Drug transport and transport-metabolism interplay in the human and rat intestine Li, Ming
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Drug Transport and Transport-Metabolism Interplay in the Human and Rat Intestine:
Ex Vivo Studies with Precision-Cut Intestinal Slices
Ming Li
This research project was financially supported by the China Scholarship Council (CSC).
Publication of this thesis was financially supported by:
The University of Groningen and the Faculty of Mathematics and Natural Sciences
Cover design: Ming Li & Xubin Liu Layout: Ming Li
Printing: Wuhui Culture Communication Co., Ltd. （武辉文化传播有限公司）
ISBN printed version: 978-90-367-8432-0 ISBN electronic version: 978-90-367-8431-3
Copyright © 2016 by Ming Li
No part of this book may be reproduced or transmitted in any form or by any means without written permission of the author and the publisher holding the copyright of the published articles.
Drug Transport and
Transport-Metabolism Interplay in the Human and Rat Intestine
Ex Vivo Studies with Precision-Cut Intestinal Slices
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. E. Sterken
and in accordance with the decision by the College of Deans.
This thesis will be defended in public on Friday 15 January 2016 at 12.45 hours
by
Ming Li
born on 17 September 1986 in Hunan, China
Prof. G.M.M. Groothuis
Co-supervisor Dr. I. A M. de Graaf
Assessment Committee Prof. P. Olinga
Prof. H.G.D. Leuvenink Prof. F.G.M. Russel
Viktoriia Starokozhko
Chapter 1 Introduction
9
Chapter 2 Rat Precision-Cut Intestinal Slices to Study P-gp Activity and the Potency of its Inhibitors Ex Vivo
(Toxicology in vitro. 2015; 29(5): 1070–1078)
21
Chapter 3 The Consequence of Drug-Drug Interactions Influencing the Interplay between P-gp and Cyp3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices
(Submitted)
43
Chapter 4 P-gp Activity and Inhibition in the Different Regions of Human Intestine Ex Vivo
(Submitted)
63
Chapter 5 The Consequence of Drug-Drug Interactions by P-gp Inhibitors on the P-gp/CYP3A4 Interplay in Human Intestine Ex Vivo
(Submitted)
83
Chapter 6 Human and Rat Precision-Cut Intestinal Slices as Ex Vivo Models to Study Bile Acid Uptake by the Apical Sodium-dependent Bile Acid Transporter
(Manuscript in preparation)
103
Chapter 7 Precision-Cut Intestinal Slices: Alternative Model for Drug Transport, Metabolism, and Toxicology Research
(Invited Review, Submitted: Expert Opinion on Drug Metabolism and Toxicology)
121
Chapter 8 General Discussion and Future Perspectives
151
Chapter 9 Samenvatting
159
Appendices Abbreviations
166
Acknowledgements
167
Curriculum Vitae
171
1
Chapter 1
Introduction
General introduction
The intest ine is the main location for drug absorption after oral administration, whereas its role in drug metabolis m, distribut ion, excretion and toxicity (ADME- tox) has been intensive ly stud ied and is well acknowledged in t he last decade s [1-3]. However, in contrast to the liver, the intest ine is a very heterogeneous organ and its invo lve ment into the ADME- tox process is considerably different in the d ifferent intestina l regions, e.g. duodenum, jejunum, ileum and colon. These differences are not merely on the tissue/organ level, e.g. different structure, blood flow, lumen pH etc. [4], but also on the cell and molecular level, e.g. different expression profiles of drug transporters (DTs) and metabolizing enzymes (DMEs) [5, 6]. Thus, the influence of DTs on the ADME- tox in the various regions of the intestine must be taken into account in drug development [7, 8].
Intestinal transporters
Alt hough the intest ina l absorption of many drugs occurs for an important part via passive diffus ion, intest ina l transporters also play important roles for a large group of compounds and is even the dominant route for certain classes of drugs [9- 11]. The most important DTs that are expressed and located in the intestine are shown in Fig. 1.
