Research Paper

Regional difference in intestinal drug absorption as a

measure for the potential effect of P-glycoprotein efflux
transporters
Shimaa M. Ashmawy , Sanaa A. El-Gizawy, Gamal M. El Maghraby and Mohamed A. Osman
Department of Pharmaceutical Technology, College of Pharmacy, University of Tanta, Tanta, Egypt

Keywords Abstract
intestinal perfusion; P-glycoprotein; piperine;
ranitidine hydrochloride Objectives The aim of this research was to assess regional difference in the
intestinal absorption of ranitidine HCl as an indicator for the potential effect of
Correspondence P-glycoprotein (P-gp) efflux transporters.
Shimaa M. Ashmawy, Department of
Methods In situ rabbit intestinal perfusion was used to investigate absorption of
Pharmaceutical Technology, Faculty of
Pharmacy, University of Tanta, Tanta 31111,
ranitidine HCl, a substrate for P-gp efflux from duodenum, jejunum, ileum and
Egypt. colon. This was conducted both in the presence and absence of piperine as P-gp
E-mail: Shim23shmawy@yahoo.com inhibitor.
Key findings Ranitidine HCl was incompletely absorbed from rabbit intestine.
Received March 6, 2018 The length normalized absorptive clearance (PeA/L) of ranitidine HCl was
Accepted September 29, 2018 ranked as colon > duodenum > jejunum > ileum. This is the reverse order of
the magnitude of P-gp expression. Coperfusion of piperine with ranitidine HCl
doi: 10.1111/jphp.13036
significantly increased the PeA/L of ranitidine HCl from jejunum and ileum with
no significant change on the absorption from duodenum and colon. This was
confirmed by significant reduction in the length required for complete ranitidine
HCl absorption from jejunum and ileum in presence piperine.
Conclusions The results indicate that P-gp transporters play a major role in
determining regional difference in intestinal absorption of ranitidine HCl. Thus,
the regional absorption of drugs may be taken as an indirect indication for the
role of P-gp in intestinal absorption.

done in the presence and absence of known inhibitors for

Introduction
The efflux transporters such as P-glycoprotein (P-gp) can
play a major role in drug absorption after oral administra-
tion. They can determine the concentration of active phar-
maceutical ingredients and/or their metabolites in the
enterocytes. This may affect the driving force of drug
absorption by back efflux.[1] This effect has been shown to
play an integral role in minimizing the oral bioavailability
of drugs.[2,3] Accordingly, researchers concentrated on
developing methods to monitor the contribution of these
efflux transporters to the intestinal absorption of drugs.[4]
Several methods have been used to study the effect of
P-gp on intestinal drug absorption. These studies employed
both in vitro to in vivo methods. Among the used methods
are vesicular systems and Caco-2 cells monolayers. In these
two techniques authors monitored the influx of drugs into
vesicular structure or into the cell, respectively. This can be
the efflux transporters.[1,4,5] Other methods include everted
intestinal sac,[6–8] rat precision-cut intestinal slices (PCIS)
as an ex vivo model.[1] In these studies, the mucosal to sero-
sal transport can be compared to the corresponding serosal
to mucosal influx with intestinal efflux being diagnosed if
the later was significantly higher than the former.
Despite of simplicity and generation of large amount of
data, the in vitro methods are not standardized and are
associated with many problems.[9] For example, in vitro
methods cannot determine location of transporters. In vitro
models may create data which underestimate effect of efflux
transporters on low permeability drugs.[4] In addition,
in vitro simulation of efflux transporter can overlook the
heterogeneous distribution and expression of the trans-
porters in the intestine.[1]
In situ intestinal perfusion technique using animal
models was introduced as an alternative strategy to monitor

© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–** 1
A new method for p-gp assessment
the effect of efflux transporters on the intestinal absorption
of drugs. This technique preserves the viability of the tissue
while eliminating the effect of food and gastric residence
time on drug absorption. In addition to these advantages,
several animal models have been employed providing
greater flexibility to the researchers.[9–13] To employ this
technique in monitoring P-gp efflux transporters, authors
perfused different drug solutions of increasing concentra-
tions of drug to eliminate possible saturation of the trans-
porters by perfusion of highly concentrated solution.[14]
The regional distribution of P-gp efflux transporters have
been investigated with the published data providing reliable
distribution of these transporters throughout the intes-
tine.[3,15] The studies revealed site specific expression P-gp
efflux transporters with this expression increasing as we
move from the proximal to distal regions in the small intes-
tine. For the large intestine greater expression was recorded
in the central region but the overall expression is signifi-
cantly lower than that extracted from any of small intestinal
segments.[3,15,16] Accordingly, exploring the regional differ-
ences in drug absorption may provide indirect indication
for the susceptibility of the given drug for efflux trans-
porters. However, this needs to be verified and the current
study is the first study to test this hypothesis.
Accordingly, the aim of this work was to investigate
regional difference in intestinal drug absorption as a
parameter reflecting the potential effect of P-gp efflux
transporters. To achieve this, rabbit was selected as the
model animal and in situ intestinal perfusion technique was
employed. The intestinal absorption of ranitidine HCl, a
known substrate for Pgp-efflux transporters,[17–20] was
monitored through the duodenum, jejunum, ileum and
colon. This was conducted both in the presence and
absence of piperine as an inhibitor for the efflux.
Materials and Methods
Materials
Ranitidine HCl was obtained from Sigma Pharmaceutical
industries S.A.E. (Quesna, Egypt). Caffeine and acetonitrile
high pressure liquid chromatography (HPLC)-grade were
obtained from BDH Chemicals Ltd (Poole, England).
Piperine was procured from Sigma-Aldrich (St. Louis, MO,
USA). Methanol (HPLC-grade), potassium dihydrogen
phosphate and phosphoric acid were ordered from Merck
Chemical Co. (Darmstadt, Germany). Sodium chloride
0.9% w/w for injection (normal saline) was purchased from
El-Nasr Pharmaceutical Chemicals Company (Cairo,
Egypt). Ketamine HCl injection (50 mg/ml) was a product
of Sigma-Tec Pharmaceutical Industries (Giza, Egypt).
Chloropromazine HCl injection (25 mg/ml) was produced
by Misr Pharmaceutical Company (Cairo, Egypt).
2 © 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
Shimaa M. Ashmawy et al.
Chromatography
Quantification of ranitidine HCl was conducted using
HPLC. This utilized a Waters HPLC system equipped with
Waters 600 flow rate controller (Milford, CT, USA), a
Waters 468, variable wavelength UV detector. Samples
(40 ll) were injected using Waters 717 Plus autosampler.
This system was remote controlled using Millennium 32
software. Chromatographic separation was performed using
C18, reversed phase column (150 9 4.6 mm). The average
particle size of the stationary phase was 5 lm (Thermo Sci-
entific Hypersil, Waltham, MA, USA). The study employed
a mixture of Acetonitrile and 20 mM potassium dihydrogen
phosphate adjusted to pH 6 (9 : 91) with the flow rate
being set at 1.2 ml/min. The concentration of drug in the
effluent was measured by monitoring at 228 nm. Caffeine
was used as an internal standard and was included at a fixed
concentration (7 lg/ml) in each sample. The calibration
curves were constructed from the drug concentrations and
the peak area ratios of the drug/internal standard. The
method was validated for linearity, range, lower limit of
quantification (LOQ) and lower limit of detection (LOD).
In situ intestinal perfusion studies
The study employed 12 rabbits (average weight of 2 kg).
This selection was based on the similarity in the anatomical
and physiological parameters of the gastrointestinal tract of
Rabbit compared to that of human.[10–12] The study proto-
col and animal manipulations were conducted as per the
approval of the Ethical Committee of College of Pharmacy,
Tanta University (Approval number, 208013).
Preparation of drug solution for intestinal
perfusion
The perfusion solutions containing 37.5 lg/ml ranitidine
HCl were prepared in normal saline in absence and pres-
