Journal of Pharmacological and Toxicological Methods 65 (2012) 13–17

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Journal of Pharmacological and Toxicological Methods

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Original article

Standardization of an ex vivo method for determination of intestinal permeability ofdrugs using everted rat intestine apparatus

Pankaj Dixit ⁎, Dinesh Kumar Jain, Jacky DumbwaniCollege of Pharmacy, IPS Academy, Rajendranagar, A.B. Road, Indore 452012 (M.P.), India

⁎ Corresponding author at: Associate Professor (Pharcy, IPS Academy, Rajendranagar, A.B. Road, Indore 4520731 4041627.

E-mail address: pankaj-dixit@hotmail.com (P. Dixit)

1056-8719/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.vascn.2011.11.001

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 13 October 2011Accepted 3 November 2011

Keywords:Absorption kineticsAtenololMetoprololPropranololPhenol redMethods

Introduction: Everted gut sac of rat intestine is a paradigm widely employed for determination of ab-sorption kinetics of drugs along with evaluation of effects of absorption enhancers. Since its inception in1954, it has been optimized to enhance tissue survival and use, but it still suffers the limitation of small se-rosal compartment size and lack of validity of single experiment. Methods: The aim of the present workwas to standardize a new ex vivomodel to study drug absorption using a specially designed glass apparatus,everted segment of rat intestine, and three absorption markers [paracellular (atenolol), transcellular (met-oprolol and propranolol)]. To validate a single experiment phenol red was used as non-absorbable marker.Results: Themean apparent permeabilities (Papp) for the markers were found to be 0.054±0.024×10−4 cm/s(atenolol), 0.84±0.14×10−4 cm/s (metoprolol), and 1.64±0.16×10−4 cm/s (propranolol); wherein datafrom only those experiment was used, which showed negligible absorption of phenol red. Discussion: The

model is simple to establish, gives excellent absorption kinetics, and most importantly provides a way to vali-date the experiment simultaneously. The proposed method can be used in all kinds of drug absorption studies,especially biopharmaceutical investigations studying absorption enhancement strategies.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

The everted gut sac of the rat small intestinewas originally describedbyWilson andWiseman (1954). It can be used to determine transport ofvarious compounds from intestine like sugars, amino acids, drugs, andevaluate the performance of novel drug delivery systems with highreliability and reproducibility (Barthe, Woodley, & Houin, 1999;Barthe, Woodley, Kenworthy, & Houin, 1998). Oxygenated tissueculture media (TC 199) and specific preparation techniques ensuretissue viability for up to 2 h (Barthe et al., 1998; Gandia, Lacombe,Woodley, & Houin, 2004). The technique can be used to study drugtransport across the intestine and into the epithelial cells. Earlierthe technique was widely used to study uptake of liposomes, pro-teins, macromolecules etc. (Rowland & Woodley, 1981a, 1981b,1981c); but recently it was reported to be useful to quantify theparacellular transport of hydrophilic molecules, and estimation ofthe effect of absorption enhancers (Leppert & Fix, 1994).

The well known limitations of the everted gut sac model are mor-phological damages to intestinal tissue while everting (Balimane,Chong, & Morrison, 2000); presence of muscularis mucosa (Le Ferrecet al., 2001); small closed serosal compartment (Gandia et al., 2004).Secondly, the validation of experiment cannot be made simultaneously

macology), College of Pharma-12 (M.P.), India. Tel./fax: +91

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as no parameters are set tomeasure the integrity ofmucosal barrier liketransepithelial electrical resistance measurement (Barthe et al., 1999).In order to overcome some of these limitations a new apparatus wasused by Appaji (1980). In the present investigation, we have attemptedto standardize the use of this apparatus, which employs everted seg-ment of rat intestine for permeability estimation.We have taken specialcare to ensure the validity of one experiment, which was one of themajor drawbacks of methods employing everted rat intestine.

For standardization,we have usedmetoprolol, propranolol, atenolol,and phenol red. These drugs represent the biopharmaceutical classifica-tion system, as they belong to class I to class IV respectively. In addition,propranolol and metoprolol are absorbed via the transcellular route(Brouwers, Mols, Annaert, & Augustijns, 2010; Hilgendorf et al.,1999); atenolol by paracellular route (Brouwers et al., 2010); and phe-nol red is employed as a non-absorbable marker (Zakeri-Milani et al.,2007).

2. Materials

2.1. Chemicals and reagents

Atenolol, propranolol, andmetoprololwere gifted by Ipca LaboratoriesLimited, Ratlam, India. Phenol redwas purchased from Loba Chemie Pvt.Ltd., Mumbai, India. All other chemicals used were of analyticalgrade. Freshly double distilled water, filtered through 0.45 mmnylon filter (47 mm) (Pall Life Sciences, Mumbai, India) in Milliporeunit (USA), was used throughout the experiments.