Influx transporters, located on the apical membrane of intestina l epithelia l cells , facilitate intest ina l absorption [5]. Many of them are members of the solute carrier (SLC) super- family. For instance, peptide transporter 1 (PEPT1; SLC15A1) is the primary uptake transporter for di- /tri- peptides; organic anion transporting polypeptides (O ATPs; SLCO s) mediate the transport of a diverse range of amphiphilic organic compounds sodium- independently; and the organic cation transporter 1 (OCT1;
SLC22A1), transports relative ly hydrophilic, low mo lecular mass organic cations. In addition, the apical sodium- dependent bile acid transporter (ASBT; SLC10A2) plays an indispensable role in the intest ina l absorption of bile acids , as part of the enterohepatic recirculat ion [12]. It reabsorbs approximately 95 % of the luminal bile acids in the ileum [13- 15]. Both conjugated and unconjugated bile acids can be taken up by ASBT but they differ in affinity for ASBT. Furthermore, malfunction of ASBT can lead to infla mmatory bowel disease, constipation and Alagille syndrome, therefore ASBT is also a determinant in these bile acid- related diseases [16, 17]. In the human sma ll intestine ASBT is solely localized on the apical membrane of the ileum whereas rat ASBT is also expressed in the caecum [18].
1
Fi g . 1 The t ranspo rte rs e xp ressed a nd lo cat ed on t he a p ic a l (lu me n s ide ) and b aso lat e ra l (b lood s ide ) me mb ra n e o f in test in a l ep ith e lia. Up ta ke t ranspo rte rs o n the ap ic a l me mb ra ne a re t he o rgan ic an ion t ranspo rt ing p o lyp ept ide (OATP) fa mily, the pe pt id e t ranspo rt e r 1 (PEPT1; SLC15A 1), t h e i lea l a p ic a l sod iu m/b ile a c id co -t ranspo rt e r (A SBT; SLC10A 2), an d mono ca rbo xy lic a c id t ranspo rt e r 1 (M CT1;
SLC16A 1). Th e ap ic a l ATP -d epe nden t e fflu x p u mps inc lude mu lt id ru g res istan ce p rote in 2 (M RP2;
A BCC2), b reast c anc e r res ista nc e p rote in (BCRP; A BCG2) a n d P-g ly cop rot e in (P-gp ; M DR1, A BCB1).
The b aso late ra l me mb rane o f intest ina l ep ithe lia c ont a ins o rgan ic c at ion t ra nspo rte r 1 (OCT1;
SLC22A 1); the hetero meric org an ic so lu te transpo rter (OSTα– OSTβ), and M RP3 (A BCC3).
Re p ro d u ce d wit h p ermis sion fro m [9].
Efflux transporters, located at both the basolateral and apical membrane, facilitate efflux o f compounds into the blood stream and the lumen respectively. The organic solute transporter (OSTα–OSTβ) mediates efflux of bile acids into the bloodstream.
P- gp, BCRP, and the members of the MRP family, belong to the ATP-binding cassette (ABC) family and can excrete their substrates out of the cells into the intestinal lumen against the concentration gradient by using the energy from ATP hydrolysis , thereby lowering the intracellular concentration and , when located at the apical membrane, decreasing intestinal absorption [19-21]. P- gp plays a key role due to its broad substrate specific it y and high expression level. P- gp activit y can significant ly influence the intracellular concentration of both the parent drug and its metabolite(s) in the enterocyte, thereby also determining the systemic exposure to these compounds. This is crucial for drugs with a narrow therapeutic window, such as digoxin, since relatively
small fluctuat ions of its blood plasma concentration can lead to either therapeutic failure or toxic effects [22]. Furthermore, the level of expression and funct ionalit y of P- gp can be modulated by induction and inhib it ion. As a result, it is of importance to evaluate for every new che mical ent ity (N CE) whether it is a substrate and/or inhib itor of P- gp and if so, its affinity and/or inhib itory potency [9]. In addition, it is a lso important to take the regional differences into the consideration for this evaluat ion, as the P- gp expression is reported to exhib it a gradient ile um > jejunum > duodenum ≥ colon [23, 24]. Since P- gp is a target for a wide spectrum of inhibitors and has broad and overlapping substrate specificit y with some of the DMEs, clinically important drug- drug interactions (DDIs) related to P - gp activity occur frequently [25, 26].
