Research Article Effects of Danshen Ethanol Extract on the ...

Research ArticleEffects of Danshen Ethanol Extract on the Pharmacokinetics ofFexofenadine in Healthy Volunteers

Furong Qiu Jin Zeng Songcan Liu Min He Leilei Zhu Yujie YePing Miao Shujiao Shen and Jian Jiang

Laboratory of Clinical Pharmacokinetics Shuguang Hospital Shanghai University of Traditional Chinese MedicineShanghai 201203 China

Correspondence should be addressed to Furong Qiu furong qiu126com and Jian Jiang jiangjiansg126com

Received 12 June 2014 Accepted 30 August 2014 Published 3 November 2014

Academic Editor Min Ye

Copyright copy 2014 Furong Qiu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study investigated the effect of multidose administration of danshen ethanol extract on fexofenadine pharmacokinetics inhealthy volunteers A sequential open-label two-period pharmacokinetic interaction design was used 12 healthy male volunteersreceived a single oral dose of fexofenadine (60mg) followed by danshen ethanol extract (1 g orally three times a day) for 10 days afterwhich they received 1 g of the danshen extract with fexofenadine (60mg) on the last dayThe plasma concentrations of fexofenadinewas measured by LC-MSMS After 10 days of the danshen extract administration the mean AUC and 119862max of the fexofenadinewas decreased by 372 and 274 compared with the control respectively The mean clearance of fexofenadine was increased by1049 The in vitro study showed that tanshinone IIA and cryptotanshinone could induce MDR1 mRNA This study showed thatmultidose administration of danshen ethanol extract could increase oral clearance of fexofenadine The increased oral clearance offexofenadine is attributable to induction of intestinal P-glycoprotein

1 Introduction

Herb used as complementary and alternative medicine(CAM) has dramatically increased over the last 20 years Dueto the widespread use of CAM in combination with propri-etary medications there is a strong possibility of herb-druginteractions (HDIs) involving absorption andor metabolismandor excretion processes Recent progress in the study ofmembrane transport has expanded our understanding ofthemechanisms underlying pharmacokineticHDIs involvingtransporters [1]

The extract from the roots of Salvia miltiorrhiza (dan-shen) is widely and traditionally used in the treatment ofangina pectoris myocardial infarction stroke and cancer inChina and other countries [2ndash5] The commercially availablepreparations from danshen extract are primarily formulatedwith the ethanol extract in which the diterpenoid tanshi-nones accounted for approximately 95 of the total amountwith cryptotanshinone tanshinone IIA and tanshinone I asthe major components [6] We found that danshen ethanolextract could induce CYP3A4 in vivo [6] and the two major

components cryptotanshinone and tanshinone IIA presentin the extract are responsible for CYP3A4 induction viathe activation of PXR [7] Because CYP3A4 and MDR1genes have PXR transcriptional binding sites and commonmolecular mechanism responsible for induction of CYP3A4and MDR1 by ligand cryptotanshinone and tanshinone IIAmay be assumed to induceMDR1 (also called P-glycoproteinP-gp) [8]

Currently there is little knowledge about whether thedanshen extract has a modulatory effect on human in vivoP-gp The aim of this study was to investigate multidoseadministration of danshen ethanol extract on in vivo MDR1activity in healthy volunteers The constituent(s) 119903 inducedto MDR1 was also investigated using human cryopreservedhepatocytes It will provide valuable information for using thedanshen preparation in clinical practice

2 Methods

21 Study Drugs The danshen ethanol extract in the form ofcapsule (250mgcapsule Lot 20090904) was manufactured

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 473213 5 pageshttpdxdoiorg1011552014473213

2 Evidence-Based Complementary and Alternative Medicine

and the quality control was established and enforced strictlyby Hebei Xinlong XiLi Pharmaceuticals Ltd according tostate drug standard (China State Food and Drug Admin-istration Ws3-B-3140-98-009) The contents of tanshinoneIIA cryptotanshinone and tanshinone I were 1062mgg880mgg and 531mgg respectively [6] Fexofenadinetablets (60mgtablet Lot 100827) were manufactured byJiangsu Hengrui Pharmaceuticals Ltd