14 P. Dixit et al. / Journal of Pharmacological and Toxicological Methods 65 (2012) 13–17

2.2. Experimental animals

Male Albino Wistar rats (150–200 g) born and reared in theAnimal House of College of Pharmacy, IPS Academy, Indore, M.P. froma stock originally purchased from Sudhakar Rao Naik Institute ofPharmacy, Pusad, Maharashtra were used for the study. Animalswere acclimatized to laboratory conditions 1 week before startingthe experiment; they were given free access to water and standardrat diet (Trimurti Industries, Maharashtra, India) except duringexperimentation.

2.3. Everted rat intestine apparatus

The design of the glass apparatus used for the present study isdepicted in Fig. 1a. The apparatus consists of two cylindrical glasstubes; one (110×17 mm) joined to other (48×17 mm) via J-shapedtapering end. Both the tubes are held together by a glass joint onthe upper end. On the lower ends of both tubes a bulge is givenfor proper mounting of tissue. The dimensions of the apparatus(110×49×17 mm) are such that it can be conveniently set up ina 250.0 ml glass beaker. After mounting the everted intestinal seg-ment on the apparatus and setting it in the beaker; the inside ofthe glass tubes serve as the mucosal compartment and the beakerserves as the serosal compartment (Fig. 1b).

3. Methods

3.1. Drug analysis by RP-HPLC

Drug analysis was carried out using an HPLC system (Shimadzu LCIOAT, Tokyo, Japan) attached to solvent delivery module with a lowpressure gradient pump (LC 10AT VP), Rheodyne injector port (7725i,20 μl loop), 15 cm C-18 column (Supelcosil®, Merck, India), and, UV/Vis photodiode array detector (SPD-M10A). The data interpretationwas done with CSW (Chromatographic Work Station) CLASS-VP ver-sion 1.6 software (Shimadzu, Tokyo, Japan). We employed a previouslyreported method with slight modification (Zakeri-Milani, Valizadeh,Azarmi, Jalali, & Tajerzadeh, 2006) for drug analysis. The mobile phase

Fig. 1. Photographs of the apparatus (a) wit

for analysis of metoprolol, propranolol, and phenol red was a mixtureof 55% methanol and 45% of 0.05 M KH2PO4 adjusted to pH 6.0, towhich 0.2% (v/v) triethylamine was added. The mobile phase for analy-sis of atenolol was a mixture of 10% methanol and 90% of 0.05 MKH2PO4 aqueous solution adjusted to pH 6.0. The mobile phase waspumped in isocratic mode at a flow rate of 1.0 ml/min at ambient tem-perature. The UV detectionwas accomplished at 223 nmand samples of20 μl were injected using Hamilton syringe on to the column.

3.2. Isolation and eversion of the intestine

The rat was sacrificed humanely by cervical dislocation and theabdomen was opened by midline incision and the intestine was care-fully maneuvered to identify the ileocaecal junction. A 7 cm segment,5–6 cm distant to the ileocaecal junction was excised, and it was re-moved of the mesenteric attachments carefully without damagingthe intestinal architecture. The intestinal segment was transferredto a petri dish containing Kreb's medium (118.0 mM Nacl, 4.7 mMKcl, 2.5 mM CaCl2, 1.2 mM MgSO4. 7H2O, 25.0 mM NaHCO3, 1.2 mMKH2PO4, and 5.5 mM glucose). It was cleaned with Kreb's solutionand then gently everted using a glass rod. A 3.0 cm everted segmentwas then used for permeability experiments.

3.3. Permeability determination

After mounting the tissue, the glass apparatus was placed in a250.0 ml beaker containing 100 μg/ml drug solutions. The mucosalside of the intestine was perfused with Kreb's solution. This assembly(beaker and apparatus with tissue) was placed on a magnetic stirrerand a magnetic bead was allowed to rotate at 25 rpm in beaker andthe temperature was maintained at 37 °C with adequate aeration.The samples were collected at different time points (at every 5 minfor 1 h) and analyzed by HPLC for estimation of permeability.

3.4. Histopathology

After completion of the experiments, a small piece of the evertedintestine was fixed with 10% formalin for 24 h and then dehydrated

h dimensions; and (b) complete set up.

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with a graded alcohol series and fixed by using paraffin wax. It wasstained by H&E stain and specimens were observed and photomicro-graphed under a confocal microscope (Leppert & Fix, 1994).