ence of piperine (28.5 lg/ml). This was achieved with the
aid of bath sonication. The drug concentration was selected
taking into consideration the daily dose of ranitidine
HCl and the average fluid input into the gastrointesti-
nal tract.[12]
Segment preparation
The surgical procedures and preparation of the target seg-
ment were conducted based on the well established
methodology.[10–12] Briefly, Rabbits were allowed a free
access to water with complete restriction of food for the
night before the experiment. On the day of the study the
rabbit was anaesthetized using chloropromazine HCl injec-
tion (2 mg/kg) as muscle relaxant with ketamine HCl being
Shimaa M. Ashmawy et al.
used as the main anaesthetic. Ketamine HCl was injected
15 min after the muscle relaxant and was delivered in two
successive doses (45 mg/kg) at 15 min intervals. Smaller
dose of the anaesthetic (25 mg/kg) was injected if required.
The anaesthetized rabbit is mounted on a thermostated
matrix with the animal being in a supine position. Abdomi-
nal hair was removed the skin cleaned before making a 6–
8 cm midline abdominal incision. The target intestinal seg-
ment was exposed and the required length was cannulated
from both sides after tying with surgical thread. This iso-
lates the segment and eliminates the effect of food while
maintaining the viability and blood supply of the segment.
The cannulated segment was cleaned using warm saline.
The measured length depended on the target segment with
length being 15, 30, 30 and 10 cm for duodenum, jejunum,
ileum and ascending colon, respectively. The perfusate was
pumped at constant rate (0.19 ml/min) for 2 h during
which the perfusate was collected at 10 min intervals.
Pumping was achieved using Harvard-22, constant rate
perfusion pump obtained from Harvard Apparatus (Millis,
MA, USA). The net weight of each perfusate sample was
recorded. This was used in estimation of the net water flux
which is determined from the difference between the
expected sample volume and the recorded sample volume.
The concentration of ranitidine HCl in the perfusate sam-
ples was quantified using the developed HPLC method.
This involved centrifugation of the perfusate samples and
loading of known volume of the perfusate sample into
tubes previously spiked with the specified amount of the
internal standard. This was subjected to vortex mixing
before loading into the HPLC vials.
Data analysis
The in situ intestinal perfusion study generates useful data
including the permeation parameters and water flux. These
data have been manipulated to predict the mechanism of
drug transfer through intestinal membrane. Detailed meth-
ods for calculation of these parameters and analysis of data
are available in literature reports.[11,12] The proceeding sec-
tions will present a briefing on this.
Absorptive clearance. The concentration of the ranitidine
HCl recorded in each sample was corrected with respect to
the net water flux to produce the actual amount of drug
remaining in each sample (Cout). The ratio between Cout
and the amount of drug perfused Cin was used to calculate
the fraction of drug remaining after perfusion. The fraction
remaining at steady state {(Cout/Cin)ss} was taken from the
average of the fractions remaining in samples collected dur-
ing the second hour of perfusion. This was used to calculate
the absorptive clearance (PeA) expressed as ml/min using
the following equation in which A represents the effective
© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
A new method for p-gp assessment
surface area (cm2), Pe denotes the apparent permeability
coefficient (cm/min) and Q is the average flow rate of the
perfusate through the target segment (ml/min)
PeA ¼ Q  lnfCðoutÞ =CðinÞ gss ð1Þ
The fraction of the drug absorbed at steady state (Fa)
was calculated using the following equation.
Fa ¼ 1  fCðoutÞ =CðinÞ g ss ¼ 1  expðPeA=QÞ ð2Þ
The length of the intestine remaining after complete drug
absorption is described as the anatomical reserve length
(ARL) and was calculated from the following equation in
which L* is the anatomical length of given intestinal seg-
ment and l* is the intestinal length along which complete
absorption is achieved. These lengths were expressed in
centimetres.
ARL ¼ ðL Þ  ðl Þ ð3Þ
Practically, it is difficult for the solute to disappear com-
pletely form the intestinal lumen especially in a logarithmic
situation. Accordingly, a 5% fraction was assumed to
remain with 95% absorption being taken as an approximate
reflection of complete absorption. Taking this into consid-
eration the length required for 95% absorption (L95%) was
calculated using the following equation.

