC-Me as a tool for kinetic analysis of hepatobiliary transport.

P501. Canalicular Mrp2 localization is reversibly regulated by the phosphorylation status of its an-choring protein

Shuichi Sekine1, Kousei Ito2 and Toshiharu Horie3

1Department of Biopharmaceutics, Graduate school of pharmaceutical sciences, Chiba university, Chiba, Japan, 2Department of Pharmacy, the University of Tokyo Hospital, Faculty of Medicine, the University of Tokyo, Tokyo, Japan, 3Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

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The multidrug resistance-associated protein 2 (Mrp2) belongs to ATP-binding cassette transporter family expressed on the bile canalicular membrane of hepatocytes involved in bile flow formation. Cholestasis accompanied by internaliza-tion of Mrp2 from the canalicular membrane surface to intracellular compartment was observed by acute oxidative stress condition accompanied by the decreased intracellular reduced form of glutathione (GSH). This internalization process was regulated by a series of signaling pathway, finally leading to activation of novel protein kinase C (nPKC). Besides, we have reported that internalized Mrp2 was recycled to the canalicular membrane surface in a protein kinase A (PKA)-dependent manner when intracellular GSH was replenished1. However, putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. We have recently demonstrated that the decrease of C-terminally phosphorylated ezrin (a family of ezrin-radixin-moesin expressed in the small intestine) as well as its reduced interaction with Mrp22. In this study, we examined if the C-terminal phosphorylation status of radixin (mainly expressed in liver) and its interaction with Mrp2 were affected by redox status in rat hepatocytes. In the cultured rat hepatocyte, PKC inhibitor (Go6850) 100 nM and PKA inhibitor 100 nM were treated prior to tert-butylhydroperoxide (t-BHP) 100 mM and GSH ethyl ester (GSH-EE; cell permeable form of GSH) 3 mM treatment. C-terminally phosphorylated radixin (p-radixin) co-immunoprecipitated with Mrp2 was detected with p-radixin spe-cific antibody. As a result, canalicular membrane localization of Mrp2 was decreased and recovered by t-BHP treatment and subsequent GSH-EE treatment, respectively, although Mrp2 expression in total cell lysates was not affected. On the other hand, p-radixin in co-immunoprecipitant with Mrp2 were significantly decreased by t-BHP treatment and returned to control level by subsequent GSH-EE treatment. These data indicated that phosphorylation status of radixin and its association with Mrp2 were reversibly regulated by the intracellular redox status. PKC inhibitor prevented the t-BHP induced decrease of p-radixin, but did not affect its recovery process by GSH-EE treatment. On the other hand, PKA inhibitor did not affect the t-BHP induced decrease of p-radixin, but did affect its recovery by GSH-EE treatment. These results suggested that the interaction of p-radixin with Mrp2 was decreased by the activation of PKC under oxidative stress condition which subsequently leads to Mrp2 internalization, while the interaction between p-radixin and Mrp2 was increased by the activation of PKA during the recovery process from oxidative stress. In conclusion, balance of activation of PKA and PKC accompanied by the variation of intercellular GSH content determines the phosphorylation status of radixin and this change regulates redox sensitive canalicular Mrp2 localization.

References

1. Sekine S et al., Canalicular Mrp2 localization is reversibly regulated by the intracellular redox status. Am J Physiol Gastrointest Liver Physiol. 295: G1035-41, 2008

2. Nakano T et al., Correlation between apical localization of Abcc2/Mrp2 and phosphorylation status of ezrin in rat intestine. Drug Metab Dispos. 37: 1521-7, 2009

P502. Ezrin has a key role in the expression profile of MRP2 and MDR1 along intestinal tract

Shuichi Sekine1, Kousei Ito2, Takafumi Nakano3 and Toshiharu Horie3

1Department of Biopharmaceutics, Graduate school of pharmaceutical sciences, Chiba university, Chiba, Japan, 2Department of Pharmacy, the University of Tokyo Hospital, Faculty of Medicine, the University of Tokyo, Tokyo, Japan, 3Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Multidrug resistance-associated protein 2 (Mrp2/Abcc2) and multidrug resistance-protein 1 (Mdr1/Abcb1) are well known efflux transporters located on the brush border membrane of small intestinal epithelia to limit absorption of a broad range of their substrates. The expression of Mrp2 and Mdr1 along the intestinal tract is well regulated. Mrp2 expression is the highest at the upper region of small intestine and decreases along the tract. On the other hand, Mdr1 increases along the tract. It has been well known there is an inconsistency between relative mRNA and protein expression profiles of Mrp2 and Mdr1 along the intestinal tract. These reports imply the presence of post-transcriptional and/or post-translational regulatory mechanism. We have also reported the post-translational regulation of Mrp2 in small intestine that the membrane surface localization of Mrp2 is physically supported by ezrin, which acts as a molecular anchor con-necting Mrp2 to F-actin cytoskeleton, in the C-terminus phosphorylation of ezrin dependent manner1. Therefore, in this study, we examined whether phosphorylation status of ezrin contributes to Mrp2, and Mdr1 which is also regulated by ezrin, expression profile along the small intestinal tract. The western blot analysis revealed that there are good correlation between the phosphorylation profile of ezrin and the Mrp2 expression profile, and correlation between the expression

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profile of ezrin and the Mdr1 expression profile along the rat small intestinal tract. To confirm the causality of these cor-relations, we established ezrin T567A mutant (mimic for dephosphorylated form) expressing Caco-2 cells and investigated the effect on MRP2 and MDR1. As a result, both major decrease of membrane surface and minor decrease of total MRP2 expression were observed in T567A expressing Caco-2 cells compared with wild type expressing Caco-2 cells. In contrast to MRP2, both wild-type and T567A expression increase membrane surface and total expression of MDR1 compared to parent Caco-2 cells. These findings suggest that phosphorylation and dephosphorylation profiles of ezrin regulate MRP2/Mrp2 and MDR1/Mdr1 expression profile along small intestinal tract in a different manner, respectively.