4. Data analysis

4.1. Permeability calculation

The apparent permeability (Papp) value was calculated accordingto the following equation:

Papp ¼ dQdt

� 1A � C0 ¼ v � dt � A�C0

(Le Ferrec et al., 2001)where,

Papp: apparent permeability co-efficientdQ/dT: the cumulative amount of drug (Q) appearing in the acceptor

(serosal) compartment as a function of time, and wasobtained from the slope of the linear portion of the amounttransported-versus-time plot;

A: surface area of the intestine (cm2) [0.18 cm as radius(Shirasaka, Masaoka, Kataoka, Sakuma, & Yamashita, 2008)]

C0: the initial concentration of drug in the donor compartment(μg/ml)

V: volume of sample (ml)

Fig. 2. Absorption kinetics of marker drugs using everted intestine apparatus. Eachpoint represents the mean±SD of n=4–7.

5. Results

5.1. Permeability assay optimization

The everted rat intestine glass apparatus originally used by Appaji(1980) was larger in dimension and required a 5–7 cm long intestinalsegment. However, the length of ileum and duodenum in rat is re-portedly 3 cm and 10 cm respectively (Suckow, Sharp, & La Regina,1998). Hence, in order to make the estimations of permeability sitespecific for drugs having predefined absorption windows the lengthof intestine was selected as 3 cm. Secondly, this alteration reducedthe size of the apparatus, made it portable, and hence only 250 mlof mucosal compartment size was rendered feasible, which furtherreduced the amount of drug required for estimation of permeability.In addition, if required mouse intestine can also be mounted on thisapparatus making it more versatile. A slight increase in the openingwhere tissue is mounted can make it suitable for large size intestinealso; rendering it suitable for use with any size of intestinal tissuelumen viz. rabbit/goat.

We have performed the permeability estimations in the presenceof phenol red, as it was reported to be a zero permeability marker(Gorham, 1923), which was verified in our preliminary experiments.It is very difficult to measure the transmembrane electrical resistancein case of everted gut sacs as marker of tight junction integrity. Hence,the incorporation of phenol red was considered, as it is easy to analyzeby HPLC-UV/Vis spectroscopy along with drug of interest. The results ofa single experiment were considered valid only when the levels of phe-nol red in the serosal compartment were below quantitation limit forphenol red.

Further, instead of TC 199 as culture media we have chosen Kreb'sbuffer for incubation as the use of Kreb's buffer widens the incorpora-tion of many drugs which are also water insoluble and renders theHPLC analysis easy. Further, we have reduced the incubation timefrom 2 h to 1 h, so that the tissue viability issues, if any are takencare of.

5.2. HPLC analysis

The representative chromatograms of samples containing pro-pranolol, metoprolol, atenolol, and phenol red are given as Supple-mentary data. The retention times were 13.0, 4.5 and 7.3, 8.6 minfor propranolol, metoprolol, atenolol, and phenol red respectively.The chromatographic run time of 15 min which was sufficient forsample analysis allows analyzing a large number of samples in ashort period of time.

5.3. Permeability estimations

The absorption rate of various drugs across the everted intestinalsegment is depicted in Fig. 2. The absorption of all three drugs waslinear after initial lag phase and then had a plateau. The slope of thelinear phase was used to determine the dQ/dt and then the apparentpermeability co-efficients. The Papp values were found to be 0.84±0.014×10−4, 1.64±0.16×10−4, and 0.054±0.024×10−4 cm/s re-spectively for metoprolol, propranolol, and atenolol respectively.The slope from only those experiments was used where phenol redpermeation was negligible.

5.4. Histopathology

Tissue was examined microscopically to visualize any alterationsin mucosal morphology. Photomicrographs of representative tissuesections are shown in Fig. 3. Panels A (10×) and B (40×) show con-trol mucosal tissue in which the intestine was cut immediately afterthe intestine was everted and then placed in fixative. Panels C(10×) and D (40×) show a section which was treated the same as

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in A and B except that it was incubated for 1 h in Krebs. In B, the villusepithelium remained intact and the lamina propria was dense andcompact. The section shown in D was fixed 60 min after the intestinewas everted. The mucin-producing goblet cells appeared larger andmore indistinct or diffuse, as compared to A and B. Similarly, the lam-ina propria is not as dense as it appears in control. The integrity of thevillus epithelium with the brush border membrane appeared intact,although the nuclei of the epithelial cells were arranged less regularlythan in A and B.

6. Discussion

In this study we present a new and relatively simple ex vivo modelstandardization for studying absorption kinetics and apparent perme-abilities using the rat small intestine. In this paper we have describedthe transport of three model marker compounds, selected to exempli-fy the major routes of drug absorption. In our laboratory, the instru-ment was in use since long and used for studying P-gp mediateddrug interactions (Salunke, 2006); effect of various gastrointestinalailments on permeability of drugs. However, the need for its valida-tion was constantly felt, as we wanted to increase the use of this ap-paratus for biopharmaceutical work for evaluation of variousapproaches in increasing the drug bioavailability.