22 Subjects and Ethical Approval Twelve male healthyChinese volunteers participated in this study (age range 25ndash30 years BMI range 19ndash25 kgm2) These volunteers wereenrolled in the study after obtaining written informed con-sent The clinical protocol and informed consent form wereapproved by the independent medical ethics committee ofShuguang Hospital affiliated with the Shanghai University ofTraditional Chinese Medicine

All subjects were nonsmokers and were judged to behealthy by a medical history a physical examination electro-cardiogram and laboratory tests (including complete bloodcount blood biochemistry testing and urinalysis) beforeentering the study Subjects abstained from consuming herbaland citrus fruit products for 2 weeks before the study andfrom alcohol and medications for 2 weeks before and duringthe study period and caffeine-containing foods orange juicegrapefruit juice and beverages were also excluded during thestudy period

23 Study Design The study design was a sequential open-label two-period trial conducted at the Shuguang Hospitalphase I clinical trial ward [6] On the morning of day 1the volunteers took a single dose of 60 mg of fexofenadineBeginning on day 2 they received the danshen extract (1 gthree times a day) for 10 days On day 12 the volunteersreceived 1 g of the danshen extract together with 60mg offexofenadine The volunteers fasted overnight before eachdosing The subjects were provided a light standard mealat 4 h after medication intake and at 6 pm on the two testdays of intaking probe drugs Blood samples (4mL each)were drawn before and at 025 05 1 15 2 3 4 6 8 1012 and 24 hours after fexofenadine administration and keptin heparinized Eppendorf tubes The blood samples werecentrifuged and plasma was separated and stored at minus80∘Cuntil the time of analysis

24 Sample Analysis Plasma samples were spiked withan internal standard (diazepam) and extracted with ethylacetate After evaporation of the organic solvent under nitro-gen reconstituted residues of the organic phase were ana-lyzed using a SciexAPI 4000 coupled liquid chromatography-tandem mass spectrometry (LC-MSMS) system (AppliedBiosystemsSCIEX CA) Chromatographic separation ofthe compounds was accomplished using a C

18column

(5 120583m 46mm times 150mm Agilent America) with waterphase (ammonium acetate 4mmolL and methanoic acid008) methanol (10 90 v v) as the mobile phase at aflow rate of 080mLmin The mass spectrometer was oper-ated in the MRM mode under positive ionization The ion

transitions monitored were mass-to-charge ratios of 5022ndash4665 (fexofenadine) and 2852ndash1931 (internal standard)The collision energy (CE) declustering potential (DP) andcollision cell exit potential (CXP) were set as follows fexofe-nadine 3632V 10032V and 1609V respectively diazepam4300V 9806V and 1145V respectively A set of 7 nonzerocalibration standards ranging from 1 to 500 ngmL and 3quality controls at concentrations of 2 50 and 400 ngmLwere performed during each day of analysis The interdayCVs for the lowmiddle and high quality controls were 167334 and 310 respectivelyThe intraday CVs were 3361284 and 033 respectively The recovery for the lowmiddle and high quality controls was 10091 9611 and9211 respectively

25 Induction of MDR1 mRNA by Tanshinones in HumanHepatocytes Cryopreserved human hepatocytes (Lot ONOand JYM) provided by the Research Institute for Liver Dis-ease Co (Shanghai China) were thawed in plating mediumand transferred to collagen I precoated 24-well plates at adensity of 30 times 105 viable cells The cells were maintained at37∘C in a humidified incubator with 90 atmospheric air and5 CO