0:05 ¼ expfðPeA: l Þ=Qg
ð4Þ
where PeA is absorptive clearance normalized to length and
l* is L95% for the given drug.
Effect of solvent drag on intestinal absorptive clearance. To
investigate the pathway of drug transport across the intesti-
nal membrane it was essential to monitor the drug absorp-
tion as a function of water flux. This was achieved by
plotting the PeA as a function of net water flux Jw (ml/min)
which was calculated from the difference between the vol-
ume of the perfusate pumped during a given time period
(Qin) and the volume of perfusate recovered from the
segment during this time (Qout). This is simplified by the
following equation.
Jw ¼ Qin  Qout ð5Þ
The rate of drug absorption (Js) which is calculated in
lg/min depends on the contribution of diffusive and con-
vective processes. The net amount of drug absorbed per
unit time can by driven from the following equation is then
given by[21,22]:
Js ¼ Ks ðC  Cp Þ þ [s Jw C ð6Þ
where the diffusive process is described by [Ks (C-Cp)] in
which Ks is the diffusive permeability coefficient of the
drug. The concentration of the drug in the intestinal lumen
is expressed as C with that in the plasma being defined as
3
A new method for p-gp assessment
Cp. and the convective process is represented by (Øs Jw C)
in which Øs is the sieving coefficient of drug. Equation (6)
can be approximated to Equation (7) at steady state, taking
into consideration the existence of sink conditions in
blood.
Jss ¼ DAKp=DxðCss Þ þ [s Jw ðCss Þ ð7Þ
where Jss is the steady state solute flux (lg/min), D is the
diffusion coefficient of the drug, A is the effective surface
area of drug absorption Kp is the (octanol/water) partition
coefficient of the compound. Dx is the path length. Css is
the length averaged steady state concentration of the solute
in the lumen (lg/ml). Rearrangement of Equation (7)
gives:
Jss =Css ¼ DAKp=Dx þ [s Jw ð8Þ
The term Jss/Css represents the overall PeA of the given
solute (ml/min) which is achieved via different permeation
pathways and can be practically calculated as permeability
surface area product, PeA.
Statistical analysis
The calculated membrane transport parameters were sub-
jected to statistical treatment to test for the significance of
the recorded differences. This employed Kruskal–Wallis test
with multiple pairwise comparisons using the Conover-
Iman procedure to compare between individual segments
and groups. P-value <0.05 was considered significant.
Results
Membrane transport parameters of
ranitidine HCl in rabbit intestine
The intestinal perfusion study involved calculation of the
membrane transport parameters at steady state. The last six
samples were taken as the samples corresponding to the
steady state. Figure 1 presents a plot of the PeA per unit
length as a function of time. This plot confirms the steady
state pattern of the data. Table 1 presents the membrane
transport parameters of ranitidine HCl after perfusion of
its aqueous solution through different intestinal segments
of rabbit. The calculated membrane transport parameters
reflect the dependence of the intestinal absorption of rani-
tidine HCl on the absorption site. The extent of drug
absorption decreases as we move distally through the small
intestine before increasing again at the ascending colon.
This is reflected from the PeA per unit length which was
ranked as duodenum > jejunum > ileum < ascending
colon. Statistical analysis revealed that the existence of sig-
nificant differences among different segments according to
the above rank (P < 0.05). This pattern was further
4 © 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
Shimaa M. Ashmawy et al.
Figure 1 The absorptive clearance of ranitidine HCl normalized to
intestinal length (PeA/L) during the perfusion of different anatomical
sites of rabbit intestine. (Data at steady state are presented as
mean + SEM, n = 3).
reflected from the values of L95 which showed as opposite
ranking with the differences being significant (Table 1).
The recorded L95 value in any of the tested segment was
significantly longer than the actual length of the segment
indicating incomplete absorption of the drug after oral
administration. The incomplete absorption is even clearer
from the recorded negative values for the ARL (Table 1).
The water flux normalized to segment length was also
monitored. The results indicated nonsignificant differences
(P > 0.05) between different segments of the small intestine
with respect to the water flux. With respect to the colon
there was a significant increase in the water flux with water
flux approaching 3-fold that is recorded in the tested small
intestinal segments (Table 1).
The effect of net water flux on the PeA of ranitidine HCl
was assessed. Figure 2 shows a plot of the PeA of ranitidine
HCl from different intestinal segments as a function of the
net water flux, both normalized to the segment length. The
regression parameters of these plots are presented in
Table 2. The regression data indicated that the slope of the
plots was significantly different from zero in cases of duo-
denum, jejunum and ascending colon. The slope of the plot
was not significantly different from zero in case of the
ileum. The intercept of the line passing through the data
was used to calculate the relative contribution of the tran-
scellular and paracellular pathways in the overall absorp-
tion of ranitidine HCl. For the duodenum 52.9% of the
absorbed drug was due to the transcellular route with com-
parable contribution being shown in case the jejunum in
which 53.8% of the PeA was due to transcellular route. This
route was responsible for 62.5% of the PeA in the ileum.
For the colon the absorption was mainly via the paracellu-
lar pathway which was responsible for absorption of 92.3%
of the amount of drug absorbed (Table 2, Figure 2).
Shimaa M. Ashmawy et al. A new method for p-gp assessment