References

1. Nakano T et al., Correlation between apical localization of Abcc2/Mrp2 and phosphorylation status of ezrin in rat intestine. Drug Metab Dispos. 37: 1521-7, 2009

P503. hiBMEC/kyas, A NEWLY IMMORTALIZED HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELL LINE, IS A PROMISING TOOL FOR AN IN VITRO BLOOD-BRAIN BARRIER MODEL

Satoshi Kishida1, Tomomi Furihata1, Atsuko Kamiichi1, Yuki Ohta1, Kosuke Saito1 and Kan Chiba2

1Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University., Chiba-Shi, Japan, 2Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMEC) and strictly regulates drug pen-etration into the brain. Until now, many in vitro BBB models with animal BMEC have been developed and used to predict drug penetration into human brain. However, the results obtained from these models cannot be always extrapolated to the human due to the species-difference between human and animals. In the present study, we have established and characterized novel human immortalized BMEC, termed hiBMEC/kyas-1, as a promising tool for in vitro human BBB model. hiBMEC/kyas-1 were established by immortalization of human primary BMEC (hprBMEC) through introduc-tion of the temperature-sensitive simian virus 40 large T antigen (tsSV40T-ag) gene and the human telomerase reverse transcriptase (hTERT) gene via a lentiviral vector system. hiBMEC/kyas-1 expressed tsSV40T and hTERT proteins, and they showed temperature-dependent proliferation ability. The results of reverse transcription-polymerase chain reaction (RT-PCR) showed that hiBMEC/kyas-1 expressed many mRNAs of the junctional genes and the pharmacokinetics-related genes, such as P-glycoprotein (P-gp), at the levels similar to those in hprBMEC. As for the function of tight junctions (TJs), the results of the sucrose permeability assay showed that permeability coefficient for [14C]-sucrose was 1.02 ± 0.0725 × 10−3 cm/min. The functional activity of P-gp was determined by Rhodamine-123 (R-123) permeability assay. Amount of R-123 transported basolateral side (B) to apical side (A) was 1.6-fold higher than that of A to B. The uptake activity of 1-methyl-4-phenylpyridinium, estrone-3-sulfate and adenosine were also observed in hiBMEC/kyas-1, which were presumably attributed to organic cation transporter 3, plasma membrane monoamine transporter, organic anion transporter polypep-tides and equilibrative nucleoside transporter 1 and 2, respectively, on the basis of the results of RT-PCR. In addition, the function of aryl hydrocarbon receptor (AhR) in hiBMEC/kyas-1 was observed in hiBMEC/kyas-1 with marked induction of cytochrome P450 1A1 and 1B1 gene expression upon the response to treatment with benzo [a] pyrene. In conclusion, our results show that hiBMEC/kyas-1 have a high proliferating ability with the morphologies similar to that of hprBMEC and that they possess the functional activities of TJs and drug transporters as well as the function of AhR, which are important regulators of drug penetration into the brain. Therefore, hiBMEC/kyas-1 may be useful for the in vitro BBB model which accurately predicts drug penetration into human brain.

P504. Phylogenetic Analysis and Cross Species Characterisation of Drug Transporters in the Intestine

Ciaran Fisher1, Tanya Coleman2 and Nick Plant1

1Molecular Toxicology, University of Surrey, Guildford, United Kingdom, 2Global Development DMPK, AstraZeneca Ltd, Macclesfield, Cheshire, United Kingdom

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The accurate prediction of the behaviour of drugs and xenobiotics in humans is essential to the safe use of such compounds and the development of new drug therapies. These compounds are tested in model pre-clinical species and in vitro systems that in many cases do not offer an accurate analogy of the human condition. Drug metabolis-ing enzymes and transporters play a key role in the body′s reaction to drugs/xenobiotics and have been shown to differ in their expression and activity between pre-clinical species and in vitro systems. The increasing availability of genomic data, coupled with accessible tools to analyse this data, offers the opportunity to study the evolution of important gene families. The ATP-binding cassette (ABC) super-family of genes encodes 48 proteins in humans, divided into seven sub-families. Of these seven sub-families, five encode for membrane exporter proteins with an extensive and diverse range of substrate specificities including both endobiotics and xenobiotics. Indeed, these exporters are implicated in a multiple drug resistance phenotype that is associated with reduced clinical efficacy of a number of current therapies. To examine the inter-relationship of this super-family we have constructed phylogenetic trees over an extended evolutionary distance representing each of the seven sub-families, plus the first complete phylogeny within humans across all sub-families. The results identify a number of novel orthologues in pre-clinical species, and are thus important for the extrapolation of data to humans. Using the generated phylogenetic trees we have also been able to demonstrate for the first time that genes associated with a multiple drug resistance phenotype cluster separately from other genes within the same sub-family, suggesting a conserved, fundamental, difference in these proteins. Such data provides insight into potential biological rationales underlying multiple drug resistance phenotypes, and allows the identification of the central transporters in both humans and pre-clinical species that must be further studied. We have then gone on to characterise a range of drug transporters along the gastrointestinal tract, at the transcript and protein level, in both rat and dog. This coupled with kinetic data will allow construction of an in silico model of intestinal absorption, representing a starting point for more accurate prediction of human intestinal ADME processes from pre-clinical species.

P505. Examining the comparative roles of influx and efflux in determining the life cycle of chemicals within the cell

Samantha L. Forster1, Steve R. Hood2 and Nick J. Plant3

1University of Surrey, Guildford, United Kingdom,2Dmpk-MET, GlaxoSmithKline R&D, Ware, United Kingdom, 3Centre for Toxicology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom

Drug transporters are increasingly recognized as important drivers in the pharmaco- and toxico- kinetic characteristics of therapeutic agents. Preclinical assays are used to determine the effects of transport on the pharamcokinetics and toxicology of new chemical entities early in the drug discovery/development programme. However, extrapolation of data derived from in vitro and in vivo animal models to the human situation can often be confounded by differences between the systems: Such differences may include the differential expression and/or functionality of drug transport proteins. Such a problem lends itself well to examination using in silico modelling, with regard to both extrapolation between model systems and increased understanding of the underlying design principles for the ADME network. Current models to address such questions tend to apply a reductionist approach, whereby multiple parameters are described within a single mathematical term. Whereas such an approach is highly suited for the first of these approaches (extrapolation), it is less well suited to mechanism dissection, due to the lack of individual parameter details. In such cases, the use of a more detailed, data-rich, model covering a smaller aspect of the biology may be of more value1. We have created a deterministic model of the life cycle of the ABCB1 (MDR1) substrate rho123 within human hepatocytes, using the systems biology markup language (SBML)-compliant programs CellDesigner and COPASI. This model is populated with quantity and kinetic parameters taken from existing literature or derived de novo. We demonstrate that such a model is capable of replicating the in vitro situation for rhodamine 123 disposition within hepatocytes, validat-ing its use as a model. The first utility for such a model, as described above, is to aid extrapolation between different biological concepts. We have measured the expression level of important proteins within different in vitro systems, and demonstrate how these changes may impact upon the disposition of chemicals. In addition, we show how knowledge of these different levels can be used to aid extrapolation between different test systems. Finally, we wished to use this model to increase our understanding of the key determinants for compound life cycle within a cell. To examine this important concept, we next undertook sensitivity analysis to determine which proteins or parameters were most likely to cause a significant alteration in rho123 disposition. We demonstrate that the most sensitive node, with respect to