2 After the initial 24-hour conditioning culture the

medium was removed and the hepatocytes were treated withvehicle which contained the same amount of DMSO (01)tanshinones (2 120583M 10 120583M) and rifampicin (25 120583M) for 72hours All drugs were dissolved in DMSO and then added tothe culture medium (final DMSO concentration 01) TotalRNA was isolated from cells using TRIzol reagent (Invitro-gen) according to themanufacturer-supplied protocolQuan-titative real-time PCR was performed using gene-specificprimers and the SYBR Green PCR kit (Invitrogen) in an ABI7900 system (Applied Biosystems) The relative quantity ofthe target MDR1 gene compared with the endogenous con-trol (glyceraldehyde-3-phosphate dehydrogenase) was deter-mined by the ΔΔCT method The following primer sets wereused in this studyMDR1 (51015840CGGACATCCCAGTGCTTCA-31015840 and 51015840-GTCGCTTTATTTCTTTGCCATC-31015840)

26 Pharmacokinetic Determination The plasma concentra-tion-time data of analyteswere analyzed by compartment-independent approaches The maximum plasma drug con-centration (119862max) and time to 119862max (119879max) were directlyobtained from the plasma concentration-time data Theelimination half-life (119905

12) was calculated as 0693Ke

where Ke the elimination rate constant was calculated viasemilog regression on the terminal phase of the plasmaconcentration-time curve The AUC from time 0 to infinity(AUC

0minusinfin) was estimated as AUC

0minus119905+ 119862119905Ke where 119862

119905

is the plasma concentration of the last measurable sampleand AUC

0minus119905was calculated according to the linear trape-

zoidal rule Total plasma clearance (CLF) was calculated asdoseAUC

0minusinfin

27 Statistical Analysis Statistical comparisons between con-trol and drug treatments were initially performed by two-way ANOVA for repeated measures performed with SAS forWindows software (version 92 SAS Institute Inc) For the

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Pharmacokinetic parameters of fexofenadine after a single dose administration of 60mg fexofenadine in 12 healthy volunteers beforeand after multiple-dose coadministration of danshen ethanol extract

PK parameter Control Multiple dose Ratio to control (multiple doses)119862max (ngmL) 22392 plusmn 7436 14805 plusmn 6093lowast 065 (051ndash083)119879max (h) 3 (15ndash60) 20 (05ndash30) mdashAUC0ndash24 (ngsdothmL) 161223 plusmn 77358 84457 plusmn 33967lowast 055 (042ndash071)AUC0ndashinfin (ngsdothmL) 171766 plusmn 81521 88999 plusmn 35311lowast 054 (041ndash070)11987912

(h) 597 plusmn 082 596 plusmn 100 099 (089ndash111)CLF (Lh) 4435 plusmn 2557 7788 plusmn 3120lowast 186 (143ndash242)Data are presented as mean plusmn SD Data of ratio to control are shown as mean (95 confidence interval) 119879max data are shown as median (range)lowast119875 values are given for the differences with respect to control

ANOVA analyses of treatment effect with 119875 lt 005 post hoca priori comparisons were performed between control andthe treatment with the paired 119905-tests119879max was analyzed usingWilcoxonrsquos signed rank test 119875 lt 005 indicated statisticalsignificance 90 confidence intervals (CIs) were constructedfor the ratios of with- to without-danshen treatment usingthe log-transformed data for the geometric means of 119862maxAUC0ndash24 AUC0ndashinfin 11990512 and CLF The resulting confidence

limits were transformed by exponentiation and reported onthe original measurement scale The statistical limits were setat 080ndash125 The DAS statistical analysis system (version 10)was also used

3 Results

No clinically undesirable signs or symptoms attributable tothe administration of fexofenadine and the danshen extractwere recognized during the study All subjects completed thestudy according to the protocol

The mean plasma concentration-time profiles of fexofe-nadine before and after multidose administration of thedanshen extract are presented in Figure 1 Table 1 summarizesthe pharmacokinetic parameters of fexofenadine before andafter multidose of the danshen extract treatment

After 10 days of danshen ethanol extract administrationAUC of fexofenadine was decreased by 372 (161223 plusmn77358 ngsdothmL versus 84457 plusmn 33967 ngsdothmL) comparedwith the controlThe clearance of fexofenadine was increasedby 1049 (4435plusmn2557 Lh versus 7788plusmn3120 Lh) and 12volunteersrsquo individual data of CLF are shown in Figure 2The119862max was decreased by 274 (22392 plusmn 7436 ngmL versus14805 plusmn 6093 ngmL) compared with the control 90 CIsof 119862max and AUC

