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 of

drugs using everted rat intestine apparatus
Pankaj Dixit ⁎, Dinesh Kumar Jain, Jacky Dumbwani
College of Pharmacy, IPS Academy, Rajendranagar, A.B. Road, Indore 452012 (M.P.), India

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: Everted gut sac of rat intestine is a paradigm widely employed for determination of ab-
Received 13 October 2011 sorption kinetics of drugs along with evaluation of effects of absorption enhancers. Since its inception in
Accepted 3 November 2011 1954, 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 work
Keywords:
was to standardize a new ex vivo model to study drug absorption using a specially designed glass apparatus,
Absorption kinetics
everted segment of rat intestine, and three absorption markers [paracellular (atenolol), transcellular (met-
Atenolol
Metoprolol
oprolol and propranolol)]. To validate a single experiment phenol red was used as non-absorbable marker.
Propranolol Results: The mean apparent permeabilities (Papp) for the markers were found to be 0.054 ± 0.024 × 10− 4 cm/s
Phenol red (atenolol), 0.84 ± 0.14× 10− 4 cm/s (metoprolol), and 1.64± 0.16× 10− 4 cm/s (propranolol); wherein data
Methods from 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 intestine was originally described
by Wilson and Wiseman (1954). It can be used to determine transport of
various compounds from intestine like sugars, amino acids, drugs, and
evaluate the performance of novel drug delivery systems with high
reliability and reproducibility (Barthe, Woodley, & Houin, 1999;
Barthe, Woodley, Kenworthy, & Houin, 1998). Oxygenated tissue
culture media (TC 199) and specific preparation techniques ensure
tissue viability for up to 2 h (Barthe et al., 1998; Gandia, Lacombe,
Woodley, & Houin, 2004). The technique can be used to study drug
transport across the intestine and into the epithelial cells. Earlier
the 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 the
paracellular transport of hydrophilic molecules, and estimation of
the 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 Ferrec
et al., 2001); small closed serosal compartment (Gandia et al., 2004).
Secondly, the validation of experiment cannot be made simultaneously
⁎ Corresponding author at: Associate Professor (Pharmacology), College of Pharma-
cy, IPS Academy, Rajendranagar, A.B. Road, Indore 452012 (M.P.), India. Tel./fax: + 91
731 4041627.
E-mail address: pankaj-dixit@hotmail.com (P. Dixit).
as no parameters are set to measure the integrity of mucosal barrier like
transepithelial electrical resistance measurement (Barthe et al., 1999).
In order to overcome some of these limitations a new apparatus was
used by Appaji (1980). In the present investigation, we have attempted
to standardize the use of this apparatus, which employs everted seg-
ment of rat intestine for permeability estimation. We have taken special
care to ensure the validity of one experiment, which was one of the
major drawbacks of methods employing everted rat intestine.
For standardization, we have used metoprolol, 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, and metoprolol were gifted by Ipca Laboratories
Limited, Ratlam, India. Phenol red was purchased from Loba Chemie Pvt.
Ltd., Mumbai, India. All other chemicals used were of analytical
grade. Freshly double distilled water, filtered through 0.45 mm
nylon filter (47 mm) (Pall Life Sciences, Mumbai, India) in Millipore
unit (USA), was used throughout the experiments.