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compound life-cycle within the cell, is uptake through active transport proteins. Such information is important as it may further guide investigations as to the accurate cellular targeting of therapeutics, or the further understanding of multiple drug resistance phenotype.

References

Howe, K., G. G. Gibson, T. Coleman and N. Plant. In silico and in vitro modelling of hepatocyte drug transport processes: Importance of ABCC2 expres-sion levels in the disposition of carboxydichlorofluroscein. Drug Met. Disp. 2009; 37(2): 391-399.

P506. Uptake mechanism of ochratoxin A and citrinin through human liver

Ge Gao1, Maria Karlgren1 and Per Artursson2

1Department of Pharmacy, Uppsala University, Uppsala, Sweden, 2Department of Pharmacy, Uppsala University, Uppsala, Sweden

Purpose: Liver is one of the most important organs for accumulation and elimination of toxins. However, the transport mechanisms through human liver for ochratoxin A and citrinin are still little known. The aim of this study was to screen out effective uptake transporters that are highly expressed on liver membrane for ochratoxin A and citrinin uptake, and also figure out ochratoxin A and citrinin′s inhibition or stimulation effects on these transporters.

Methods: Uptake 1μM ochratoxin A or citrinin by HEK 293 cells stably transfected with human OATP1B1, OATP1B3, OATP2B1 and NTCP to identify effective transporters. With different toxin incubation time, time dependant curve was plotted. Finally, transporters uptake their specific substrates inhibited by ochratoxin A or citrinin was studied to see the inhibition effect.

Results: All the transporters studied are active uptake transporters for ochratoxin A but none of them is effective in citrinin uptake. With assistance of modeling, OATP1B1 is shown to be the most efficient for ochratoxin A uptake. Besides, ochratoxin A showed big inhibition effect on OATP1B1, OATP1B3, and OATP2B1 but stimulation effect on NTCP. Citrinin only has inhibition effect for OATP1B1.

Conclusion: In conclusion, this study showed that ochratoxin A uptake was more than citrinin by human liver under the same condition, which may present more toxicity. OATP1B1 was proved to be the main transporter during uptake. Moreover, both ochratoxin A and citrinin are inhibitors for OATP1B1, and ochratoxin A has more interactions with other transporters.

P507. Human Cryopreserved Precision-Cut Liver Slices as An Integrated Tool for Xenobiotics Uptake and Metabolism Studies in Short Term Cultur Conditions

Julian Bursztyka, Marie-Michèle Trancart, Fabrice Guillet and Françoise Brée

Xenoblis, Saint Grégoire, France

Despite precision-cut tissue slices are presented as an interesting tool for in vitro studies because they retain the organ architecture with all cell types present in their original tissue-matrix configuration, few data are available on their uptake capabilities, even for liver slices. As drug transporters, beside phase I and phase II metabolism enzymes, have gained major interest in drug discovery, we have evaluated uptake capabilities of human cryopreserved precision-cut liver slices by OATPs, OCT and NTCP transporters. In order to give an overview of the metabolic capabilities of this in vitro system, phase I and phase II metabolism were also evaluated. Slices were obtained from Biopredic International (Rennes, France). Human liver sample was from a diabetic, non smoker Caucasian male, 78 years old, suffering from an hepatocellular myeloma due to alcohol. Thawing and preincubation of the slices were managed according to provider′s recommendations. For all studies, incubations were done at +37°C under an O2/CO2 (95/5 % v/v) atmosphere, in MW12 (1 slice per well in 600 µL of medium) under gentle shaking (40 rpm). Uptake studies were done in duplicate using radiolabeled estradiol 17beta-glucuronide, Mpp+ or taurocholate for OATPs, OCT or NTCP

316

uptake, respectively. Incubations were managed in parallel at +4°C to take into account passive diffusion. CYP activi-ties were measured in triplicate using a cocktail including phenacetin (CYP1A), bupropion (CYP2B6), tolbutamide (CYP2C9), dextrometorphan (CYP2D6) and midazolam (CYP3A4). Phase II metabolism was assessed in triplicate using paracetamol (SULTs and UGTs). At least, testosterone, used as a non-specific substrate of CYP enzymes, was incubated at 200 µM in triplicate. Its clearance was measured, as its hydroxylated metabolites. Uptake assays indicated that OATPs, OCT and NTCP transporter were maintained despite cryopreservation. Uptake was time and temperature dependent, with linearity between 5 and 30 minutes, depending on the considered transporter. Testosterone was readily biotransformed mainly into androstenedione and 6beta-hydroxytestosterone (CYP3A4), but also in 6alpha-, 16beta-, 16alpha- and 2beta-hydroxytestosterone, and was almost completely metabolized after a 3 hours incubation. As a conclusion, cryopreserved precision-cut human liver slices is an easy to handle in vitro system allowing both uptake and phase I and II metabolism studies.