0ndashinfin of fexofenadine were under low limitof bioequivalence (lt080) 90 CIs of CLF of fexofenadinewere beyond upper limit of bioequivalence (gt125) But 90CIs of 119905

12were within range of bioequivalence (080ndash125)

A Wilcoxon signed rank test for fexofenadine indicated that119879max was nonsignificant difference (119875 gt 005)

The effects of the tanshinones on MDR1 mRNA wereshown in Figure 3 Rifampicin can upregulate MDR1 tran-scripts 228-fold at 25 120583M Cryptotanshinone increasedMDR1 transcripts by 143- and 225-fold at 2 120583M and 10 120583Mrespectively Tanshinone IIA increased MDR1 transcripts by

0

50

100

150

200

250

0 4 8 12 16 20 24Time after administration (h)

Fexo

fena

dine

(ng

mL)

BaselineMultiple-dose DSEE

Figure 1 Mean (plusmnSE 119899 = 12) plasma concentration of fexofenadineafter the administration of a single dose of 60mg of fexofenadinebefore and after multiple-dose coadministration of danshen ethanolextract

172- and 214-fold at 2 120583M and 10 120583M respectively Tanshi-none I did not increaseMDR1 transcripts (103- and 113-fold)at 2 120583M and 10 120583M

4 Discussion

This study examined the effect of multiple doses of danshenethanol extract on the pharmacokinetics of fexofenadine inhealthy subjects To our knowledge this is the first report toevaluate the effect of danshen ethanol extract on fexofenadinein healthy volunteers

Fexofenadine an orally active nonsedating H1-receptor

antagonist prescribed for oral treatment of allergic rhinitisand chronic idiopathic urticaria has been shown to be asubstrate of P-glycoprotein (P-gp) [9ndash11] MDR1 inhibitors(itraconazole ritonavir and verapamil) can increase whileinducers (carbamazepine rifampicin and St Johnrsquos wort)decrease fexofenadine AUC and 119862max [12ndash18] Furthermoreit had good clinical safety over a wide dose range Sofexofenadine was possibly a superior clinical drug probe toevaluate the effect of the danshen extract on MDR1 activity


Before After0

20

40

60

80

100

Danshen extract

CLF

(Lh

)

Figure 2 Individual value of oral clearance for fexofenadine beforeand after multidose of the danshen extract treatment (119899 = 12)

0

1

2

3

mRN

A ex

pres

sion

(fold

of N

C) lowast

lowast

lowast

lowast

lowast

NC

Rif (25120583

M)

Cry

(2120583

M)

Cry

(10120583

M)

Tan

I (2120583

M)

Tan

I (10120583

M)

Tan

IIA (2

120583M

)

Tan

IIA (10120583

M)

Figure 3 Induction of MDR1 mRNA by tanshinone I (TanI)cryptotanshinone (Cry) and tanshinone IIA (Tan IIA) Humanhepatocytes were exposed to tanshinone I (2 120583M 10120583M) cryptotan-shinone (2120583M 10 120583M) tanshinone IIA (2120583M 10 120583M) or 25 120583Mrifampin (PC) for 3 days MDR1 mRNA levels were measured withreverse transcription real-time PCRThese data were obtained fromtwo independent experiments and each experiment was performedin triplicate Each columnwith bar represents themean and SDThemean is expressed as fold induction over vehicle control (NC)

Although fexofenadine is a widely used probe drug of MDR1activity in vivo whichmediated the cellular efflux of fexofena-dine to reduce the absorption of its substrates [9ndash13] thisnonsedating antihistamine drug is also known to be asubstrate of drug uptake transporters Among the investi-gated uptake transporters OATP1A2 was the only OATPtransporter capable of fexofenadine uptake in intestine [19]OATP1A2 colocalized with MDR1 to the brush borderdomain of enterocytes Unlike P-glycoprotein enteric OATPacts to facilitate the absorption of its substrates [19]

