University of Groningen Drug transport and transport ......transporters, P-gp plays a key role due to its broad substrate specificity and high expression level. Furthermore, since

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Drug transport and transport-metabolism interplay in the human and rat intestineLi, Ming

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21 Rat Precision-Cut Intestinal Slices to Study P-gp Activity

and the Potency of its Inhibitors Ex Vivo

2

Chapter 2

Rat Precision-Cut Intestinal Slices

to Study P-gp Activity and the Potency of its Inhibitors

Ex Vivo

Ming Li, *Inge A.M. de Graaf, Marina H. de Jager, Geny M. M. Groothuis

Toxicology in vitro. 2015; 29(5): 1070–1078

Affiliations:

Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713

AV Groningen, the Netherlands

Corresponding author:

Inge A. M. de Graaf

22 Chapter 2

Abstract

Rat Precision-Cut Intestinal Slices (PCIS) were evaluated as ex vivo model to study the

regional gradient of P-gp activity, and to investigate whether the rank order of inhibitory

potency of P-gp inhibitors can be correctly reproduced in this model with more accurate IC50

values than with current in vitro models. PCIS were prepared from small intestine (duodenum,

jejunum, ileum) and colon. Rhodamine 123 (R123) was used as P-gp substrate, while

verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 were employed

as P-gp inhibitors. Increase in tissue accumulation of R123 in the presence of the inhibitors

was considered as an indication of the inhibitory effect. The P-gp inhibitors increased the

tissue accumulation of R123 in a concentration dependent manner. Fluorescence microscopy

elucidated that this increase occurred predominantly in the enterocytes. The rank order of the

corresponding IC50 values agreed well with reported values from cell lines expressing rat P-gp.

The activity of and inhibitory effects on P-gp were significantly higher in ileum compared to

the other regions. These data suggest that rat PCIS are a reliable ex vivo model to study the

activity of intestinal P-gp and the inhibitory effect of drugs. PCIS have potential as ex vivo

model for the prediction of transporter-mediated drug-drug interactions.

Keywords P-glycoprotein; IC50; P-gp inhibitor; precision-cut intestinal slices; ex vivo



2

1. Introduction

The intestine is the ma in location for drug absorption after oral administration. Its role

in drug metabolism, excretion and toxicity has been intens ive ly studied and is well

acknowledged in the last decade [1-3]. Although many drugs are ma inly absorbed by

passive diffus ion, intestinal transporters are also invo lved in this process. Located on

the apical membrane of intestina l epithe lial ce lls, influx transporters, such as members

of the solute carrier (SLC) super-family, facilitate intestina l absorption. Efflux

transporters on the apical membrane of the enterocytes, mainly belonging to the

ATP-bind ing cassette (ABC) family, excrete their substrates from the cells into the

intestina l lumen, thereby lowering the intracellular concentration and decreasing

intestina l absorption [4-6]. Thus, this influence of drug transporters on the oral

absorption must be taken into account in drug development. Particular ly the apical

efflux transporters with broad substrate specific ity limit absorption thereby exerting a

large impact on bioavailability [7, 8]. The three major types of ABC transporters

expressed on the apical side of the epithelia l cells, namely mult idrug resistance protein

(MDR1/P-gp), mult idrug resistance- associated protein 2 (MRP2) and breast cancer

resistance protein (BCRP), actively excrete their substrates, including drugs, back into

the gut lumen and thus form a protective barrier in the intestine [5, 7]. Among these

transporters, P-gp plays a key role due to its broad substrate specific ity and high

expression level. Furthermore, since many drugs are P-gp substrates and/or inhibitors,

drug-drug interactions (DDIs) related to P-gp activity occur frequently [9, 10]. Since

P-gp activity influences both the intracellular concentration of xenobiotics and its

metabolites in the intestine but also determines the systemic exposure to these

compounds, prediction of inhib itory potency of (new) drugs is highly important for the

safety assessment of P-gp substrates. Furthermore, it is important to take into account

the regional d ifferences in P-gp expression and activity in the various parts of the

intestine, because this is an important determinant of local exposure to xenobiotics.

To study the activity of intestina l transporters, several in v itro models have been

developed, such as membrane vesicles, cell lines, everted gut sac, intestinal perfus ion

and Ussing chamber [4, 11-13]. However, none of these models can fully represent the

properties of the intestine with respect to the in v ivo gradient of expression along the

length of the intestine, and at the same time, serve as a fast and effic ient screening

method. To meet these challenges, we invest igated whether the rat precision-cut

24 Chapter 2

intestina l slices (PCIS) model could be used to study the activity of intest ina l

transporters. Precisely sliced from the intest ine immed iately after harvesting the tissue,

PCIS represent an ex v ivo mini-organ model which contains all types of intestinal cells

in the ir natura l environment, i.e. in v ivo 3D structure with intact intercellular and

cell-matr ix interactions. They have been successfully used in the investigat ion of drug

metabolism and toxicity in the intestine [14-20]. The localizat ion along the length of

the intestine and the regulat ion of some of the transporters were studied on mRNA

leve l before [21]. To date, studies on the functional activity of these transporters ex

v ivo is limited to the study of Possidente et al. who investigated the interactions of

xenobiotics with Mrp2 and P-gp in rat PCIS with calcein-AM as a probe and concluded

that rat PCIS are a reliable system to study interaction of xenobiotics with these

transporters [22]. However in this paper only jejunum slices were studied and

information on the activity in the different regions of the intestine was lacking. In

addition the cells involved in the accumulation of the substrates were not identified.