P508. Regulation of ABC-Transporters and Solute Carrier Transporters under Diabetic Conditions at the Blood-Brain Barrier in vitro

Melanie Ott1, Valeska Reichel2 and Gert Fricker2

1Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy and Molecular Biotechnology, Heidelberg, Germany, 2Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy and Molecular Biotechnology, Heidelberg, Germany

More than 220 million people worldwide are diagnosed with diabetes mellitus (DM). This metabolic disorder, char-acterized by high blood glucose and insulin resistance, is not only a major risk factor for heart disease and stroke, but also a predisposing factor for Alzheimer′s disease (AD). Much is known about diabetes-induced endothelial cell dysfunction in tissues like heart, kidney or retina, but changes in the endothelial cells of the blood-brain barrier are understudied. Gaining a better understanding of the pathophysiological consequences of diabetes on blood-brain bar-rier function may lead to potential new molecular drug targets. Chronic hyperglycemia in diabetic patients stimulates the formation of Advanced Glucose Endproducts (AGEs) and increases production of Reactive Oxygen Species (ROS). AGEs are now known to play a role as proinflammatory mediators and have also been implicated in AD. Overall, oxida-tive stress and protein glycation are important causes involved in both, DM and AD. Plasma levels of Methylglyoxal (MG), a precursor to AGEs, 4-Hydroxynonenal (4-HNE), a by-product of lipid peroxidation as well as Homocysteine and Endothelin-1 (ET-1) are markers that are often increased in diabetic patient plasma and/or brain cells with AD. For this purpose we investigated effects of high glucose, MG, 4-HNE, Homocysteine and ET-1 on expression levels of members of the solute carrier family, multidrug resistance-associated proteins (MRPs), breast cancer resistance protein (BCRP) and P-glycoprotein (P-GP) in a human brain endothelial cell line hCMEC/D3 as a model of the blood-brain barrier. These membrane transporters at the luminal and abluminal side of brain endothelial cells are critical for the uptake as well as efflux of xenobiotics and by-products of metabolism from brain to blood. They are important determinants of drug disposition, since changes in their expression can modulate drug pharmacokinetics. We quan-tified mRNA levels of 16 different uptake and efflux transporters in hCMEC/D3 using quantitative real-time PCR. MCT1 (SLC16A1) was one of the genes that was most strongly up-regulated by high glucose, Homocysteine, 4-HNE and ET-1. In parallel to the changes in gene expression, western blot analyses, immunohistochemistry and confocal scanning laser microscopy indicated that the protein expression of MCT1 was significantly altered after treatment with high glucose, Homocysteine, 4-HNE and ET-1. The potential for regulation of expression of MCTs by diabetes or other stress-induced diseases at the blood-brain barrier may have important consequences for the progression and treatment of cerebrovascular disorders.

P509. Effect of Steroids on Transport Characteristics of the Organic Anion Transporting Polypeptides (OATP) 1A2, 1B1, 1B3 and 2B1

Anna B. Koenen, Kathleen Köck, Markus Keiser, Werner Siegmund, Markus Grube and Heyo K. Kroemer

Pharmacology, Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany

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Background: The family of organic anion transporting polypeptides (OATP) consists of 11 members that mediate the cellular uptake of endo- and exogenous compounds like bile acids, steroid conjugates, cardiac glycosides and even peptides. It has been demonstrated that these transporters play an important role in drug disposition and elimination. Of this family OATP1B1 and 1B3 are predominantly liver-specific while the expression of members like OATP2B1 and OATP1A2 demonstrate a wider tissue distribution. Previously, we have demonstrated that progesterone is able to stimulate OATP2B1-mediated E

1S, dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate uptake

in a concentration-dependent manner (1), but no comparable data exist for other OATP2B1 substrates as well as other OATPs. In this study we therefore investigated whether this progesterone effect is transporter and substrate specific.

Methods: For an initial screening approach HEK293-cells stable-expressing OATP1A2, OATP1B1, OATP1B3 and OATP2B1 were incubated with radiolabeled DHEAS – a shared substrate for all of these transporters – in the presence of progesterone and other steroid hormones. Based on these results, progesterone and dexamethasone, which dem-onstrated the highest stimulatory effect on OATP2B1-mediated DHEAS uptake, were further characterized concerning their effects on other OATP2B1 substrates.

Results: In the initial screening approach progesterone and the glucocorticoide dexamethasone significantly stimulated OATP2B1-mediated DHEAS transport (315% and 235% as well as 185% and 493% of control in concentrations of 10 and 100 µM, respectively). In contrast, DHEAS uptake mediated by OATP1B1, OATP1B3 and OATP1A2 was inhibited. Following, the interaction of progesterone and dexamethasone on the uptake of E

1S, BSP, DHEAS and atorvastatin into

OATP1A2, 1B1, 1B3 and 2B1 transfected cell lines was studied. Thereby only the OATP2B1-mediated transport of E1S

(EC50

values: 6.9 µM for progesterone and 9.3 µM for dexamethasone) and DHEAS (EC50

values of 15.4µM for proges-terone and 40.3 µM for dexamethasone) was stimulated by the respective substances. OATP2B1-mediated uptake of BSP and atorvastatin as well as the transport of other OATPs was either inhibited or not affected.

Conclusion: Our data demonstrate that the stimulatory effect of progesterone and dexamethasone is specific for OATP2B1 and restricted to sulfated steroids like E

1S and DHEAS suggesting that OATP-mediated drug transport is

not affected by either of these substances. However, stimulation of OATP2B1-mediated transport may influence the distribution of endogenous steroid conjugates.

References

1. Grube, M., Kock, K., Karner, S., Reuther, S., Ritter, C. A., Jedlitschky, G., and Kroemer, H. K. (2006) Mol.Pharmacol. 70, 1735-1741

P510. Drug clearance in different in vitro systems as a preliminary indicator of hepatic uptake

Eleanor J. Guest, Amin Rostami, J. Brian Houston and Aleksandra Galetin

School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom

The active hepatic uptake of inhibitors may increase the extent of drug-drug interactions (DDIs) due to higher concentra-tions of inhibitor available at the enzyme active site. This is particularly evident in cases when the higher concentration at the active site does not lead to higher clearance of, and consequently lower exposure, to the inhibitor. The ability to assess this phenomenon in vitro represents a valuable aid in the DDI prediction models. Current study examines differ-ent methods for the early assessment of hepatic uptake using well- known inhibitors, namely fluoxetine, fluvoxamine, fluconazole, itraconazole and ketoconazole. Comparison of clearances obtained in rat microsomes and hepatocytes was performed with the assumption that higher unbound CL

int in hepatocytes compared to microsomes may be used as an

indicator of hepatic uptake. In addition, uptake was assessed through comparison of CLint

determined in hepatocytes using the conventional (analysis of loss of drug in the cells and media) and media loss (analysis of media data only) assays. The CL

int for each individual inhibitor was determined using depletion approach at an inhibitor concentration of 0.1μM