This study showed that multiple doses of the danshenextract significantly increased the oral clearance (CLF) and

decreased the bioavailability of fexofenadine without affect-ing the terminal half-life Because of a negligible hepatic first-pass effect the bioavailability of fexofenadine is attributableto its absorption in the intestine Given that the danshenextract reduces the bioavailability of fexofenadine the induc-tion of intestinal efflux (MDR1) can be the underlyingmechanism The in vivo findings do not support inductionof OATP1A2 because this may contribute to increasing thebioavailability of fexofenadine not to decreasing the bioavail-ability But inhibition of the danshen extract to OATP1A2was not supported because single dose administration ofthe danshen extract can increase systemic drug exposureof fexofenadine (data not shown) So the danshen ethanolextract was found to markedly reduce the systemic exposureof fexofenadine mainly via the induction of intestinal MDR1proteins that led to enhanced clearance

The induction of CYP3A4 and MDR1 is thought to bemediated by the nuclear receptor PXR PXR is predominantlyexpressed in the human liver and intestine [20 21] so thein vitro induction studies were conducted in cryopreservedhuman hepatocytes as reported previously [22] to evaluatethe relevance of the induction of MDR1 by the danshenextract In in vitro studies we found that tanshinone IIAand cryptotanshinone could induce MDR1 mRNA usingthe primary human hepatocytes The induction of MDR1by tanshinone IIA and cryptotanshinone at 10 120583M can becomparable to the effect of rifampin at 25 120583M

We also found that danshen ethanol extract could induceCYP3A in healthy Chinese volunteers [6] MDR1 and CYP3Atogether constitute a highly efficient barrier for many orallyabsorbed drugs which are substrates for both P-gp andCYP3A4 [23 24] This synergistic effect of danshen ethanolextract ismore evident for cosubstrates for P-gp andCYP3A4

Conflict of Interests

The authors declared no conflict of interests

Acknowledgments

This work was supported by NSFC (Grant 81173118) andGrants for Building Research Technology and Develop-ment of Innovative Drug (Grant 2012ZX09303009-001) theShanghai Key Lab of Traditional Clinical Medicine (GrantC10dZ2220200) and the Project of Three-year Action PlanforDevelopment of TCM in Shanghai(ZYSNXDCC-YJXYY)

References

[1] K M Giacomini S-M Huang D J Tweedie et al ldquoMembranetransporters in drug developmentrdquo Nature Reviews Drug Dis-covery vol 9 no 3 pp 215ndash236 2010

[2] T O Cheng ldquoDanshen A popular chinese cardiac herbal drugrdquoJournal of the American College of Cardiology vol 47 no 7 pp1498ndash1500 2006

[3] Y Y Xu R Z Wan Y P Lin L Yang Y Chen and C X LiuldquoRecent advance on research and application of Salvia miltior-rhizardquoAsian Journal of DrugMetabolism and Pharmacokineticsvol 7 pp 99ndash130 2007


[4] X Y Ji B K H Tan and Y Z Zhu ldquoSalvia miltiorrhiza andischemic diseasesrdquo Acta Pharmacologica Sinica vol 21 no 12pp 1089ndash1094 2000

[5] M Song T-J Hang Z Zhang and H-Y Chen ldquoEffects of thecoexisting diterpenoid tanshinones on the pharmacokinetics ofcryptotanshinone and tanshinone IIA in ratrdquo European Journalof Pharmaceutical Sciences vol 32 no 4-5 pp 247ndash253 2007

[6] F Qiu J Jiang Y Ma et al ldquoOpposite effects of single-dose andmultidose administration of the ethanol extract of danshen onCYP3A in healthy volunteersrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 730734 8 pages2013

[7] C Yu S Ye H Sun et al ldquoPXR-mediated transcriptionalactivation of CYP3A4 by cryptotanshinone and tanshinoneIIArdquo Chemico-Biological Interactions vol 177 no 1 pp 58ndash642009