The aims of the present study were: (1) to verify the applicat ion of rat PCIS to the study

the activity o f rat intest ina l P-gp using a P-gp-specific substrate; (2) to identify the

cells invo lved (3) invest igate the intestinal regional difference with respect to P-gp

inhib it ion; and (4) to verify whether rat PCIS correctly reflect the inhibitory potency,

i.e. rank order and IC 5 0 values, of different P-gp inhibitors in the rat intestine ex v ivo;

In this study, P-gp was chosen as the targeted transporter for its key role in efflux

transport in the intestine. R123 was selected as P-gp substrate, because it is only

excreted by P-gp, but not metabolized by CYP-enzymes, as that might interfere with

the transport results [23]. Moreover, it is easy to detect with high sensit ivity due to its

intensive fluorescence [24], which also makes it possible to identify by microscopy the

increased accumulat ion of this substrate in the presence of a P-gp inhibitor. Two

strong and specific third generation P-gp inhib itors, CP100356 and PSC833, were

employed [25], as well as several drugs well known as classical P -gp inhibitors,

namely verapamil, cyclosporine A, ketoconazole, and quinidine [26-28].

2. Materials and Methods

2.1. Chemicals

Rhodamine 123, verapamil hydrochloride, cyclosporine A, ketoconazole, quinidine and low

gelling temperature agarose (type VII-A) were purchased from Sigma-Aldrich (USA).

PSC833 and CP100356 were from Tocris Bioscience (UK). Gentamicin, Williams Medium E



2

(WME) with glutamax-I, and amphotericin B (fungizone) solution were obtained from

Invitrogen (UK). HEPES was obtained from MP Biomedicals (Germany).

The stock solutions were prepared in ethanol (R123), methanol (quinidine) or DMSO

(verapamil, cyclosporine A, ketoconazole, PSC833 and CP100356).

2.2. Animals

Male Wistar (HsdCpb:WU) rats weighing ca. 300 - 350 g were purchased from Harlan (Horst,

the Netherlands). Rats were housed in a temperature- and humidity- controlled room on a

12/12 h light/dark cycle with free access to food and tap water, and acclimatized at least 7

days before use. All the animal experiments were approved by the animal ethical committee

of the University of Groningen.

2.3. Preparation and incubation of rat precision-cut intestinal slices

Precision-cut intestinal slices were prepared from the three different regions of the rat small

intestine and from the colon as previously described [15, 29]. Briefly, the rat was anesthetized

by isofurane around 9 am. After the small intestine and colon were excised and put into

ice-cold, oxygenated Krebs-Henseleit buffer (containing 10 mM HEPES and 25 mM

D-glucose, pH 7.4), the rat was sacrificed by bleeding by dissection of aorta. The whole

intestine was divided into four parts: duodenum, jejunum, ileum and colon (duodenum was

the segment between 2 and 12 cm and jejunum was between 20 and 40 cm from the pylorus,

while ileum was the segment of the last 20 cm before the ileocecal junction and colon was the

segment after the ileocecal junction). A 3-cm-segment was cut from the required part and

flushed with ice-cold buffer. With one end tightly closed, it was filled with 3 % (w/v) agarose

solution in 0.9 % NaCl (37 °C) and then cooled in ice-cold buffer, allowing the agarose

solution to gel. Subsequently, the filled segment was embedded in 37 °C agarose solution in a

precooled tissue embedding unit (Alabama R&D, USA). After the agarose solution had gelled,

precision-cut slices (thickness about 300 µm and wet weight about 3-5 mg) were made using

a Krumdieck tissue slicer (Alabama R&D, USA).

The slices were incubated individually in a 12-well culture plate (Greiner Bio-One GmbH,

Austria) with 1.3 ml WME (with glutamax-I), supplemented with D-glucose, gentamicin and

amphotericin B (final concentration: 25 mM, 50 µg/ml, and 2.5 µg/ml, respectively). The

culture plates were placed in plastic boxes in a pre-warmed cabinet (37 °C) under humidified

carbogen (95 % O2 and 5 % CO2) and shaken back and forth approximately 90 times per

26 Chapter 2

minute (Reciprocating Shaker 3018, Gesellschaft für Labortechnik GmbH, Germany).

2.4. Viability of the intestinal slice

Intracellular ATP levels in the intestinal slices were evaluated to judge the overall viability of

the tissue during incubation in parallel groups [17, 30]. After the intestine was excised from

the abdomen, three tiny pieces were cut and stored as control of untreated tissue, which was

used to evaluate the quality of intestine as it is the source of intestinal slices. In addition, three

freshly prepared slices were stored as controls of 0 h. ATP content was measured in slices

after 5 hours of incubation with or without R123 (highest concentration: 10 µM). Furthermore,

to estimate the viability of the slices during incubation with R123 and inhibitors, the ATP

levels of slices co-incubated with a P-gp inhibitor and/or R123 were measured and compared

with the levels in control groups. All the ATP samples were snap-frozen in 1 ml of

preservation solution (70 % ethanol containing 2 mM EDTA, pH 10.9) in liquid N2

immediately after sampling and then stored at -80 °C until further analysis. ATP was analyzed

by using the ATP Bioluminescence Assay Kit CLS II (Roche Applied Sciences, Germany) and

by measuring the luminescence in a Spectramax micro-plate reader (Molecular Devices, USA)

as described before [17, 31].

2.5. Kinetics of R123 uptake

After slices were pre-incubated for 30 min, R123 was added to the incubation medium. To

study the time-course of the uptake, slices were harvested at 15, 30, 60 and 120 minutes after

the addition of the substrate, respectively. Furthermore, the effect of the medium

concentration of R123 on its cellular accumulation was tested by using 0.5, 2 and 10 µM

R123 that were chosen as low, medium and high concentration based on literature studies. To

select a suitable concentration to show the P-gp inhibition effect, slices were also incubated

with these R123 concentrations in the absence or presence of CP100356 (2 µM) for 120 min.

After incubation, slices were collected and rinsed in blank PBS for 5 min and stored at -20 °C

until further analysis.