(n=3) and corrected for the corresponding experimentally determined extent of nonspecific binding in microsomes and hepatocytes. No CL

int was detectable for predominantly renally-cleared fluconazole. A 5-7-fold hepatocytes:microsomes

unbound CLint

ratio was observed for fluoxetine and fluvoxamine; this fold difference in clearance was consistent for the media loss vs. conventional assay data. Conflicting results were obtained for the azole inhibitors, with no indication of uptake from the hepatocytes:microsomes CL

int ratio. However, a 4-5-fold difference was obtained for ketoconazole

and itraconazole from the CLint

ratio via comparison of media loss and conventional assays in hepatocytes, suggesting

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active uptake of these inhibitors. The current findings do not correspond to the previous analysis of the unbound Ki ratios

between rat hepatocytes and microsomes where no uptake was indicated for any of these inhibitors [1]. All the inhibitors investigated are lipophilic and tissue binding may confound the interpretation of the data. Preliminary analysis of the inclusion of estimated hepatic uptake in the dynamic DDI prediction model in Simcyp® was performed; the impact on the DDI prediction accuracy is discussed.

References

[1] Brown, H.S., Chadwick, A. and Houston, J.B. Use of Isolated Hepatocyte Preparations for Cytochrome P450 Inhibition Studies: Comparison with Microsomes for K

i Determination. Drug Metab Dispos. 2007: 35(11): 2119-2126.

P511. Characterization of Placental SLC22A4 (OCTN1) and SLC22A5 (OCTN2) Expression: Influence of Genotype and Gestation Age

Markus Grube, Sebastian Reuther, Kathleen Köck, Karen Salje, Werner Siegmund and Heyo K. Kroemer

Pharmacology, Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany

Background: L-carnitine plays an essential role in fetal development and there is evidence that it is translocated from the maternal to the fetal circulation by the sodium-dependent high affinity L-carnitine transporters SLC22A5 (OCTN2) and the closely related SLC22A4 (OCTN1). While placental expression of these transporters is already known, only limited data are available for transcriptional regulation in this tissue. We therefore studied localization and expression of both transporters with regard to gestation age and genetic variance.

Method/Results: OCTN1 expression could be detected by immunofluorescence microscopy in fetal blood vessels, while OCTN2 was expressed in the maternal facing membrane of the syncytiotrophoblast. mRNA expression of both transport-ers as determined by real-time PCR in 22 term, 24 preterm and 21 early-preterm placenta. Expression was independent from gestation age; however, OCTN2 mRNA expression was significantly affected by the SLC22A5 -207G>C promoter polymorphism with a lower expression for the C allele (median expression and CI

25-75: 1.21 (0.79-1.67) for the GG; 0.97

(0.11-0-97) for GC and 0.88 (0.38-0.88) for CC genotype).

Conclusion: In summary, both carnitine transporter OCTN1 and 2 are expressed in human placenta, with a specific expression in fetal blood vessels for OCTN1 and a predominant localization within the apical membrane of the syncy-tiotrophoblast for OCTN2. While expression of both transporters was not altered by gestation age, OCTN2 expression was affected by the 207G>C promoter polymorphism on mRNA transcription level. These findings provide insight into the transplacental carnitine transfer and indicate a possible impact of the 207G>C promoter polymorphism in this process.

P512. In Vitro Evaluation Of P-gp, BCRP And MRP2 In Caco-2 Cells Using Probe Substrates And Inhibitors

Gayle Newbury, Samantha Ellis, Divya Mistry, David Turner, Andrew James, Andy Clarkson and Clive Dilworth

Cyprotex Discovery Limited, Macclesfield, United Kingdom

Until recently the focus of drug-transporter interactions has primarily been on P-glycoprotein (P-gp), however other transporters are now being recognized as playing an important role in clinical drug-transporter interactions. A recent FDA white paper 1 proposes seven primary transporters including P-gp and BCRP (breast cancer resistance protein). Whilst not discussed in detail in the publication the importance of MRP2 (multidrug resistance protein) as a clinically important transporter was also proposed 2. Epithelial cell systems such as the Caco-2 cell system are traditionally used for identification of potential efflux transporter substrates. Typically a compound with an efflux ratio of ≥ 2 indicates that a new molecular entity is a potential substrate for a transporter. The aim of this poster is to discuss the utility of the bidirectional Caco-2 monolayer for assessment of transport of compounds of interest by P-gp, BCRP and/or MRP2.

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The assay described here has been developed in a 96-well format and uses LC-MS/MS as an end point to quantify the compounds. The efflux ratios (basolateral-apical Papp/apical-basolateral Papp) of specific substrates for each trans-porter of interest were determined. mRNA expression levels of P-gp, BCRP and MRP2 have also been evaluated using qRT-PCR. Where possible, the assay has been further validated using selective inhibitors for each transporter giving increased confidence that the compound of interest has been correctly identified when a decrease in the efflux ratio in the presence of a probe inhibitor was observed. The Caco-2 permeability assay can be used to predict intestinal absorption or investigate the transport of compounds of interest by specific transporters such as P-gp, BCRP and/or MRP2 thus facilitating a greater understanding of mechanisms of drug efflux and can also highlight early potential issues with drug permeability.

References

1 The International Transporter Consortium, (2010) Membrane transporters in drug development, Nature Reviews 9; 215-236 2 Huang, S-M, et al (2010) The International Transporter Consortium: A Collaboration Group of Scientist From Academia, Industry, and the FDA, Nature 87; 32-36

P513. Utility of Automated Drug Transport Assays in 96-Well Format, using Permeable Support Systems

Bradley R. Larson1, Peter Banks2, Hilary Sherman3 and Mark Rothenberg3

1Applications, BioTek Instruments, Winooski, VT, USA, 2Marketing and Sales, BioTek Instruments, Winooski, VT, USA, 3Applications, Corning Life Sciences, Kennebunkport, ME, USA

Drug transport assays play an important part in determining how a compound is absorbed into the body. Therefore, the performance of these assays is essential to help determine the ADME/Tox profile of a new drug entity (NDE). Typically, these assays have been carried out using colorectal carcinoma (Caco-2) cells, or Madin-Darby Canine Kidney (MDCK) cells in 24-well plates. However, due to the fact that ADME/Tox testing is now moving further upstream in the drug discovery process, a greater number of lead compounds are now being tested in an effort to fail NDEs with negative profiles earlier and in a more cost effective manor. To meet the demands for higher through-put and reduced processing time we present an automated drug transport assay using either Caco-2 or MDCK cells in 96-well Permeable Supports. The entire assay process was automated, including cell dispensing, media exchanges, and compound addition and removal, using simple, yet robust robotic instrumentation. A two-part permeable support system, incorporating an insert plate, and receiver plate, was used in order for manipulations to be performed without the need to separate the parts of the system. The metrics used to validate the automated process were Transepithelial Electrical Resistance (TEER), and Lucifer Yellow and Rhodamine 123 permeability. All automated methods were done in parallel to manual for comparison. Results show that the automated assay is able to deliver results that are equal to, or more consistent, than manual processing, while reducing the overall experimental time. Thus, by automating the drug transport assay, one increases efficiency with out the loss of data quality or integrity.