Intestinal metabolizing enzymes
In the last two decades, the metabolic capacity of the intestine and its contribut ion to first pass metabolis m has been increasingly recognized. Studies in v ivo demonstrate that this significant intestinal metabolism has implications for the bioavailability of drugs [27]. In human intestine, the clinica lly important DMEs include cytochrome P450 (CYP) oxidases and the flavin- containing mo nooxygenase (FMO ), UDP- glucuronosyltrans ferases (UGTs) [28], glutathione S- transferases (GSTs) [29], and sulfotransferases (SULTs) [30]. This system of enzymes acts in two stages to firstly oxid ize the xenobiotic (phase I) and then conjugate water- soluble groups onto the mo lecule (phase II). Thus, the modified water- soluble xenobiotic can be pumped out of cells (considered as phase III nowadays) and can then be excreted from the body via the urine or bile.
CYP-enzymes, the most important DME family, play a central role in the hydroxylat ion, dealkylatio n and oxidative metabolis m (phase I) of clinically used drugs and other xenobiotics [31]. Among the CYP superfamily, the CYP3A subfamily accounts for about 30 % of total CYP-enzymes and invo lves in the metabolis m of approximately 50 % of all therapeutic drugs on the current market [31]. Also CYP3A has a broad substrate specific it y, is a target for many inhibitors and its expression can be induced by many compounds. CYP3A shows the highest expression in the proxima l intest ine, which decreases towards the ile um w ith t he lowest expression in colon [32]. Data on metabolic activit y in the different regions are scarce as it is difficult to obtain these data in man in v iv o.
1
Transport-metabolis m interplay
In the intestine, DTs, especially efflux transporters, and DMEs share a physio logical funct ion, which is to decrease the absorption of xenobiotics inc luding drugs.
Furthermore, they can work coordinately, which is currently known as the transport- metabolis m interplay, to complete this barrier funct ion in an efficient way, either based on the overlapping substrate specific it y (parallel interplay) or based on the fact that products of DMEs are substrates of DTs (concatenated interplay) [33].
Due to the fact that both P- gp and CYP3A have a significant influence on the ADME- tox process, the P- gp/CYP3A interplay raised a lot of interest and was widely studied. Such interplay has been postulated based on experiments in Caco- 2 cells for shared P- gp and CYP3A substrates [34], and it is believed that their coordinated activit ies can further reduce the intracellular concentrations of xenobiotics and therefore lower the intestina l absorption of drugs [35]. However, the consequence of the P- gp/CYP3A interplay in v ivo is still under debate till now as controversial results have been found. Watkins et al. hypothesized that P- gp may prolong the residence time of its substrates in the intest ine, therefore increasing t he possibilit y for intest ina l metabolis m [36]. Supporting t he same conclusio n, other explanations also exist. For instance, P- gp is believed to excrete also the CYP3A metabolites, as they are often also P- gp substrates, from the enterocytes, thereby reducing the chance of product inhib it ion. Consequently, P- gp inhib it ion would decrease the total intest ina l metabolis m. O n the contrary, Pang et al. stated that P- gp efflux limits metabolis m due to the competit ion between P- gp and CYP3A within the cell and that their interplay is independent of the mean residence time of drug in the system [37]. Thus, they conclude that P- gp inhibit ion w ill increase metabolis m, since the intracellular substrate available to CYP3A will be increased [38]. Similar ly, when P- gp limits the absorption in the proxima l s mall intestine, the absorption is shifted to more distal, less catalytically effic ient segments that contain lower amounts of CYP3A [36, 39], thus P- gp inhib it ion will increase metabolis m, but only under conditions where the CYP3A is not saturated . However, the effect of the interplay in the different regions of t he intestine has not been studied in detail and evidence of P- gp/CYP3A4 interplay in t he human intest ine ex v iv o is also lacking.
Transport- metabolism interpla y was also suggested for MRP2/UGT, MRP2/GST, and
BCRP/SULT but has been much less investigated. This interplay is not based on a common substrate but in these cases the metabolite is the substrate for the transporter.
Phase II DMEs, such as UGT, GST, and SULT, produce drug- conjugates which have much higher hydrophilicit y and show high affinity for MRP2 and BCRP. Thus, the intracellular concentration of the absorbed drug quickly decreases by the metabolis m and excretion of the metabolite prevents accumulatio n and associated product inhib it ion of the enzyme. Therefore, inhib it ion of MRP or BCRP may increase the concentration of the drug-conjugates in the cell, thereby exerting feedback inhibit ion of the metabolism [33].