2.6. Inhibition study

To study the inhibitory effects of P-gp inhibitors, a range of concentrations was used to be

able to depict the inhibition vs concentration curve and calculate the IC50 value. Therefore,

several concentrations below and above effective concentration of inhibitors were employed

for each inhibitor. The inhibitors were pre-incubated for 30 minutes before adding R123 to



2

allow sufficient uptake to ensure the presence of the inhibitors in the enterocytes at the

moment the substrate was added. The incubation time of 2 hours and R123 concentration of 2

µM were selected based on the results of kinetics study above.

To study the differences in P-gp activity in the different intestinal regions, slices were

prepared from duodenum, jejunum, ileum and colon and pre- incubated for 30 min in the

absence or presence of inhibitor. Quinidine and CP100356 were employed as P-gp inhibitors

with final concentrations in the range of 0 - 200 µM and 0 - 5 µM, respectively. Thereafter,

R123 was added into each well, followed by 2-h incubation.

To compare the inhibitory potency of various P-gp inhibitors, slices from rat ileum were

incubated with verapamil (0 - 50 µM), cyclosporine A (0 - 20 µM), quinidine (0 - 200 µM),

ketoconazole (0 - 50 µM), PSC833 (0 - 2 µM) and CP100356 (0 - 5 µM) for 30 min,

whereafter R123 was added into each well, followed by 2-h incubation. Then, tissue samples

were harvested and stored as described above. The increase of tissue accumulation of R123

was considered as an indication of P-gp inhibition.

The final concentration of the solvents in culture medium was always lower than 1 %, which

did not influence the viability of the slice (evaluated by intracellular ATP, data not shown) and

the transport activity (compared to the control group without any of these solvent, data not

shown).

2.7. Intestinal localization of R123

Fluorescence microscopy was applied to check the localization of R123 in the intestinal

epithelial cells and to visualize the increase of its accumulation by the action of P-gp

inhibitors. Based on the inhibition study, quinidine and CP100356 were selected to represent a

weak and a strong P-gp inhibitor, respectively. The concentrations of inhibitor were selected

as such, that at these concentrations >50% inhibition occurred, i.e. quinidine (50 µM) and

CP100356 (2 µM). After pre- incubation for 30 min with or without P-gp inhibitor, intestinal

slices made from each region were incubated with R123 (final concentration: 2 μM) for 2 h.

Then the slices were washed in ice-cold PBS, embedded in Tissue-tek (3 slices in one core)

and snap-frozen in isopentane placed on dry ice within 30 s. They were stored at -80 °C until

sections of 8 μm were cut perpendicular to the surface of the slice in a Cryostat (CryostatTM

NX70, Thermo Scientific, USA) at -20°C. The sections were attached and dried on a glass

slide, and examined unmounted under a fluorescence microscope (Leica DM4000 B, Leica

Microsystems, Germany) with a +L5 filter (excitation 480/40 nm, emission 527/30 nm) by

28 Chapter 2

using a DC350FX digital camera with QWin software (Leica) at a fixed exposure time (1.2 s).

Thereafter, they were stained with hematoxylin and eosin as described previously [32].

Pictures were taken with light microscope (Olympus BX41, Olympus America Inc., USA) at

approximately the same areas where pictures of the fluorescence were made. A comparison

between these two groups of images was made to confirm the localization of R123 in the

intestine in the absence or presence of P-gp inhibitors.

2.8. R123 measurement

The slices were homogenized using a Mini-BeadBeater-8 (BioSpec, USA) with 200 µl blank

WME and 400 µl acetonitrile. After centrifugation for 5 min at 13000 rpm and 4 °C, 150 µl

supernatant was transferred into a 96-well plate, and the fluorescence of R123 was measured

with a fluorescence plate reader (Molecular Devices, USA) (excitation/emission wavelength:

485 nm / 530 nm). The intracellular content of R123 was calculated using a calibration curve

prepared in a homogenate of blank intestinal slices.

2.9. Protein determination

The pellet remaining after the ATP assay and the R123 measurement was dried overnight at

37 °C and dissolved in 200 µl of 5 M NaOH for 30 min. After dilution with H2O to 1 M

NaOH, the protein content of the samples was determined using the Bio-Rad DC Protein

Assay (Bio-Rad, Germany) with a calibration curve prepared from bovine serum albumin.

The protein content of each slice was used to normalize for the size variation of the intestinal

slices.

2.10. Statistical analysis

All the experiments were performed with at least three different rats and within each

experiment all incubations were carried out in triplicate and the results were expressed as

mean ± SEM. Two-tailed paired Student’s t-test was employed for a two-group comparison,

while one way ANOVA and two way ANOVA were used to compare multiple groups with one

factor and two factors, respectively. A difference of p



2

3. Results

3.1. Viability of the intestinal slices

As shown in Fig. 1, the ATP level in the control of untreated tissue was 2.7 nmol/mg protein

whereas the slices directly after preparation had an ATP content of 4.0 nmol/mg protein. After

incubation for 5 h, the ATP content decreased slightly, but not significantly by 12.5 %

compared to that in the fresh slices (0 h), until 3.5 nmol/mg protein. This level is comparable

with that in the untreated tissue and also with that in the fresh slices (P >0.05), indicating that

the intestinal slices are viable during a 5-hour incubation. In addition, no significant

difference in ATP content of the slice was observed when R123 (2 or 10 µM) was added,

suggesting that R123 did not influence the viability of the slice during incubation.

Furthermore, the slice ATP content after co- incubation for 3 h or 5 h with the P-gp inhibitors

at the highest concentration used for the inhibition study with or without R123 was found to

be retained at a similar level as that in control group (data not shown). This indicates that the

intestinal slices are also viable during the incubation with P-gp inhibitors.