Table 1 Membrane transport parameters of ranitidine HCl after perfusion through rabbit intestinal segments in absence and presence of
piperine

Segment PeA (ml/min) Rout/Rin PeA/L (ml/min.cm) l*(L95%) (cm) ARL (cm) JW (ml/min) JW/L (ml/min.cm)

Control
Duodenum 0.0241 (0.0020) 0.8734 (0.0120) 0.0017 (0.0001) 370.38 (38.45) 350.38 0.0189 (0.0058) 0.0013 (0.0004)
Jejunum 0.0380 (0.0019) 0.8023 (0.0132) 0.0013 (0.0001) 538.01 (37.59) 418.01 0.0382 (0.0138) 0.0014 (0.0005)
Ileum 0.0237 (0.0023) 0.8708 (0.0106) 0.0008 (0.0001) 2432.76 (720.38) 2372.76 0.0403 (0.0101) 0.0014 (0.0003)
Colon 0.0369 (0.0015) 0.8065 (0.0065) 0.0039 (0.0003) 146.21 (10.57) 131.21 0.0383 (0.0011) 0.0040 (0.0002)
Ranitidine HCl with piperine
Duodenum 0.0228 (0.0025) 0.8742 (0.0144) 0.0016 (0.0002) 597.41 (139.1) 577.41 0.0129 (0.0014) 0.0009 (0.0001)
Jejunum 0.0708 (0.0013) 0.6735 (0.0027) 0.0024 (0.0000) 326.10 (28.30) 206.10 0.0473 (0.0156) 0.0016 (0.0005)
Ileum 0.0476 (0.0073) 0.7546 (0.0288) 0.0017 (0.0002) 346.83 (30.571) 286.83 0.0480 (0.0106) 0.0018 (0.0004)
Colon 0.0382 (0.0030) 0.8047 (0.0120) 0.0036 (0.0002) 270.08 (62.98) 255.08 0.0322 (0.0040) 0.0030 (0.0004)
Values between brackets are SEM, n = 3. PeA is the overall absorptive clearance, Rout/Rin is the fraction remaining to be absorbed, PeA/L is the
absorptive clearance per unit length, L95% is the length required for 95% absorption, ARL is the anatomical reserve length which is the length of
intestine remaining after complete drug absorption, JW is the water flux and JW/L is the water flux normalized to the segment length.