Models for the intestinal transport and/or metabolism
Many methods, as shown in Fig. 2, have been developed and applied to characterize and predict the ADME- tox properties in the intestine of N CEs [9, 19, 40-43]. In vit ro cell cult ures and in vivo anima l models are the conventiona l methods which are widely used in both academia and industry, and they are well characterized. However, in vit ro cell cultures do not reflect the tissue mult i- cellularit y, 3D structure, physio logical expression leve ls of DTs and DMEs, and some cell types lose their polar izat ion dur ing cult ure. O n the other hand, the in vivo models retain the proper physio logical conditions, the ir screening capacity is too low and the cost, both in animal lives and in money, is much higher. Moreover applicat ions of these in v ivo models in human are extremely difficult due to technical and ethical constraints and exceptionally high costs.
To fill this gap, alternative methods us ing intact tissue, such as everted sac, perfused intestina l loops, Ussing chamber, intestinal punches and, more recently, precision- cut intestina l s lices (PCIS) have been developed and are increasingly used, to provide additional infor mation on the intestina l hand ling of N CEs during pre -clinical investigation.
Precision-cut intestinal slices
Precision-cut intestinal slices (PCIS) have been well-established for drug metabolism, induction and toxicity in the intestine ex vivo [44-48]. However at the start of this study no paper had been published on drug transport or transport- metabolism interplay studies in PCIS yet.
The applications of precision-cut intestinal slices in drug metabolism, transport and toxicity, including the studies described in the thesis, are introduced and extensively discussed in
1
Chapter 7.
Fi g . 2 Sche ma t ic c lass ific at io n o f th e cu rrent mo de ls to st udy t he A DM E -to x p ro pe rt ies d rugs and xe n o b io t ics in t h e in t estin e. Re p rod uce d wit h p ermis sion fro m [49]
The scope of the thesis
The aim of the research described in this thesis is to investigate the transport and transport- metabolism interplay in human and rat intestine ex vivo using precision-cut intestinal slices.
The following research questions were studied:
Can PCIS from the rat and human intestine serve as a model to study uptake and efflux transporters?
Are the regional differences in transporter expression as reported in the literature reflected in functional differences?
What is the effect of P-gp inhibitors on the P-gp/CYP3A interplay?
What are the species differences between human and rat intestine with respect to transport function and P-gp/CYP3A interplay?
In Chapter 1, the background of the research is introduced; including the transport, metabolism and their interplay in the intestine as well as precision-cut intestinal slices PCIS as ex vivo model.
In Chapter 2 and 4 PCIS are validated as an ex vivo model to investigate P-gp activity and inhibition in rat and human intestine, respectively. In addition, the potencies of several P-gp inhibitors in the different regions of the intestine are presented and a broader range of
inhibitors was investigated in PCIS of rat ileum and human jejunum. The species differences of P-gp activity and inhibition are discussed in Chapter 4.
Based on the work of Chapter 2, the consequences of DDIs influencing the P- gp/Cyp3a interplay are further explored in the different regions of the rat intestine in PCIS in Chapter 3.
Similarly, the P-gp/CYP3A4 interplay and its related DDIs with P-gp inhibitors in human PCIS are presented in Chapter 5. In addition, the correlation between the magnitudes of the response and the absolute P-gp abundance is discussed.
In Chapter 6, the use of PCIS to study the function of uptake transporters is validated. The function of the apical sodium-dependent bile acid transporters (ASBT), an important influx transporter in the intestine, is presented by analyzing the uptake of three different bile acids in rat and human PCIS.
In Chapter 7, the PCIS technology is reviewed and discussed as an alternative method for drug transport, metabolism and toxicology research.