Fig. 1 ATP content of the intestine (tissue untreated), fresh PCIS at the start of the incubation, and PCIS after 5 h

of incubation in the absence or presence of 2 and 10 µM of R123 (n=7 to 9). Data are presented as Mean ± SEM

of each group. One way ANOVA with Tukey’s comparison as post-hoc test was employed for the comparison

between every two groups, no statistical significance was found.

3.2. Intestinal localization of R123

In jejunum and ileum, the R123 content in the epithelial cells was very low, resulting in a faint,

30 Chapter 2

nearly invisible line, contrary to the bottom of villi, crypt cells and muscle layer where the

staining was more intense (Fig. 2 c&d). This is probably caused by the active efflux of R123

by P-gp which is mainly expressed in the mature epithelial cells on the villi, resulting in a

gradient of P-gp expression along the villus axis. In line with this, when P-gp was inhibited by

quinidine or CP100356, the fluorescence intensity of the epithelium lining clearly increased.

As shown in Fig. 2, the increase of fluorescence intensity of R123 in the jejunum and ileum

due to the presence of the P-gp inhibitor occurred predominantly in the villi and not in the

other intestinal structures. In contrast in duodenum and colon, the effect of P-gp inhibition

was less noticeable, as there was only a small increase of the fluorescence intensity, which

probably indicates that much less P-gp is expressed in these segments.

3.3. R123 uptake assays

3.3.1. Time-dependent uptake

To determine the time-course of R123 uptake during incubation, intestinal slices from ileum

were incubated with 0.5 or 2 µM R123 for 15, 30, 60 or 120 min. A relatively low R123

concentration was chosen in order to avoid saturation of P-gp efflux. As shown in Fig. 3a, the

R123 uptake in both concentrations was linear between 15 min and 120 min. The extrapolated

tissue concentration at t=0 min is probably due to the non-specific tissue binding of R123.

3.3.2. Concentration-dependent uptake and the effect of P-gp inhibition

Based on the results above, we investigated the effects of P-gp inhibition on accumulation of

R123 at different concentrations during 120 min of incubation (shown in Fig. 3b). When

incubated with R123 (0.5, 2 or 10 µM), the tissue accumulation of R123 increased linearly

with the R123 concentration (38.7 ± 10.3, 154.0 ± 33.9, and 797.4 ± 272.7 pmol/mg protein,

respectively). When P-gp efflux was blocked with CP100356, the R123 accumulation in the

tissue was enhanced to 77.9 ± 14.4, 330.0 ± 51.4, and 1556.8 ± 189.5 pmol/mg protein,

respectively, which was significant when the concentration of R123 was 2 µM. These data

indicate that the R123 uptake in PCIS is concentration dependent and its efflux by P-gp is not

saturated up to10 µM R123.



2

32 Chapter 2

Fig. 2 The localizat ion of R123 in the intestine (A, duodenum; B, jejunum; C, ileum; D, co lon) (left : no inhibitor;

middle: quin idine 50 µM; right: CP100356 2 µM). Pictures were taken at approximately the same areas of the

sections using the fluorescent microscope (upper panels) and the light microscope (lower panels). Scale bar =

100 µm.

Fig. 3a (left panel) The time course of R123 uptake in PCIS at 0.5 and 2 µM R123 (n=3). Results were

expressed as mean ± SEM. The fluorescence value of blank slice, which was very low and negligible, was

included by the calibration curves using an homogenate of untreated slices.

Fig. 3b (right panel) Concentration-dependent uptake of R123 and the effect of P-gp inhibition by CP100356 (2

µM) (n=3). Results were expressed as mean ± SEM. Two-tailed paired Student’s t-test was employed for

comparison between the control and CP100356 group at each R123 concentration . * indicates p



2

The regional difference of P-gp inhibition was studied with intestinal slices made from

duodenum, jejunum, ileum and colon co- incubated with R123 and at a range of concentrations

of quinidine or CP100356. As shown in Fig. 4a&b, the maximum effect of P-gp inhibition by

quinidine was highest in the ileum (approximately 2.5-fold increase) compared to the

duodenum, and jejunum (approximately 1.5-fold increase). No effect (CP100356) or highly

variable (quinidine) P-gp inhibition was shown for colon. However, the IC50 values of

quinidine and CP100356 were similar for each of the intestinal regions.

When the absolute concentrations of R123 (without inhibitor) in the slices of the different

regions are depicted it becomes clear that P-gp is differently expressed in the different regions

resulting in a lower tissue concentration of R123 in the ileum (high efflux activity) and a high

tissue concentration in the colon (low efflux activity) (Fig. 5). After P-gp efflux was inhibited

by either CP100356 or quinidine, R123 accumulation in ileum slices was significantly

enhanced, eliminating the regional differences in accumulation of the substrate caused by the

efflux pump.

Fig. 4 a&b Effects of P-gp inhibit ion by quinidine and CP 100356 on R123 accumulation in PCIS of the

different regions along the intestine. The results are expressed as mean ± SEM which are relat ive to the control

group (no inhibitor), n=3 for duodenum, jejunum and colon, n=6 for ileum (data for ileum include the data for

CP 100356 and quinidine obtained in the experiments where the six inhibitors were tested). NI, no inhibition .

3.4.2. Inhibitory potencies of various P-gp inhibitors

The inhibitory potency of six P-gp inhibitors on R123 accumulation in ileum was compared

using various concentrations of the inhibitors. As shown in Fig. 6, all six inhibitors enhanced

R123 accumulation in the PCIS concentration-dependently, indicating their inhibition of P-gp.