[8] A Geick M Eichelbaum and O Burk ldquoNuclear receptorresponse elements mediate induction of intestinal MDR1 byrifampinrdquo Journal of Biological Chemistry vol 276 no 18 pp14581ndash14587 2001

[9] M D Perloff L L von Moltke and D J Greenblatt ldquoFexofe-nadine transport in Caco-2 cells inhibition with verapamil andritonavirrdquo Journal of Clinical Pharmacology vol 42 no 11 pp1269ndash1274 2002

[10] S Drescher E Schaeffeler M Hitzl et al ldquoMDR1 gene poly-morphisms and disposition of the P-glycoprotein substratefexofenadinerdquo British Journal of Clinical Pharmacology vol 53no 5 pp 526ndash534 2002

[11] Q Zhou Z Ye Z Ruan and S Zeng ldquoInvestigation onmodulation of human P-gp bymultiple doses of Radix Astragaliextract granules using fexofenadine as a phenotyping proberdquoJournal of Ethnopharmacology vol 146 no 3 pp 744ndash749 2013

[12] M Garrett J Smeraglia X Lin and L Tan ldquoA pilot study toassess simultaneous administration of oral midazolam (MDZ)and fexofenadine (FEX) for the evaluation of cytochrome (CYP)3A4 and P-glycoprotein (P-GP) activitiesrdquo Clinical Pharmacol-ogy ampTherapeutics vol 77 p 45 2005

[13] M Shimizu TUno K Sugawara andT Tateishi ldquoEffects of sin-gle and multiple doses of itraconazole on the pharmacokineticsof fexofenadine a substrate of P-glycoproteinrdquo British Journal ofClinical Pharmacology vol 62 no 3 pp 372ndash376 2006

[14] R P G van Heeswijk M Bourbeau P Campbell et alldquoTime-dependent interaction between lopinavirritonavir andfexofenadinerdquo Journal of Clinical Pharmacology vol 46 no 7pp 758ndash767 2006

[15] T Sakugawa M Miura N Hokama T Suzuki T Tateishi andT Uno ldquoEnantioselective disposition of fexofenadine with theP-glycoprotein inhibitor verapamilrdquo British Journal of ClinicalPharmacology vol 67 no 5 pp 535ndash540 2009

[16] Y Akamine M Miura N Yasui-Furukori M Kojima and TUno ldquoCarbamazepine differentially affects the pharmacokinet-ics of fexofenadine enantiomersrdquoThe British Journal of ClinicalPharmacology vol 73 no 3 pp 478ndash481 2012

[17] M A Hamman M A Bruce B D Haehner-Daniels and S DHall ldquoThe effect of rifampin administration on the dispositionof fexofenadinerdquo Clinical Pharmacology ampTherapeutics vol 69no 3 pp 114ndash121 2001

[18] Z Wang M A Hamman S-M Huang L J Lesko and SD Hall ldquoEffect of St Johnrsquos wort on the pharmacokenetics offexofenadinerdquo Clinical Pharmacology and Therapeutics vol 71no 6 pp 414ndash420 2002

[19] M Shimizu K Fuse K Okudaira et al ldquoontribution of OATP(organic anion-transporting polypeptide) family transportersto the hepatic uptake of fexofenadine in humansrdquo DrugMetabolism and Disposition vol 33 no 10 pp 1477ndash1481 2005

[20] M Nishimura S Naito and T Yokoi ldquoTissue-specific mRNAexpression profiles of human nuclear receptor subfamiliesrdquoDrug Metabolism and Pharmacokinetics vol 19 no 2 pp 135ndash149 2004

[21] M Nishimura and S Naito ldquoTissue-specific mRNA expressionprofiles of humanATP-binding cassette and solute carrier trans-porter superfamiliesrdquo Drug Metabolism and Pharmacokineticsvol 20 no 6 pp 452ndash477 2005

[22] BWilliamson K E Dooley Y Zhang D J Back and A OwenldquoInduction of influx and efflux transporters and cytochromeP450 3A4 in primary human hepatocytes by rifampin rifabutinand rifapentinerdquo Antimicrobial Agents and Chemotherapy vol57 no 12 pp 6366ndash6369 2013