In Chapter 8, the findings in Chapter 2 - 6 are summarized and discussed, followed by suggestions for future research and future perspectives
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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
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 d ependent 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
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1. Introduction
The intestine is t he 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 me mbrane 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 bioavailabilit y [7, 8]. The three major types of ABC transporters expressed on the apical side of the epithelia l cells, name ly 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 pro tective 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 activit y 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 it ro models have been developed, such as membrane vesicles, cell lines, everted gut sac, intestinal perfus ion and Ussing c hamber [4, 11-13]. However, none of these models can fully represent the properties of the intestine w ith respect to the in v ivo gradient of expression along the length of t he 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
intestina l slices (PCIS) model could be used to study the activit y of intest ina l transporters. Precisely sliced from the intest ine immed iately after harvesting t he tissue, PCIS represent an ex v ivo mini- organ model which contains all types of intestinal cells in the ir natura l environme nt, 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 metabolis m and toxicit y in the intestine [14-20]. The localizat ion along the lengt h of the intestine and the regulat ion of some of the transporters were studied on mRN A leve l before [21]. To date, studies on the functio nal activity of these transporters ex v ivo is limited to the study of Possidente et al. who investigated the interactions of xenobiotics wit h 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 wit h these transporters [22]. However in this paper only jejunum slices were studied and infor mation on the activity in the different regio ns of the intestine was lacking. In addition the cells involved in the accumulation of the substrates were not identified.
The aims of the present st udy 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 w hether rat PCIS correctly reflect the inhibitory potency, i.e. rank order and IC5 0 values, of different P - gp inhibitors in the rat intestine ex v iv o;
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 ivit y 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
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(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
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 K it 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
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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. Q uinidine 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
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 (O lympus BX41, O lympus 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 <0.05 was considered as the level of significance.
In the inhibition study with various concentrations of inhibitors, data were normalized to their control groups (100 %) and fitted into a log [inhibitor] vs. response curve. The IC50 value of each P-gp inhibitor was calculated using GraphPadPrism 5 (GraphPad Inc., USA).
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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 a fter 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 A NOVA with Tukey’s co mparison as post-hoc test was employed for the co mparison 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,
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.
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Fig. 2 The loca lizat ion of R123 in the intestine (A, duodenum; B, je junum; C, ileu m; D, co lon) (left : no inhibitor;
middle : quin idine 50 µM; right: CP100356 2 µM ). Pictures we re taken at appro ximate ly the same a reas of the sections using the fluorescent mic roscope (upper panels) and the light microscope (lowe r panels). Sca le 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 e xpressed as mean ± SEM. The fluorescence value of blan k slice, wh ich was very lo w and negligible , was included by the calibration curves using an homogenate of untreated slices.
Fig. 3 b (right panel) Concentration-dependent uptake of R123 and the effect of P-gp inhibition by CP100356 (2 µM) (n=3). Results were e xp ressed as mean ± SEM. Two-ta iled pa ired Student’s t-test was employed for comparison between the control and CP100356 group at each R123 concentration . * indicates p<0.05.
3.4. Inhibition study
3.4.1. Regional difference of P-gp inhibition
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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 accumu lation 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, je junum and colon, n=6 for ileu m (data for ileu m inc lude 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
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 CP 100356 on R123 accu mulation in PCIS of the different intestinal regions. The results were e xp ressed as mean ± SEM. N = 3 for duodenum, je junum and colon, n = 6 for ileu m (data for ileu m include the data for CP 100356 and quin idine obtained in the e xperiments where the six inhibitors we re tested). Two-way A NOVA 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 accu mulat ion was found significantly d iffe rent between ileu m and colon, howeve r, with P-gp inhibit ion, no regional difference in R123 accumulat ion was found. Besides, there was a significant increase of R123 accu mulat ion after P -gp inhibit ion in ileum slices. ns = not significant; ** significant with p<0.01, *** significant with p<0.001
4. Discussion
Intestina l transporters are major determinants of the absorption, and thus of the efficacy and safety profile of drugs and in addition to that, determine the local concentration in the intestina l epithelium. There is no doubt that they need to be taken into account in drug development, as reviewed by the internationa l transporter
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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 accu mulat ion in PCIS. The results are e xpressed 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 develop ed PCIS as ex v ivo model to study drug metabolis m 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 w ithin one experiment, thus, the number of anima ls needed is largely reduced, which is in good accordance with the 3Rs (Reduction, Replacement and Refineme nt). 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
infor mation on gradients along the lengt h of t he intestine and on the cellular localization of the accumulated substrates.
Table 1 Co mparison of IC50 values of the tested P-gp inhibitors and their rank order a mong diffe rent 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
Cycl osporine 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 funct ion 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