1056-8719/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.vascn.2011.11.001
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 the
Animal House of College of Pharmacy, IPS Academy, Indore, M.P. from
a stock originally purchased from Sudhakar Rao Naik Institute of
Pharmacy, Pusad, Maharashtra were used for the study. Animals
were acclimatized to laboratory conditions 1 week before starting
the experiment; they were given free access to water and standard
rat diet (Trimurti Industries, Maharashtra, India) except during
experimentation.
2.3. Everted rat intestine apparatus
The design of the glass apparatus used for the present study is
depicted in Fig. 1a. The apparatus consists of two cylindrical glass
tubes; one (110 × 17 mm) joined to other (48 × 17 mm) via J-shaped
tapering end. Both the tubes are held together by a glass joint on
the upper end. On the lower ends of both tubes a bulge is given
for proper mounting of tissue. The dimensions of the apparatus
(110 × 49 × 17 mm) are such that it can be conveniently set up in
a 250.0 ml glass beaker. After mounting the everted intestinal seg-
ment on the apparatus and setting it in the beaker; the inside of
the glass tubes serve as the mucosal compartment and the beaker
serves 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 LC
IOAT, Tokyo, Japan) attached to solvent delivery module with a low
pressure 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 interpretation
was done with CSW (Chromatographic Work Station) CLASS-VP ver-
sion 1.6 software (Shimadzu, Tokyo, Japan). We employed a previously
reported method with slight modification (Zakeri-Milani, Valizadeh,
Azarmi, Jalali, & Tajerzadeh, 2006) for drug analysis. The mobile phase
Fig. 1. Photographs of the apparatus (a) with dimensions; and (b) complete set up.
for analysis of metoprolol, propranolol, and phenol red was a mixture
of 55% methanol and 45% of 0.05 M KH2PO4 adjusted to pH 6.0, to
which 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 M
KH2PO4 aqueous solution adjusted to pH 6.0. The mobile phase was
pumped in isocratic mode at a flow rate of 1.0 ml/min at ambient tem-
perature. The UV detection was accomplished at 223 nm and samples of
20 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 the
abdomen 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 damaging
the intestinal architecture. The intestinal segment was transferred
to a petri dish containing Kreb's medium (118.0 mM Nacl, 4.7 mM
Kcl, 2.5 mM CaCl2, 1.2 mM MgSO4. 7H2O, 25.0 mM NaHCO3, 1.2 mM
KH2PO4, and 5.5 mM glucose). It was cleaned with Kreb's solution
and then gently everted using a glass rod. A 3.0 cm everted segment
was then used for permeability experiments.
3.3. Permeability determination
After mounting the tissue, the glass apparatus was placed in a
250.0 ml beaker containing 100 μg/ml drug solutions. The mucosal
side of the intestine was perfused with Kreb's solution. This assembly
(beaker and apparatus with tissue) was placed on a magnetic stirrer
and a magnetic bead was allowed to rotate at 25 rpm in beaker and
the temperature was maintained at 37 °C with adequate aeration.
The samples were collected at different time points (at every 5 min
for 1 h) and analyzed by HPLC for estimation of permeability.
3.4. Histopathology
After completion of the experiments, a small piece of the everted
intestine was fixed with 10% formalin for 24 h and then dehydrated
 P. Dixit et al. / Journal of Pharmacological and Toxicological Methods 65 (2012) 13–17 15

with a graded alcohol series and fixed by using paraffin wax. It was
stained 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 according
to the following equation:
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 min
for propranolol, metoprolol, atenolol, and phenol red respectively.
The chromatographic run time of 15 min which was sufficient for
sample analysis allows analyzing a large number of samples in a
short period of time.
5.3. Permeability estimations

The absorption rate of various drugs across the everted intestinal

dQ 1
Papp ¼  ¼ v  dt  AC0 segment is depicted in Fig. 2. The absorption of all three drugs was
dt A  C0
linear after initial lag phase and then had a plateau. The slope of the
linear phase was used to determine the dQ/dt and then the apparent
(Le Ferrec et al., 2001)where, permeability 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-
Papp: apparent permeability co-efficient spectively for metoprolol, propranolol, and atenolol respectively.
dQ/dT: the cumulative amount of drug (Q) appearing in the acceptor The slope from only those experiments was used where phenol red
(serosal) compartment as a function of time, and was permeation was negligible.
obtained from the slope of the linear portion of the amount
transported-versus-time plot; 5.4. Histopathology
A: surface area of the intestine (cm 2) [0.18 cm as radius
(Shirasaka, Masaoka, Kataoka, Sakuma, & Yamashita, 2008)] Tissue was examined microscopically to visualize any alterations
C0: the initial concentration of drug in the donor compartment in mucosal morphology. Photomicrographs of representative tissue
(μg/ml) sections are shown in Fig. 3. Panels A (10×) and B (40 ×) show con-
V: volume of sample (ml) trol mucosal tissue in which the intestine was cut immediately after
the intestine was everted and then placed in fixative. Panels C
(10×) and D (40×) show a section which was treated the same as