P514. Taurocholate or Glycocholate – Which is the More Appropriate Substrate for Assessing Hepatic Bile Salt Transport?

Erica Deibert, Jonathan Jackson, Joy Mitchell, Kirsten Amaral, Rafal Witek, Stephen Ferguson and Jasminder Sahi

ADME/Tox, CellzDirect/Life Technologies, Durham, NC, USA

Purpose: Interactions with the hepatic bile salt transporters NTCP (basolateral uptake) and BSEP (apical efflux) can contribute to cholestasis and hypercholesterolemia. The initial studies to evaluate uptake and biliary excre-tion via these transporters were developed by Brouwer and LeCluyse (1998) in primary sandwich cultured rat

320

hepatocytes, using the bile salt taurocholate as a substrate. Since then, a majority of in vitro biliary transporter studies have been conducted with taurocholate for preclinical and human extrapolations. While taurine conju-gated bile acids (e.g. taurocholate) are the major bile salts in rat, human plasma predominantly contains glycine-conjugated bile acids (e.g. glycocholate). To identify the appropriate bile salt for in vitro transporter evaluations, we compared the substrate and inhibition potential of taurocholate and glycocholate in human and rat in vitro systems.

Methods: Mechanistic studies were conducted using primary rat and human hepatocytes in suspension and in primary culture. Hepatocytes were prepared from healthy human liver resections and from male Sprague Dawley rats and used freshly isolated or after cryopreservation. The oil spin method was used to compare transporter - mediated uptake of taurocholate and glycocholate in suspension hepatocytes. Uptake was also conducted after 24 hours in primary culture on collagen coated plates. Additional hepatocytes were overlaid with Geltrex® and used at 5-7 days in culture (B-CLEAR® model) to assess uptake and biliary efflux of the bile salts. Membrane vesicles over-expressed with the human and rat BSEP transporter (Genomembrane) were also used. Inhibition studies were conducted using prototypical probe substrates and market drugs that are known inhibitors of bile salt transport.

Results: Time linearity studies with taurocholate and glycocholate indicate linear uptake at 5 minutes incubation for the rat and human hepatocytes in culture and a 1 minute incubation time for suspension hepatocytes. In 24-hour plated hepatocytes, the K

m for taurocholate uptake was similar: 13.3 microM in human and 22.1 microM in rat; while

the Km

for glycocholate demonstrated differences between the two species: 23.1 microM (human) and 166.8 microM (rat). Using sandwich hepatocytes with intact bile canaliculi, we demonstrated a biliary excretion index (BEI) for tau-rocholate of 82.5%, in rat and 49.6% in human hepatocytes. The BEI for glycocholate was 77.5% for human and 53.4% for rat hepatocytes. As a whole, in these two culture systems, glycocholate is a more efficient substrate in human vs. rat hepatocytes and taurocholate is a stronger substrate in rat hepatocytes. BSEP vesicles mimicked these trends. Inhibition studies were less conclusive and further evaluations are being conducted to understand differences if any, using a wider range of inhibitors in both species.

Conclusions: In preliminary evaluations, glycocholate is a stronger substrate for human hepatic bile salt uptake and efflux transporters while taurocholate is the appropriate substrate for assessing rat biliary transport. Mechanistic stud-ies to further understand differences between these two bile salts in rat and human hepatocytes are currently being conducted. Our data thus far indicates that serious consideration should be given to using glycocholate for in vitro studies aimed at studying human bile salt transporters.

P515. Evaluating Drug Interaction with ABC Transporters using Colorimetric ATPase Assay

Joanne Bourgea, Christopher J. Patten, Charles L. Crespi and Na Li

BD Biosciences, Bedford, MA, USA

ATP binding cassette (ABC) transporter family is localized on apical/basal membrane of organs involved in drug absorption, distribution, elimination and toxicity. With increasing number of new chemical entities associated with poor bioavailability and transporter mediated excretion, accurate estimation of potential drug interactions with transporters in vitro is necessary. The purpose of this study is to investigate drug interaction with major ABC transporters (P-gp, MRP2 and BCRP) using Colorimetric ATPase Assay. Membranes were prepared from insect cells infected with baculovirus expressing human P-gp1, MRP2, or BCRP. The amount of inorganic phosphate released from substrate-stimulated ATP hydrolysis was measured via a secondary colorimetric assay in which phosphate react with ammonium molybdate to generate blue color with absorbance at 800 nm. The reagents used for ATPase assay performed consistently with low day-to-day and lot-to-lot variance. More than twenty drug compounds were tested for vanadate-sensitive ATPase activity with human P-gp, MRP2 and BCRP membranes. K

m values were determined

for the majority of the test compounds. PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) stimulated ATPase activity of human BCRP membrane with a K

mof 1.6 ± 0.5 μM. Prazosin, Saquinavir, Ritonavir, Quinidine, Loperamide,

Loratadine and Verapamil all showed high affinity (low Km

) for human P-gp membrane. The concentration depend-ent drug stimulated ATPase activity correlated with MDCK cell-based bidirectional efflux ratio. Vanadate –sensitive ATPase activity, coupled with the K

m for ATPase stimulation, is discussed as a convenient and predictive means to

evaluate drug-interactions with ABC transporters.