The three test drugs were chosen to represent the major routes ofdrug absorption and the BCS classification as well. Two of these, met-oprolol and propranolol are transported by transcellular route(Hilgendorf et al., 1999) and represent BCS Class I and II respectively.Whereas, atenolol is transported by paracellular route (Brouwers etal., 2010) and represents BCS class III.

Atenolol is a low molecular weight drug with a log P of 0.23 (Neil-Dwyer, Bartlett, & Mcainsh, 1981), thus it is hydrophilic. In man thefraction absorbed (Fa) is 0.50 (Zakeri-Milani et al., 2007). Given thatthe paracellular route constitutes a small proportion of the total sur-face (0.01–0.1%) (Nellans, 1991) of the intestine, not surprisinglythe permeation of this highly polar low molecular weight moleculeis slower than for lipophilic molecules. It is not a P-gp substrate andso it is an appropriate marker to quantify the simple paracellularroute of absorption and the Papp value obtained with our apparatuswas 0.054±0.024×10−4 cm/s. This value is closed to that reportedby Volpe (2004) who, using a Caco-2 cell culture model, found thePeff 0.02×10−4 cm/s. A slightly higher figure of 0.16×10−4 cm/s

Fig. 3. Photographs of everted intestine of rat. (A) TS of everted intestine of rat (control), at 1exposed to Kreb's medium for 1 h at 10×; (D) TS of everted intestine of rat exposed to Kre

was obtained by Zakeri-Milani et al. (2007) using an in situ intestinalperfusion system in anesthetized rats. Using the Ussing chambertechnique, Watanabe, Takahashi, and Hayashi (2004) obtained avalue of 0.06×10−4 cm/s for atenolol transport across the rat jeju-num. Thus, with all the in vitro methods and animal perfusionmethods the permeabilities are in a similar range, with an approxi-mately 8-fold difference between highest and lowest values.

Metoprolol has been very widely used as the standard marker formeasuring transcellular permeation. It has a Fraction Absorbed (Fa) ofaround 0.95 in man (Zakeri-Milani et al., 2007). The mean Papp mea-sured for metoprolol in the present study was 0.84±0.14×10−4 cm/s.This value for an in situ intestinal perfusion by Zakeri-Milani et al.(2007) was 0.32×10−4 cm/s. Using the Ussing chamber technique,Watanabe et al. (2004) obtained a value of 0.23×10−4 cm/s. It is similarwith the Caco-2 cell monolayer system, with Papp value of0.27×10−4 cm/s. Our value is slightly higher than the reported perme-ability values; the reason for which appears intriguing.

The third drug used in this study was propranolol. Propranolol is adrug that is lipophilic (log P— 3.65) and as such is absorbed in the in-testine by transcellular passive diffusion. The mean permeability co-efficient (Papp) measured for propranolol in the present study was1.64±0.16×10−4 cm/s, which is higher than the value obtainedwith an in situ intestinal perfusion by Milani et al., with Peff value0.49×10−4 cm/s. Watanabe et al. obtained Peff value of propranololusing Ussing chamber was 0.118 and Volpe, 2004 found it 0.405using Caco-2 cell culture model. Thus, it appears that the evertedgut sac model gives higher values of permeability for drugs trans-ported via transcellular route, which may be attributed to the intactmuscle mass that remains in the intestinal segment.

Thus, the everted intestinal segment described in this study repre-sents some advantages over the previously described models. Nota-bly, we have established a way of determination of integrity of thetight junction by concomitant used of phenol red as non-absorbablemarker. Secondly, with the use of advanced techniques like LC–MS,PEG 4000 or mannitol can also be used as a zero permeability marker,which further increases the scope of use of the apparatus. Secondly,the apparatus can be connected to a reservoir of 1–2 L and the fluidcan be recirculated by peristaltic pump to mimic the sink conditionsmore accurately. Finally, with slight modifications it can be employedfor any strain of rodents or mammals also. The permeability valuesobtained by us in this study will serve as a guide to future

0×; (B) TS of everted intestine of rat (control), at 40×; (C) TS of everted intestine of ratb's medium for 1 hat 40×.

17P. Dixit et al. / Journal of Pharmacological and Toxicological Methods 65 (2012) 13–17

investigators and if used properly, this apparatus may be very handyfor researchers in the biopharmaceutical field.

Supplementary materials related to this article can be found on-line at doi:10.1016/j.vascn.2011.11.001.

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