An approximately 2.5 to 3.0-fold increase of R123 accumulation at maximum inhibition was

observed. The inhibition response vs. concentration curves were used to calculate the

34 Chapter 2

corresponding IC50 values. The results clearly indicate that the potency of P-gp inhibition,

estimated by the IC50 value, varied greatly. PSC833 and CP100356, two potent P-gp inhibitors,

showed strong inhibition with IC50 values at 0.60 µM and 0.66 µM, respectively. The other

four classical P-gp inhibitors showed a lower potency of P-gp inhibition: quinidine (23.57

µM), cyclosporine A (2.34 µM), verapamil (5.21 µM), and ketoconazole (8.22 µM) (Fig. 6

and Table 1). The IC50 values of these inhibitors found for rat PCIS were generally higher

than those reported before for rat P-gp expressed in the LLC-PK1 cell line [27]. However, the

rank order was the same. In contrast, the rank order of inhibition efficacy was different for rat

and human P-gp, with respect to the rank order of ketoconazole and verapamil, indicating

species differences in P-gp substrate specificity [25].

Fig. 5 The influence of P-gp inhibit ion by quinid ine and CP100356 on R123 accumulation in PCIS of the

different intestinal regions. The results were expressed as mean ± SEM. N = 3 for duodenum, jejunum and colon,

n = 6 for ileum (data for ileum include the data for CP 100356 and quin idine obtained in the experiments where

the six inhibitors were tested). Two-way ANOVA with Bonferron i post-hoc test was used to compare mult iple

groups with two factors, i.e. reg ion and inhibitor treatment. In the control group, R123 accumulat ion was found

significantly d ifferent between ileum and colon, however, with P-gp inhibit ion, no regional difference in R123

accumulat ion was found. Besides, there was a significant increase of R123 accumulat ion after P -gp inhibit ion in

ileum slices. ns = not significant; ** significant with p



2

consortium [33]. In this review Caco-2 or P-gp-overexpressing polar ized epithelia l cell

lines were recommended as methods to screen for drug candidates that are substrates

and/or inhibitors of transporters, especially P -gp. However, it is generally known that

cell lines do not express the transporters at physiological leve ls nor can represent the

different regions of the intestine [34, 35]. The data of the present study show that the

PCIS model can be a better and more physiologically relevant a lternative for these c ell

lines.

Fig. 6 Effect of 6 different P-gp inhib itors on R123 accumulat ion in PCIS. The results are expressed as mean ±

SEM, which was relative to the control group (no inhibitor), n=3 for each inhibitor.

In the past decade we and others have developed PCIS as ex v ivo model to study drug

metabolism and toxic ity [15-20]. Because PCIS are prepared from fresh tissue, the

expression of transporters and metabolic enzymes is at physiologica l leve ls.

Furthermore it is possible to make >100 slices from each region of the intest ine within

one experiment, thus, the number of animals needed is largely reduced, which is in

good accordance with the 3Rs (Reduction, Replacement and Refinement). In previous

studies we have shown that PCIS remain viab le and retain their metabo lic activity for

at least 8 hours of cultur ing, which is substantia lly longer than other intact tissue

preparations [15-17]. However the only study on transport up to now was the study of

Possidente et al. showing the applicability of PCIS for transport studies, but without

36 Chapter 2

information on gradients along the length of the intestine and on the cellular

localization of the accumulated substrates.

Table 1 Comparison of IC50 values of the tested P-gp inhibitors and their rank order among different models

and species

P-gp inhibitor

Rat P-gp Human P-gp

rPCIS LLC-PK1 cell

line Caco-2 cell line MDCKII cell line

IC50

(µM) rank order

Ki

(µM) rank order

IC50 (µM) rank order

IC50 (µM) rank order

Ref. 1 Ref. 2 Ref. 3 Ref. 4 Ref. 5

PSC833 0.60 1--2

0.1 0.11 1--2

CP100356 0.66 1--2 0.1 1--2

Cyclosporine A 2.34 3 0.038 3 1.2 1.3 3--4 1.6 1.6 3

Verapamil 5.21 4 0.96 4 2.2 2.1 5--6 10.7 5.9 5

Ketoconazole 8.22 5 1.41 5 1.2 3--4 3.1 3.7 4

Quinidine 23.57 6 2.24 6 2.3 2.3 5--6 14.9 14.1 6

Ref.1: [27]; Ref.2: [25]; Ref.3: [36]; Ref.4: [28]; Ref.5: [26]

In the present study, we used R123 to study P-gp function along the length of the intestine and

to investigate inhibitory potency of drugs and to identify the cells involved in the transport of

the P-gp substrate. The accumulation of R123 in PCIS was linear until 120 min, and

concentration dependent (Fig. 3 a&b). Furthermore, inhibition of P-gp resulted in a further

increase of the intracellular concentration. This increase was mainly due to increase in R123

content of the enterocytes as shown by fluorescence microscopy (Fig. 2). Interestingly, it

appeared that the estimated average concentration of R123 in the tissue was 6-8 times higher

than the medium concentration after incubation for 2 h. This might be explained by the high

affinity of R123 to mitochondria and/or the formation of R123 micelles [37]. By this

intracellular compartmentalization of R123 the cytoplasmic concentration apparently remains

low, ensuring a driving force for passive diffusion into the cell even when the efflux of R123

is inhibited.

The intestine is a heterogeneous organ in which differences in structure and function

are prominent among its regions. Each of the intestinal efflux transporters has its

unique expression profile along the intest ine [5, 38]. However, it has not been studied up



2

to now to what extent these expression levels also represent functional differences. A

discrepancy between protein expression and transport activity can be anticipated as it has been

demonstrated that the transporters are not solely localized at the plasma membrane but also

intracellularly, such as in intracellular membrane vesicles [39]. In the present study, we used

slices from the different intestinal regions to study the regional differences in P-gp activity. It

should be noted that in vivo there are also other physiological differences between the

different intestinal regions that were not accounted for in the ex vivo studies. For example the

pH in the rat intestinal lumen is different in the different regions, but was kept the same (pH