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P516. Application of Inside-out Vesicle Uptake Assay to Characterize ABC Transporters Substrates and Inhibitors

Na Li, Joanne Bourgea, Sweta N. Parikh, Charles L. Crespi and Christopher J. Patten

BD Biosciences, Bedford, MA, USA

There is an increasing number of NCEs associated with ABC transporter mediated absorption, distribution, elimina-tion and toxicity, making it important to have robust and predictive in vitro tools to characterize the potential drug interaction with ABC transporters. The aim of this study is to characterize three major ABC transporters and inhibitory effects of various drugs on these transporters using inside-out vesicle model. Vesicles were prepared from insect Sf9 cells infected with baculovirus expressing hMRP2, hBCRP and hBSEP. The MRP2 mediated uptake of 3H-LTC

4 and 3H-

E17βG, BCRP uptake of 3H-E3S and BSEP uptake of 3H-TCA were characterized using a rapid filtration system. Kinetic

analysis (Km

and Vmax

) and IC50

assays were carried out under linear conditions for time and protein concentration. Uptake activity of the test substrates in corresponding ABC transporter vesicles showed typical Michaelis-Menton kinetics. hMRP2 mediated LTC4 uptake was inhibited by MK571, benzbromarone, terfenadine and indomethacin with IC

50 values of 9.2 μM, 7.8 μM, 41.6 μM and 281.5 μM, respectively. E3S uptake in hBCRP was inhibited by Sulfasalazine,

Nobomycin, Fumitremorgin C and Ko143 with IC50

values of 0.7 μM, 0.6 μM, 0.1 μM and 0.025 μM, respectively. TCA uptake in hBSEP was inhibited by Troglitazone, Glibenclamide and Cyclosporin A with IC

50 values of 4.5 μM, 3.5 μM

and 0.6 μM, respectively. The study has identified a panel of potent inhibitors for three major ABC transporters. The inhibitory effects of the selected drugs observed in inside-out vesicle uptake assay are consistent with hepatic toxicity observed in clinical studies. All together, the data demonstrates that vesicle uptake assay using inside out recombinant membrane vesicles is a robust and predictive model in characterizing ABC transporters substrates and inhibitors.

P517. Species Difference in Inhibitory Effects of MRP2 Modulators on 5(6)-Carboxy-2,′7′-Dichlorofluorescein Transport

Na Li, Sweta N. Parikh, Joanne Bourgea, Charles L. Crespi and Christopher J. Patten

BD Biosciences, Bedford, MA, USA

MRP2/ABCC2 is the major ABC transporter localized on canalicular membrane of hepatocytes and plays an important role in hepatobiliary efflux of many drugs and drug metabolites. The purpose of this study was to investigate the species difference of drug inhibitory effects on MRP2/Mrp2 mediated transport. Vesicles were prepared from insect Sf9 cells infected with baculovirus expressing human MRP2 and rat Mrp2. Vesicle uptake assay was conducted using a rapid filtration system. 5(6)-Carboxy-2,′7′-Dichlorofluorescein (CDCF) was used as MRP2/Mrp2 substrate in the inhibitor study. Kinetic parameters, K

m and V

max, for CDCF uptake into MRP2/Mrp2 vesicles and the inhibitory effect of a series

of compounds (e.g. terfenadine, benzbromarone, indomethacin and MK571) on MRP2/Mrp2 mediated uptake of CDCF were determined under linear assay conditions for both time and protein concentration. Clearance (V

max/K

m) was great-

est for hMRP2 (Vmax

= 216 pmol/min*mg/Km

= 23 μM) than rMrp2 (Vmax

=57 pmol/min*mg/Km

= 64 μM). Benzbromarone and MK571 were potent inhibitors of both human MRP2 and rat Mrp2 with IC

50 values of 3.3 μM and 6.2 μM for human,

12 μM and 2.4 μM for rat, respectively. Terfenadine and Indomethacin were less potent with IC50

values of 45.8 μM and 90.8 μM for human, 46.3 μM and 53.8 μM for rat, respectively. The ranking order of the inhibitory effects of four selected MRP2 modulators on rat Mrp2 was nearly consistent with human MRP2. It was also observed that MK571 and Indomethacin are more potent inhibitors of rat Mrp2 than human MRP2. In contrast, Benzbromarone is less potent in inhibiting rat Mrp2 transporter than human MRP2. The data shows that CDCF is a MRP2 substrate and can be used to evaluate drug inhibitors of MPR2 using inside-out vesicle model.

P518. The Role of Mrp3 in Diclofenac Acyl Glucuronide-mediated Toxicity

Ron Scialis1, Lauren M. Aleksunes2, Ivan L. Csanaky3, Curtis D. Klaassen4 and José E. Manautou5

1Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT, USA, 2Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ, USA,

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3Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA, 4Pharmacology, Toxicology, and Therapeutics, The University of Kansas Medical Center, Kansas City, KS, USA, 5Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, USA

A number of pharmaceuticals are metabolized to highly reactive acyl glucuronide intermediates that can covalently bind to cellular targets, including proteins. Such adducts can alter function such as loss of enzymatic activity. Idiosyncratic reactions are another consequence of adduct formation by stimulating the immune system to recognize non-self moieties (i.e., adducted protein). Considering the multiple therapeutic agents that are metabolized to acyl glucuronides and the safety concerns associated with their formation, there is great interest in better characterizing the disposition of such drugs and their potential for producing toxicity. Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), is primarily metabolized in the human liver to 4′- or 5-hydroxy diclofenac (OH-D) or diclofenac-1-O-acyl glucuronide (DG). As several endogenous and xenobiotic-derived glucuronides are substrates of the ABCC family of transporters, we investigated the role of MRP3 in DG disposition and toxicity in mice. Our hypothesis was that lack of basolateral MRP3 would cause accumulation of DG in hepatocytes, enhance biliary elimination, and increase susceptibility to intestinal ulceration, which is a common adverse effect associated with NSAID use. To investigate this, male wild-type (WT) and homozygous Mrp3-null (KO) mice were dosed with a single toxic dose of diclofenac at 90 mg/kg, i.p. Mice were sacrificed 24 hours later, and blood, livers, kidneys, small intestines, and stomachs were collected for analysis. Clinical chemistry data suggested no injury occurred in kidneys or livers of either genotype. However, histopathology revealed extensive gastric ulceration in both WT and KO mice, with KO mice demonstrating more severe gastric injury. Ulcers were also seen in small intestines of all WT mice. This is in stark contrast to KO mice that were completely devoid of diclofenac-induced intestinal ulcerations. Although these observations on intestinal ulcers are exactly opposite of our expected results, compensatory changes associated with the Mrp3-null genotype may be responsible for a lack of injury to the intestinal epithelia by diclofenac treatment. To further investigate this, we have initiated additional experiments to examine the hepatobiliary disposition of diclofenac using doses that produce the differential intestinal and stomach injuries between Mrp3 WT and KO mice reported here.