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 intestinal
segment. 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 site
specific for drugs having predefined absorption windows the length
of intestine was selected as 3 cm. Secondly, this alteration reduced
the size of the apparatus, made it portable, and hence only 250 ml
of mucosal compartment size was rendered feasible, which further
reduced the amount of drug required for estimation of permeability.
In addition, if required mouse intestine can also be mounted on this
apparatus making it more versatile. A slight increase in the opening
where tissue is mounted can make it suitable for large size intestine
also; rendering it suitable for use with any size of intestinal tissue
lumen viz. rabbit/goat.
We have performed the permeability estimations in the presence
of 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 resistance
in case of everted gut sacs as marker of tight junction integrity. Hence,
the incorporation of phenol red was considered, as it is easy to analyze
by HPLC-UV/Vis spectroscopy along with drug of interest. The results of
a single experiment were considered valid only when the levels of phe-
nol red in the serosal compartment were below quantitation limit for
phenol red.
Further, instead of TC 199 as culture media we have chosen Kreb's
buffer for incubation as the use of Kreb's buffer widens the incorpora-
tion of many drugs which are also water insoluble and renders the
HPLC analysis easy. Further, we have reduced the incubation time
from 2 h to 1 h, so that the tissue viability issues, if any are taken Fig. 2. Absorption kinetics of marker drugs using everted intestine apparatus. Each
care of. point represents the mean ± SD of n = 4–7.
16 P. Dixit et al. / Journal of Pharmacological and Toxicological Methods 65 (2012) 13–17
in A and B except that it was incubated for 1 h in Krebs. In B, the villus
epithelium remained intact and the lamina propria was dense and
compact. The section shown in D was fixed 60 min after the intestine
was everted. The mucin-producing goblet cells appeared larger and
more 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 the
villus epithelium with the brush border membrane appeared intact,
although the nuclei of the epithelial cells were arranged less regularly
than in A and B.
6. Discussion
In this study we present a new and relatively simple ex vivo model
standardization for studying absorption kinetics and apparent perme-
abilities using the rat small intestine. In this paper we have described
the 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 mediated
drug interactions (Salunke, 2006); effect of various gastrointestinal
ailments 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 various
approaches in increasing the drug bioavailability.
The three test drugs were chosen to represent the major routes of
drug 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 et
al., 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 the
fraction absorbed (Fa) is 0.50 (Zakeri-Milani et al., 2007). Given that
the paracellular route constitutes a small proportion of the total sur-
face (0.01–0.1%) (Nellans, 1991) of the intestine, not surprisingly
the permeation of this highly polar low molecular weight molecule
is slower than for lipophilic molecules. It is not a P-gp substrate and
so it is an appropriate marker to quantify the simple paracellular
route of absorption and the Papp value obtained with our apparatus
was 0.054 ± 0.024 × 10 − 4 cm/s. This value is closed to that reported
by Volpe (2004) who, using a Caco-2 cell culture model, found the
Peff 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 10 ×; (B) TS of everted intestine of rat (control), at 40 ×; (C) TS of everted intestine of rat
exposed to Kreb's medium for 1 h at 10 ×; (D) TS of everted intestine of rat exposed to Kreb's medium for 1 hat 40 ×.
was obtained by Zakeri-Milani et al. (2007) using an in situ intestinal
perfusion system in anesthetized rats. Using the Ussing chamber
technique, Watanabe, Takahashi, and Hayashi (2004) obtained a
value of 0.06 × 10 − 4 cm/s for atenolol transport across the rat jeju-
num. Thus, with all the in vitro methods and animal perfusion
methods 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 for
measuring transcellular permeation. It has a Fraction Absorbed (Fa) of
around 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 similar
with the Caco-2 cell monolayer system, with Papp value of
0.27 × 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 a
drug 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 was
1.64 ± 0.16 × 10 − 4 cm/s, which is higher than the value obtained
with an in situ intestinal perfusion by Milani et al., with Peff value
0.49 × 10 − 4 cm/s. Watanabe et al. obtained Peff value of propranolol
using Ussing chamber was 0.118 and Volpe, 2004 found it 0.405
using Caco-2 cell culture model. Thus, it appears that the everted
gut sac model gives higher values of permeability for drugs trans-
ported via transcellular route, which may be attributed to the intact
muscle 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 the
tight junction by concomitant used of phenol red as non-absorbable
marker. 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 fluid
can be recirculated by peristaltic pump to mimic the sink conditions
more accurately. Finally, with slight modifications it can be employed
for any strain of rodents or mammals also. The permeability values
obtained by us in this study will serve as a guide to future
 P. Dixit et al. / Journal of Pharmacological and Toxicological Methods 65 (2012) 13–17 17

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Supplementary materials related to this article can be found on- transport modulation of absorption. Advanced Drug Delivery Reviews, 7, 339.
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Rowland, R. N., & Woodley, J. F. (1981). Uptake of free and liposome entrapped horse
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