7.4) in the ex vivo incubations. Nevertheless, the increasing gradient of P-gp activity from

jejunum to ileum and lower activity in colon was consistent with the reported P-gp expression

profile. The highest inhibition effect was found in the ileum compared to the duodenum and

jejunum (Fig. 4). Similarly, using the everted gut sac model, Yumoto et al. observed a higher

transport rate by P-gp in the ileum than in duodenum and jejunum with a similar fold

difference as found in the current study [40]. The inhibition effect was lowest in colon, which

is consistent with the lower P-gp abundance in human colon quantified by mass

spectrometry-based targeted proteomics [41]. The maximum inhibition effect of the P-gp

inhibitors was approximately 2 to 2.5 fold in ileum and 1.5 fold in the other regions, which is

similar to that in vivo [42, 43]. Fenner et al. summarized data from 123 clinical trials where

P-gp inhibitors were co-administrated with digoxin, and found that the AUC and Cmax

increased not more than 2 fold [42]. In contrast, with cell lines a 6 - 10 fold decrease in efflux

rate was commonly found [44]. This suggests that the slices ex vivo mimic the in vivo

situation better than the cell lines.

The inhibition potency of P-gp inhibitors, usually estimated by the IC50 value, varies in

different in vitro models, mainly because of the different intracellular concentrations of the

inhibitors and the different P-gp substrates used as well as different calculation methods [45].

On the other hand, the rank order of inhibition ability is expected to be the same among

models and different substrates [28, 46]. In the present study, the rank order of the IC50 values

of the used inhibitors agreed very well with published data on the K i of these compounds for

rat P-gp. Compared with human cell data, we found a difference in the rank order of

verapamil and quinidine, which is probably due to a species difference, which is in line with

the data of Suzuyama et al. who found different relative IC50 values for different animal

species using quinidine and verapamil as P-gp inhibitors [47]. Since the PCIS technique can

be applied to human intestine as well [17, 29], it will be very interesting to study the

38 Chapter 2

inhibitory potency of inhibitors in human PCIS prepared from human intestine. These

experiments are currently in progress.

Nevertheless, the absolute IC50 value is important for correct prediction of DDIs and

according to the FDA, if the ratio of the systemic steady-state Cmax, [I]1 to its in vitro

inhibitory potency (Ki or IC50), is ≥ 0.1 a clinical trial should be performed to indicate the risk

of a DDI for potential P-gp inhibitors. Since the absolute IC50 value is guiding for these

predictions, it is important that in vitro systems do not only rank the potency of possible P-gp

inhibitors correctly. If the in vitro IC50 values are lower than in vivo, the predicted values of

the ratios of [I]1/IC50 are too high. This might be one of the reasons why many false positive

interactions were found [48].

The IC50 values of P-gp inhibitors found in our study were substantially higher than found

with cell lines and P-gp overexpressing cells (Table 1). The cause of the different IC50 values

could be a different concentration at the target (the transporter) in the different systems. Since

target concentrations of substrate and inhibitors are influenced by metabolism and

influx/efflux in the target cells it can be expected that IC50 values are closer to in vivo when

predicted in a system where transporters and drug metabolizing enzymes are expressed at

physiological levels and original tissue/organ structure is preserved, such as in PCIS.

Therefore predictions for DDIs made with the PCIS are possibly more relevant for the in vivo

situation in the intestine than those obtained with cell lines.

In the FDA guidelines for DDI prediction, the influence of regional differences in the intestine

and the overexpression of P-gp in Caco-2 or other P-gp-expressing polarized epithelial cell

lines are ignored. However, the influence of inhibition on the absorption and as such on the

AUC of drugs is largely determined by P-gp expression and regional differences are therefore

relevant. As the majority of drugs is already largely absorbed in duodenum and jejunum

where P-gp expression is low, systems that highly express P-gp, such as Caco-2 cells might

overestimate the effect of P-gp inhibitors on AUC in vivo. Furthermore, it should be noted that

the local exposure to a P-gp substrate in the presence of an inhibitor is largely determined by

the local P-gp expression itself. The use of PCIS of different intestinal regions can reflect

these regional differences, and thus represent the physiology of the intestine to a larger extent.

In conclusion, the present study shows that rat PCIS are a reliable and efficient ex vivo model

to study the activity of intestinal transporters, and to assess the inhibitory potential of new

drugs in each of the different regions of the intestine. In addition we confirmed for the first

time that in the PCIS this inhibitory action is exerted in the enterocytes specifically. The IC 50

values of P-gp inhibitors measured ex vivo in PCIS show the same rank order as those



2

measured in cell lines, but the absolute values are much higher and expected to be closer to

the in vivo values. Therefore, the PCIS model has the potential to make a better prediction of

transporter-mediated DDIs in the intestine and the toxicity derived from this type of DDIs.

Furthermore, its future application in human intestinal slices could overcome difficulties in

extrapolation from experimental animals to humans.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

One of the authors, Ming Li, is very grateful to be granted by the China Scholarship Council

(CSC) for his PhD scholarship.

References

1. Fisher MB, Lab issiere G. The role of the intestine in drug metabolis m and pharmacokinetics: an industry

perspective. Current drug metabolism 2007 Oct;8(7):694-9.

2. Laffont CM, Toutain P-L, Alv inerie M, Bousquet-Mélou A. Intestinal secretion is a major route for parent

ivermectin elimination in the rat. Drug Metabolism and Disposition 2002 June 1, 2002;30(6):626-30.

3. Chosidow O, Delch ier J-C, Chaumette M-T, Wechsler J, Wolkenstein P, Bourgault I, et al. Intestinal

involvement in drug-induced toxic epidermal necrolysis. The Lancet 1991;337(8746):928.

4. Kis O, Zastre J, Hoque MT, Walms ley S, Bendayan R. Role of drug efflux and uptake transporters in

atazanavir intestinal permeability and drug-drug interactions. Pharm Res 2013;30(4):1050-64.