maintaining the circulatory blood flow and tissue viabil-

Effect of piperine on intestinal absorption
of ranitidine HCl in situ
Table 1 presents the membrane transport parameters of
ranitidine HCl after co-perfusion with piperine trough dif-
ferent segments of rabbit intestine. Perfusion of piperine
along with ranitidine HCl modulated the membrane trans-
port parameters of ranitidine HCl. This was manifested as
significant increase (P < 0.05) in the PeA per unit length
(PeA/L) in case of the jejunum and ileum compared with
the corresponding values recorded for the drug in absence
of piperine. There was no significant change (P > 0.05) in
the PeA/L after coperfusion of piperine with ranitidine HCl
through the duodenum and the colon (Table 1). The same
trend was manifested in the L95 values which were reduced
significantly (P < 0.05) after coperfusion of piperine with
ranitidine HCl in case of jejunum and ileum compared
with the corresponding values recorded after perfusion of
ranitidine HCl alone (Table 1).
With regard to the intestinal transport pathway of rani-
tidine HCl, coperfusion of piperine increased the percent-
age contribution of the transcellular route from 62.5 to
76.5% in case of the ileum. There were no changes in the
relative contribution of the transport pathways in other
segments after coperfusion of piperine (Table 2, Figure 3).
Discussion
The aim of this work was to probe the feasibility of moni-
toring site dependent PeA of drugs as a possible tool to
identify the role of efflux transporters in intestinal absorp-
tion of drug. This was achieved using in situ intestinal
absorption technique with rabbit being used as model ani-
mal. The selection of the technique was based on its sim-
plicity, elimination of the effect of food, possibility of
investigating regional difference in drug absorption while
ity.[12] The selection of rabbit depended on the physiologi-
cal similarity between this animal and human intestine
with respect to surface area change from one location to
another.[23,24] Ranitidine HCl was selected on the base of
the published data which reflected the influence of P-gp
efflux transporters on its intestinal absorption.[19] The per-
fusion solution contained ranitidine HCl at a concentration
of 37.5 lg/ml. This concentration was calculated based on
the daily dose and average fluid intake and influx into the
GIT. This principle is well-documented in literature to
investigate the in situ intestinal absorption of drugs.[12]
The results of intestinal perfusion of ranitidine HCl
reflected its incomplete absorption as shown from the calcu-
lated L95 and ARL values. This incomplete absorption com-
plies with the reported poor bioavailability (50%) after oral
administration.[25,26] In addition to the incomplete absorp-
tion, the PeA of ranitidine HCl depended on the absorption
site with the absorption from different segments being in
the order of duodenum > jejunum > ileum < ascending
colon. This order can be explained taking into consideration
various factors. The first possible reason for this order can
be due to the difference in the surface area available for drug
absorption at different anatomical site. Considering this
parameter the surface area can be ranked as jejunum >
duodenum > ileum > colon.[27,28] This rank order does
not correlate well with the recorded rank of the PeA from
the tested anatomical sites. Accordingly, the surface area dif-
ference cannot be considered as the main factor contribut-
ing to site dependent PeA of ranitidine HCl. This is
particularly clear taking into consideration the higher PeA
per unit length and lower L95 recorded in case of the colon.
The relative contribution of diffusive and convective
absorption processes to drug absorption can be consid-
ered in explaining the regional differences in intestinal
absorption of the drug. The recorded results reflected

© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–** 5
A new method for p-gp assessment Shimaa M. Ashmawy et al.

Figure 2 Absorptive clearance of ranitidine HCl as a function of water flux in different intestinal segments: (a) duodenum, (b) jejunum, (c) ileum
and (d) ascending colon. Parameters are normalized to segment length.

Table 2 Effect of water flux (ml/min.cm) on the overall absorptive clearance (ml/min.cm) of Ranitidine HCl in absence and presence of piperine:
Regression parameters are obtained by fitting data to Equation (8)

Regression parameters Duodenum Jejunum Ileum Ascending colon

Control
Intercept (DAKp/Δx) 0.0009* (0.0002) 0.0007* (0.0003) 0.0005** (0.0003) 0.0003** (0.0007)
Slope (∅) 0.5647* (0.1191) 0.4513* (0.1539) 0.2523** (0.1784) 0.8883* (0.1694)
% Transcellular 52.9 53.8 62.5 7.7
% Paracellular 47.1 46.2 37.5 92.3
Ranitidine HCl with piperine
Intercept (DAKp/Δx) 0.0007* (0.0003) 0.0012* (0.0003) 0.0013* (0.0004) 0.0009** (0.0005)
Slope (∅) 0.944* (0.2527) 0.712* (0.1348) 0.216** (0.2130) 0.874* (0.1474)
% Transcellular 43.8 50.0 76.5 25.0
% Paracellular 56.2 50.0 23.5 75.0
Values in parentheses are  the standard error values, n = 3. (DAKp/Δx) is the permeability coefficient and (∅s) is the sieving coefficient. *Signifi-
cantly different from zero (P < 0.05). **Not significantly different from zero (P > 0.05).

comparable contribution of the two pathways in rani-
tidine HCl absorption from small intestinal segments
with colonic absorption being mainly through convective
pathway. This was indicated from the relationship
between the PeA and net water flux, both normalized to
segment length. This finding is expected taking into con-
sideration the polar nature of ranitidine HCl and the
reported water absorption capacity of different intestinal
segments.[29–31] Accepting these findings, ranitidine HCl
absorption can be affected by any factor affecting water
flux with those affecting transcellular absorption domi-
nate in the small intestinal absorption. Accordingly, the
effect of P-gp efflux can be a determining factor in the
overall PeA of ranitidine HCl. This supposition is sup-
ported by previous investigations on ranitidine HCl
which suggested a significant role for p-gp efflux in
reduced oral absorption of the drug.[17–20] Studying the
regional distribution of P-gp in intestinal segments