[23] D Pal and A K Mitra ldquoMDR- and CYP3A4-mediated drug-herbal interactionsrdquo Life Sciences vol 78 no 18 pp 2131ndash21452006

[24] G K Dresser U I Schwarz G R Wilkinson and R B KimldquoCoordinate induction of both cytochrome P4503A and MDR1by St Johnrsquos wort in healthy subjectsrdquoClinical Pharmacology andTherapeutics vol 73 no 1 pp 41ndash50 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and the quality control was established and enforced strictlyby Hebei Xinlong XiLi Pharmaceuticals Ltd according tostate drug standard (China State Food and Drug Admin-istration Ws3-B-3140-98-009) The contents of tanshinoneIIA cryptotanshinone and tanshinone I were 1062mgg880mgg and 531mgg respectively [6] Fexofenadinetablets (60mgtablet Lot 100827) were manufactured byJiangsu Hengrui Pharmaceuticals Ltd

22 Subjects and Ethical Approval Twelve male healthyChinese volunteers participated in this study (age range 25ndash30 years BMI range 19ndash25 kgm2) These volunteers wereenrolled in the study after obtaining written informed con-sent The clinical protocol and informed consent form wereapproved by the independent medical ethics committee ofShuguang Hospital affiliated with the Shanghai University ofTraditional Chinese Medicine

All subjects were nonsmokers and were judged to behealthy by a medical history a physical examination electro-cardiogram and laboratory tests (including complete bloodcount blood biochemistry testing and urinalysis) beforeentering the study Subjects abstained from consuming herbaland citrus fruit products for 2 weeks before the study andfrom alcohol and medications for 2 weeks before and duringthe study period and caffeine-containing foods orange juicegrapefruit juice and beverages were also excluded during thestudy period

23 Study Design The study design was a sequential open-label two-period trial conducted at the Shuguang Hospitalphase I clinical trial ward [6] On the morning of day 1the volunteers took a single dose of 60 mg of fexofenadineBeginning on day 2 they received the danshen extract (1 gthree times a day) for 10 days On day 12 the volunteersreceived 1 g of the danshen extract together with 60mg offexofenadine The volunteers fasted overnight before eachdosing The subjects were provided a light standard mealat 4 h after medication intake and at 6 pm on the two testdays of intaking probe drugs Blood samples (4mL each)were drawn before and at 025 05 1 15 2 3 4 6 8 1012 and 24 hours after fexofenadine administration and keptin heparinized Eppendorf tubes The blood samples werecentrifuged and plasma was separated and stored at minus80∘Cuntil the time of analysis

24 Sample Analysis Plasma samples were spiked withan internal standard (diazepam) and extracted with ethylacetate After evaporation of the organic solvent under nitro-gen reconstituted residues of the organic phase were ana-lyzed using a SciexAPI 4000 coupled liquid chromatography-tandem mass spectrometry (LC-MSMS) system (AppliedBiosystemsSCIEX CA) Chromatographic separation ofthe compounds was accomplished using a C

18column

(5 120583m 46mm times 150mm Agilent America) with waterphase (ammonium acetate 4mmolL and methanoic acid008) methanol (10 90 v v) as the mobile phase at aflow rate of 080mLmin The mass spectrometer was oper-ated in the MRM mode under positive ionization The ion

transitions monitored were mass-to-charge ratios of 5022ndash4665 (fexofenadine) and 2852ndash1931 (internal standard)The collision energy (CE) declustering potential (DP) andcollision cell exit potential (CXP) were set as follows fexofe-nadine 3632V 10032V and 1609V respectively diazepam4300V 9806V and 1145V respectively A set of 7 nonzerocalibration standards ranging from 1 to 500 ngmL and 3quality controls at concentrations of 2 50 and 400 ngmLwere performed during each day of analysis The interdayCVs for the lowmiddle and high quality controls were 167334 and 310 respectivelyThe intraday CVs were 3361284 and 033 respectively The recovery for the lowmiddle and high quality controls was 10091 9611 and9211 respectively