5. Takano M, Yumoto R, Murakami T. Expression and function of efflux drug transporters in the intestine.

Pharmacology & Therapeutics 2006;109(1–2):137-61.

6. Yokooji T, Yumoto R, Nagai J, Takano M, Murakami T. Role of intestinal efflux t ransporters in the

intestinal absorption of methotrexate in rats. Journal of Pharmacy and Pharmacology 2007;59(9):1263 -70.

7. Oostendorp RL, Beijnen JH, Schellens JHM. The biological and clinical role of d rug transporters at the

intestinal barrier. Cancer Treatment Reviews 2009;35(2):137-47.

8. Varma MV, Ambler CM, Ullah M, Rotter CJ, Sun H, Litchfield J, et al. Targeting intestinal transporters for

optimizing oral drug absorption. Current drug metabolism 2010 Nov;11(9):730-42.

9. König J, Müller F, Fromm MF. Transporters and drug-drug interactions: important determinants of drug

disposition and effects. Pharmacological Reviews 2013 July 1, 2013;65(3):944-66.

10. Kiv istö KT, Niemi M, Fromm MF. Functional interaction of intestinal CYP3A4 and P-g lycoprotein.

Fundamental & Clinical Pharmacology 2004;18(6):621-26.

11. Rozehnal V, Nakai D, Hoepner U, Fischer T, Kamiyama E, Takahashi M, et a l. Human s mall intestinal and

colonic tissue mounted in the Ussing chamber as a tool for characterizing the intestinal absorption of drugs.

European Journal of Pharmaceutical Sciences 2012;46(5):367-73.

40 Chapter 2

12. Ellis LCJ, Hawksworth GM, Weaver RJ. ATP-dependent transport of statins by human and rat MRP2/Mrp2.

Toxicology and Applied Pharmacology 2013;269(2):187-94.

13. Li M, Si L, Pan H, Rabba AK, Yan F, Qiu J, et al. Excipients enhance intestinal absorption of ganciclovir by

P-gp inhib ition: Assessed in vitro by everted gut sac and in situ by improved intestinal perfusion. International

Journal of Pharmaceutics 2011;403(1–2):37-45.

14. Groothuis GM, de Graaf IA. Precision-cut intestinal slices as in vitro tool for studies on drug metabolis m.

Current drug metabolism 2012;14(1):112-19.

15. van de Kerkhof EG, de Graaf IAM, de Jager MH, Meijer DKF, Groothuis GMM. Characterization of rat

small intestinal and colon precision-cut slices as an in vitro system for drug metabolis m and induction studies.

Drug Metabolism and Disposition 2005;33(11):1613-20.

16. van de Kerkhof EG, de Graaf IAM, de Jager MH, Groothuis GMM. Induction of phase I and II d rug

metabolism in rat small intestine and colon in vitro. Drug Metabolism and Disposition 2007;35(6):898-907.

17. van de Kerkhof EG, de Graaf IAM, Ungell A -LB, Groothuis GMM. Induction of metabolis m and transport

in human intestine: validation of precision-cut slices as a tool to study induction of drug metabolis m in human

intestine in vitro. Drug Metabolism and Disposition 2008;36(3):604-13.

18. Niu X, de Graaf IAM, Groothuis GMM. Evaluation of the intestinal toxicity and transport of xenobiotics

utilizing precision-cut slices. Xenobiotica 2013;43(1):73-83.

19. de Kanter R, Tuin A, van de Kerkhof E, Mart ignoni M, Draaisma AL, de Jager MH, et al. A new technique

for preparing precision-cut slices from small intestine and colon for drug biotransformation studies. Journal of

Pharmacological and Toxicological Methods 2005;51(1):65-72.

20. Martignoni M, Groothuis G, de Kanter R. Comparis on of mouse and rat cytochrome P450-mediated

metabolism in liver and intestine. Drug Metabolism and Disposition 2006;34(6):1047-54.

21. Khan AA, Chow ECY, Porte RJ, Pang KS, Groothuis GMM. The role of lithocholic acid in the regulation

of bile acid detoxication, synthesis, and transport proteins in rat and human intestine and liver slices. Toxicology

in Vitro 2011;25(1):80-90.

22. Possidente M, Dragoni S, Franco G, Gori M, Bertelli E, Teodori E, et al. Rat intestinal precision -cut slices

as an in v itro mode l to study xenobiotic interaction with t ransporters. European Journal of Pharmaceutics and

Biopharmaceutics 2011;79(2):343-48.

23. Sweatman TW, Seshadri R, Israel M. Metabolis m and elimination of rhodamine 123 in the rat. Cancer

Chemother Pharmacol 1990;27(3):205-10.

24. Forster S, Thumser AE, Hood SR, Plant N. Characterization of rhodamine-123 as a tracer dye for use in in

vitro drug transport assays. PloS one 2012;7(3):e33253.

25. Wandel C, B. Kim R, Kajiji S, Guengerich FP, Wilkinson GR, Wood AJJ. P-glycoprotein and cytochrome

P-450 3A inhibit ion: dissociation of inhibitory potencies. Cancer Research 1999 August 15,

1999;59(16):3944-48.

26. Keogh JP, Kunta JR. Development, validation and utility of an in vitro technique for assessment of potential

clin ical drug–drug interactions involving P-glycoprotein. European Journal of Pharmaceutical Sciences

2006;27(5):543-54.

27. Sugimoto H, Hirabayashi H, Kimura Y, Furuta A, Amano N, Moriwaki T. Quantitative investigation of the

impact of P-glycoprotein inhibit ion on drug transport across blood-brain barrier in rats. Drug Metabolis m and

Disposition 2011 January 1, 2011;39(1):8-14.