6 © 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
Shimaa M. Ashmawy et al. A new method for p-gp assessment

Figure 3 Absorptive clearance of ranitidine HCl in presence of piperine as a function of water flux in different intestinal segments: (a) duode-
num, (b) jejunum, (c) ileum and (d) ascending colon. Parameters are normalized to segment length.

revealed that its expression is site dependent. The magni-
tude of expression is ranked as ileum > jejunum > duo-
denum.[3,32] This rank order is the reverse of the rank
order of the PeA of ranitidine HCl which means that
drug absorption is reduced in segments containing high
expression of P-gp transporters. This suggests that P-gp
transporters play a major role in determining regional
difference in the absorption of ranitidine HCl. Taking
the findings of the current study together with the con-
firmed role of P-gp efflux in oral absorption of ranitidine
HCl, the regional absorption of drugs may be taken as
an indirect indication for the role of P-gp in its intestinal
absorption. This is of particular importance if the
recorded difference was in the small intestinal segments
in which transcellular pathway play significant role in
drug transport. It is important to highlight that existence
of P-gp transporters in the large intestine has been
noticed mainly in the central portion, but the absolute
amount much less than that present in the small intes-
tine.[16] For the current drug the absorption of ranitidine
HCl from the ascending colon was mediated by the con-
vective process with the role of water flux dominating.
To further confirm the correlation between regional
differences in drug absorption and the role of P-gp efflux
transporters, ranitidine HCl was perfused simultaneously
with piperine, a known P-gp inhibitor. The results
reflected significant changes in the membrane transport
parameters of ranitidine HCl in presence of piperine
compared with perfusion of the drug alone (Table 1).
This change depended on the site of absorption with the
PeA increasing in case of the jejunum and ileum. This
increase suggests that the inhibitory effect of piperine on
the P-gp transporters was a factor in the enhanced
absorption of ranitidine HCl and can suggest that the
regional difference in absorption of ranitidine HCl can
be explained on the base of the relative extent of expres-
sion of the efflux transporter in each segment. This sup-
position is supported by the previous findings which
classified piperine as an inhibitor for intestinal efflux of
drugs,[33–37] in addition to the published work on the
role of P-gp efflux in reduced intestinal absorption of
ranitidine HCl.[17–20] Considering the recorded effect of
piperine together with relative distribution of efflux
transporters in different intestinal segments, regional dif-
ference in intestinal drug absorption can be taken as a
measure for the potential role of efflux transporters in
intestinal absorption of given compounds. However, fur-
ther investigations are required using different drug
molecules. This can provide the pharmaceutical formula-
tor with a reliable method for identification of potential

© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–** 7
A new method for p-gp assessment Shimaa M. Ashmawy et al.

P-gp substrates. This identification can allow for opti-
mization of the absorption of P-gp substrates. The tech-
nique can be extended to identify inert P-gp inhibitors
which can be used as pharmaceutical excipients to
enhance the intestinal absorption of drugs suffering from
P-gp efflux. Optimization of intestinal absorption can
enhance oral bioavailability providing a chance for reduc-
tion in the dose and minimization of the side effects.
coperfusion of the drug with P-gp inhibitor resulted in
significant increase in drug absorption from segments
expressing the P-gp efflux transporters. The study thus
highlighted the role of P-gp transporters in determining
regional difference in intestinal absorption of ranitidine
HCl. Accordingly, the regional absorption of drugs may
be taken as an indirect indication for the role of P-gp in
intestinal absorption.

Conclusion Declarations
The regional difference in rabbit intestinal absorption of
ranitidine HCl correlated with the magnitude of expres- Conflict of interest
sion of P-gp transporters in each segment with drug The Author(s) declare(s) that they have no conflicts of
absorption reducing in segments having greater extent of interest to disclose.
P-gp expression. This was further confirmed by

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