25 Induction of MDR1 mRNA by Tanshinones in HumanHepatocytes Cryopreserved human hepatocytes (Lot ONOand JYM) provided by the Research Institute for Liver Dis-ease Co (Shanghai China) were thawed in plating mediumand transferred to collagen I precoated 24-well plates at adensity of 30 times 105 viable cells The cells were maintained at37∘C in a humidified incubator with 90 atmospheric air and5 CO

2 After the initial 24-hour conditioning culture the

medium was removed and the hepatocytes were treated withvehicle which contained the same amount of DMSO (01)tanshinones (2 120583M 10 120583M) and rifampicin (25 120583M) for 72hours All drugs were dissolved in DMSO and then added tothe culture medium (final DMSO concentration 01) TotalRNA was isolated from cells using TRIzol reagent (Invitro-gen) according to themanufacturer-supplied protocolQuan-titative real-time PCR was performed using gene-specificprimers and the SYBR Green PCR kit (Invitrogen) in an ABI7900 system (Applied Biosystems) The relative quantity ofthe target MDR1 gene compared with the endogenous con-trol (glyceraldehyde-3-phosphate dehydrogenase) was deter-mined by the ΔΔCT method The following primer sets wereused in this studyMDR1 (51015840CGGACATCCCAGTGCTTCA-31015840 and 51015840-GTCGCTTTATTTCTTTGCCATC-31015840)

26 Pharmacokinetic Determination The plasma concentra-tion-time data of analyteswere analyzed by compartment-independent approaches The maximum plasma drug con-centration (119862max) and time to 119862max (119879max) were directlyobtained from the plasma concentration-time data Theelimination half-life (119905

12) was calculated as 0693Ke

where Ke the elimination rate constant was calculated viasemilog regression on the terminal phase of the plasmaconcentration-time curve The AUC from time 0 to infinity(AUC

0minusinfin) was estimated as AUC

0minus119905+ 119862119905Ke where 119862

119905

is the plasma concentration of the last measurable sampleand AUC

0minus119905was calculated according to the linear trape-

zoidal rule Total plasma clearance (CLF) was calculated asdoseAUC

0minusinfin

27 Statistical Analysis Statistical comparisons between con-trol and drug treatments were initially performed by two-way ANOVA for repeated measures performed with SAS forWindows software (version 92 SAS Institute Inc) For the







3 Results








0

50

100

150

200

250


Fexo

fena

dine

(ng

mL)




4 Discussion





Before After0

20

40

60

80

100

Danshen extract

CLF

(Lh

)


0

1

2

3

mRN

A ex

pres

sion

(fold

of N

C) lowast

lowast

lowast

lowast

lowast

NC

Rif (25120583

M)

Cry

(2120583

M)

Cry

(10120583

M)

Tan

I (2120583

M)

Tan

I (10120583

M)

Tan

IIA (2

120583M

)

Tan

IIA (10120583

M)









Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results








0

50

100

150

200

250


Fexo

fena

dine

(ng

mL)




4 Discussion





Before After0

20

40

60

80

100

Danshen extract

CLF

(Lh

)


0

1

2

3

mRN

A ex

pres

sion

(fold

of N

C) lowast

lowast

lowast

lowast

lowast

NC

Rif (25120583

M)

Cry

(2120583

M)

Cry

(10120583

M)

Tan

I (2120583

M)

Tan

I (10120583

M)

Tan

IIA (2

120583M

)

Tan

IIA (10120583

M)









Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Before After0

20

40

60

80

100

Danshen extract

CLF

(Lh

)


0

1

2

3

mRN

A ex

pres

sion

(fold

of N

C) lowast

lowast

lowast

lowast

lowast

NC

Rif (25120583

M)

Cry

(2120583

M)

Cry

(10120583

M)

Tan

I (2120583

M)

Tan

I (10120583

M)

Tan

IIA (2

120583M

)

Tan

IIA (10120583

M)









Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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