28. Rautio J, Humphreys JE, Webster LO, Balakrishnan A, Keogh JP, Kunta JR, et al. In vitro p -glycoprotein

inhibit ion assays for assessment of clinical drug interaction potential of new drug candidates: a recommendation

for probe substrates. Drug Metabolism and Disposition 2006 May 1, 2006;34(5):786-92.



2

29. de Graaf IAM, Olinga P, de Jager MH, Merema MT, de Kanter R, van de Kerkhof EG, et al. Preparat ion

and incubation of precision-cut liver and intestinal slices for applicat ion in d rug metabolis m and toxicity studies.

Nat Protocols 2010;5(9):1540-51.

30. Hadi M, Chen Y, Starokozhko V, Merema MT, Groothuis GMM. Mouse precision -cut liver slices as an ex

vivo model to study idiosyncratic drug-induced liver injury. Chemical Research in Toxicology 2012

2012/09/17;25(9):1938-47.

31. van de Kerkhof EG, Ungell A-LB, Sjöberg ÅK, de Jager MH, Hilgendorf C, de Graaf IAM, et al.

Innovative Methods to Study Human Intestinal Drug Metabolism in Vit ro: Precision-Cut Slices Compared with

Ussing Chamber Preparations. Drug Metabolism and Disposition 2006;34(11):1893-902.

32. de Graaf IAM, van der Voort D, Brits JHFG, Koster HJ. Increased post -thaw viability and phase I and II

biotransformation activity in cryopreserved rat liver slices after improvement of a fast -freezing method. Drug

Metabolism and Disposition 2000 September 1, 2000;28(9):1100-06.

33. The International Transporter Consortium. Membrane transporters in drug development. Nature Reviews

Drug Discovery 2010;9(3):215-36.

34. Kamiyama E, Sugiyama D, Nakai D, Miura S-i, Okazaki O. Culture period-dependent change of function

and expression of ATP-Binding Cassette transporters in caco-2 Cells. Drug Metabolism and Disposition 2009

September 1, 2009;37(9):1956-62.

35. Sun D, Lennernas H, Welage LS, Barnett JL, Landowski CP, Foster D, et al. Comparison of human

duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation w ith permeability

of 26 drugs. Pharm Res 2002 Oct;19(10):1400-16.

36. FDAwebsite. Drug Development and Drug Interactions: Table of Substrates, Inhibitors and Inducers.

[cited; Availab le from: http://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/

druginteractionslabeling/ucm093664.htm#PgpTransport

37. Johnson LV, Walsh ML, Chen LB. Localization of mitochondria in living cells with rhodamine 123.

Proceedings of the National Academy of Sciences 1980 February 1, 1980;77(2):990-94.

38. MacLean C, Moenning U, Reichel A, Fricker G. Closing the Gaps: A full scan of the intestinal expression

of P-g lycoprotein, breast cancer resistance protein, and mult idrug resistance-associated protein 2 in male and

female rats. Drug Metabolism and Disposition 2008;36(7):1249-54.

39. Sekine S, Ito K, Saeki J, Horie T. Interaction of Mrp2 with radixin causes reversible canalicular Mrp2

localization induced by intracellular redox status. Biochimica et Biophysica Acta (BBA) - Molecular Basis of

Disease 2011;1812(11):1427-34.

40. Yumoto R, Murakami T, Nakamoto Y, Hasegawa R, Nagai J, Takano M. Transport of rhodamine 123, a

P-glycoprotein substrate, across rat intestine and caco-2 cell monolayers in the presence of cytochrome P-450

3A-related compounds. Journal of Pharmacology and Experimental Therapeutics 1999 April 1,

1999;289(1):149-55.

41. Drozdzik M, Gröer C, Penski J, Lapczuk J, Ostrowski M, Lai Y, et al. Protein Abundance of Clinically

Relevant Multidrug Transporters along the Entire Length of the Human Intestine. Molecular Pharmaceutics

2014;11(10):3547-55.

42. Fenner KS, Troutman MD, Kempshall S, Cook JA, Ware JA, Smith DA, et al. Drug-drug Interactions

mediated through P-glycoprotein: clin ical relevance and in vitro-in vivo correlation using digoxin as a probe

drug. Clin Pharmacol Ther 2008;85(2):173-81.

43. Bansal T, Mishra G, Jaggi M, Khar RK, Talegaonkar S. Effect of P-glycoprotein inhibitor, verapamil, on

oral b ioavailab ility and pharmacokinetics of irinotecan in rats. European Journal of Pharmaceutical Sciences

2009;36(4–5):580-90.

42 Chapter 2

44. Mease K, Sane R, Podila L, Taub ME. Differential selectivity of efflux transporter inhib itors in Caco-2 and

MDCK–MDR1 monolayers: A strategy to assess the interaction of a new chemical entity with P-gp, BCRP, and

MRP2. Journal of Pharmaceutical Sciences 2012;101(5):1888-97.

45. Balimane P, Marino A, Chong S. P-gp inhib ition potential in cell-based models: which “calculat ion”

method is the most accurate? AAPS J 2008 2008/12/01;10(4):577-86.

46. Weiss J, Haefeli W E. Evaluation of inhib itory potencies for compounds inhibiting P-glycoprotein but

without maximum effects: f2 values. Drug Metabolism and Disposition 2006 February 1, 2006;34(2):203-07.

47. Suzuyama N, Katoh M, Takeuchi T, Yoshitomi S, Higuchi T, Asashi S, et al. Species differences of

inhibitory effects on P-glycoprotein-mediated drug transport. Journal of Pharmaceutical Sciences

2007;96(6):1609-18.

48. Zhang L, Zhang Y, Strong JM, Reynolds KS, Huang S-M. A regulatory viewpoint on transporter-based drug

interactions. Xenobiotica 2008;38(7-8):709-24.

Documents

University of Groningen Drug transport and transport ......transporters, P-gp plays a key role due to its broad substrate specificity and high expression level. Furthermore, since