P519. Comparison of Inhibition Potencies of Cefem Antibiotics Between MRP4 and Basolateral Organic Anion Transporters (OAT1 And OAT3) (NEDO MicroDose-PJ)

Kayoko Kanamitsu, Hiroyuki Kusuhara and Yuichi Sugiyama

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

[Purpose]: MRP4 is an ATP binding cassette transporter showing broad substrate specificity. MPR4 is expressed in the apical membrane of the polarized endothelial cells and kidney epithelial cells. Animal studies have shown that MRP4 mediates luminal efflux into the urine in the kidney, and active efflux in the blood-brain barrier. We reported that Mrp4 knockout mice show higher concentrations of Ro64-0802 in the brain and kidney compared with wild-type mice1. The directional transport of Ro64-0802 across the proximal tubules and brain capillaries involves OAT3 in the uptake process1. Therefore, Ro64-0802 can be used as in vivo probe for OAT3 and MRP4. There is now growing interest in in vivo inhibitors of drug transporters since it is now encouraged to identify the transporters involved in the disposition clinically. Unlike OAT3, in vivo inhibitor for MRP4 has not been established. Our Microdose Project has prompted clinical studies examining drug-drug interactions to validate our prediction method. The present study applied the method to find drugs which is able to inhibit MRP4, without affecting OAT3, at therapeutic dose for future microdose studies using Ro64-0802. Based on the previous report2, the present study examined their inhibition potencies with regard to MRP4, OAT1 and OAT3.

[Methods]: ATP-dependent uptake of [3H]dehydroepiandrosterone sulfate by membrane vesicles, prepared from HEK293 cells infected with adenovirus containing human MRP4 cDNA, was determined by the rapid filtration technique in the presence of various concentrations of cefem antibiotics (cefotaxime, cefmetazole and cefalotin). Cellular accumulation of [3H]p-aminohippurate and [3H]estradiol 17beta-glucuronide was determined in HEK293 cells expressing human OAT1 and OAT3, respectively, in the presence of various concentrations of the cefem antibiotics.

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[Results and discussion]: The Ki values (μM) of cefotaxime, cefmetazole and cefalotin for MRP4 were determined to be 8.53 ± 1.00, 14.3 ± 1.5 and 23.1 ± 4.1, respectively, while those for OAT1 and OAT3 were 1280 ± 240, 4.36 ± 1.43 and 380 ± 100, and 1930 ± 550, 275 ± 132 and 141 ± 72, respectively. The maximum unbound concentration of cefotaxime (ca. 200μM), achievable at a clinical dose (2 g) is greater than the Ki value for MRP4. Therefore, cefotaxime is likely able to inhibit MRP4 at a concentration where they have minimal effect on OAT1 and OAT3.

[References]

1) Ose A, et al. Drug Metab. Dispos., 37: 315-321, 2009.2) Ci L, et al. Mol Pharmacol. 71(6):1591-7. 2007.

[Acknowledgement]

This study was carried out as a part of “Research Project for Establishment of Evolutional Drug Development with the Use of Microdose Clinical Trial (NEDO MicroDose-PJ)”, sponsored by the New Energy and Industrial Technology Development Organization (NEDO).

P520. Evaluation of the Applicability of the OATP1B1/MRP2 Double-Transfected Cell Line to in vitro Hepatic Vectorial Transport

Miki Fujishima, Teruhiko Yanagisawa, Shinsuke Aoyama, Yasuhisa Adachi and Shin-ichi Ninomiya

ADME & Tox. Research Institute, Sekisui Medical Co., Ltd., Ibaraki, Japan

Recent progress in drug transporter studies has shown a number of drug-drug interactions (DDI) mediated by sev-eral uptake transporters from the solute carrier (SLC) superfamily and efflux transporters from ATP-binding cassette (ABC) superfamily. In March 2010, a concept paper on transporter studies in new drug development was given from International Transporter Consortium (ITC). Before this publication, P-glycoprotein was the only transporter that was recommended for studying its implications for DDI in the drug development process. The ITC concept paper embodied recommendable in vitro assay methods and criteria of interpretation of assay results for SLC and ABC transporters that DDI evidences were reported. OATP1B1 and MRP2, two major transporters expressed in the liver, mainly transport organic anions. Because of similar substrate specificity between OATP1B1 and MRP2, it is predicted that these transport-ers play a significant role in hepatic vectorial transport of organic anions. When considering the hepatic clearance of a drug that is a substrate of two or more transporters in the vectorial transport, the rate-limiting step is an important factor. In the present study, a double-transfected cell line expressing human OATP1B1 and MRP2 was studied in order to evalu-ate the applicability of this cell line to in vitro hepatic vectorial transport model. A typical substrate for OATP1B1 and MRP2, estradiol-17b-d-glucuronide (E

217bG) was used to determine the effects of various drugs on E

217bG transport

in the OATP1B1-expressing cell line, MRP2-expressing vesicle and OATP1B1/MRP2 double-transfected cell line. In the OATP1B1-expressing cell line, E

217bG transport was inhibited by benzbromarone and the IC

50 value was 0.59 µmol/L. On

the other hand, E217bG transport was facilitated by low concentration of benzbromarone in MRP2-expressing vesicle.

The double-transfected cell line showed inhibition of E217bG transport at a low concentration of benzbromarone and

the IC50

value was 0.25 µmol/L. The inhibitory effect of benzbromarone on the transport of E217bG by the double-

transfected cell line was similar to that by the OATP1B1-expressing cell line. These results suggest a rate-limiting step in the inhibition of E

217bG transport by benzbromarone lies in the uptake transporter, OATP1B1. The consideration of

a rate-limiting step is important when studying the impact of drug transporters in the drug development process. The present double-transfected cell line was, therefore, revealed to be of use to in vitro studies for new drug development.

References

1) Membrane transporters in drug development. International Transporter Consortium, Giacomini KM, et al., Nat Rev Drug Discov. 2010 Mar;9(3):215-36

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