Predicting oral drug absorption and hepatobiliary clearance: Human intestinal and hepatic in vitro cell models

Environmental Toxicology and Pharmacology 21 (2006) 168–178

Predicting oral drug absorption and hepatobiliary clearance: Human intestinal and hepatic in vitro cell models Richard A. Fearn, Barry H. Hirst ∗ Institute for Cell and Molecular Biosciences, University of Newcastle, Medical School, Newcastle upon Tyne NE2 4HH, UK Available online 2 August 2005

Abstract Membrane transport proteins control the uptake and efflux of many drugs in tissues including the intestine, liver and kidneys and thus play important roles in drug absorption, distribution and excretion. With the development of high throughput screening in an industrial environment, the importance of having appropriate in vitro systems to study drug transporter function, regulation, and interactions are invaluable. Cell lines are efficient tools in screening individual transport processes. In this review, we focus on the processes involved in the absorption and hepatobiliary clearance of drugs and the potential of cell lines to model such process, paying particular attention to the use of Caco-2 and HepG2 cells. © 2005 Elsevier B.V. All rights reserved. Keywords: Intestine; Liver; Drug absorption; Hepatobiliary clearance; Bioavailability; Membrane transport

1. Oral drug delivery The oral route is preferred for routine administration of drugs required to have systemic actions, as it is the most convenient and cost-effective. In order for a drug to reach the systemic circulation and its site of action, it must have chemical and physical properties that allow it to withstand the hostile environment within the gut lumen. In particular, the gut lumen has evolved to provide an optimum environment for both digestion of ingested food, while simultaneously providing an environment hostile to ingested pathogens, such as bacteria. Thus, gastrointestinal tract luminal contents are highly acidic in the stomach, have powerful detergents added in biliary secretions (bile salts), while a plethora of digestive enzymes are added in gastric and pancreatic secretions, including proteases, lipases and nucleases. Resistance to these luminal chemical assaults may be due to intrinsic properties of drug compounds, and/or drugs may be protected by specialised formulations. Drug molecules must also show appropriate solubility and lipophilicity characteristics within the luminal contents, such that they ∗

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can be available for absorption. The quantity and quality of food in the stomach and gut motility will influence intestinal absorption (Doherty and Pang, 1997; Burton et al., 2002). Absorption of active compounds into the systemic circulation from the gut lumen requires traversing the physical and selective barrier of the small or large intestines, surviving enterocyte metabolism, as well as possible first pass metabolism by the liver. Once in the systemic circulation, drugs can distribute throughout the body and have pharmacological effects in the target tissues. However, the body aims concurrently to remove such xenobiotics, primarily via metabolism in the liver and intestine, as well as through the excretion of parent compounds and their metabolites, into bile, the intestinal lumen, and urine, via the liver, the intestine and the kidneys, respectively (Fig. 1). 1.1. Intestinal drug absorption Compounds may cross the intestinal epithelium by paracellular or transcellular routes (Fig. 2). Transport across the intestinal epithelium via the paracellular route (A), is minimal due to the presence of tight junctions between adjacent enterocytes. Therefore, this route is generally restricted to small hydrophilic molecules. Junctional modulators may enhance

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Fig. 1. The absorption, distribution and secretion of drugs. Drug (D) absorption occurs across the small intestine, after which they are presented to the liver where they can be converted into metabolites (M). Parent and drug metabolites can re-enter the GI tract from the liver through excretion in the bile, after which they can be reabsorbed or excreted into faeces. Parent and drug metabolites that enter the vascular compartment are then susceptible to excretion into urine by the kidney.

the permeability of the paracellular route and offer possibilities as absorption adjuvants or enhancers. Transcellular transport of compounds may be passive (B) or mediated by specific transporters at apical and basolateral membrane surfaces (C). A significant contribution of passive (diffusive) mechanisms to overall absorption requires appropriate molecular size, charge, lipophilicity and hydrogen bond potential, i.e. appropriate physiochemical properties to allow passage through both the apical and basolateral plasma membranes of the enterocyte. Lipinski formulated ‘the rule of 5’ as criteria for oral bioavailability. Good oral bioavailability may be achieved by a molecule with not more than five hydrogen bonds, not

Fig. 2. Pathways of intestinal absorption. (A) Paracellular diffusion; (B) transcellular passive diffusion; (C) transporter-mediated transport; (D) active apical efflux; (E) intracellular modification prior to entering the blood; (F) co-ordinated metabolism and apical excretion.

more than 10 hydrogen bond acceptors (notably N and O), a molecular weight less than 500, and a log P < 5 (Lipinski et al., 1997). Therefore, passive mechanisms are recognised as an important route for the absorption of several classes of drug molecules. In contrast to the properties relevant for passive absorption, compounds that utilise specific transporter systems tend to be hydrophilic and are recognised as substrates along with nutrients and micronutrients. Facilitative transporters enhance the movement of substrates (e.g. glucose, amino acids, and urea) across membranes with the electrochemical gradient. Active transporters may generate gradients across membranes with various energy-coupling mechanisms. Secondary active transport is indirectly coupled to the energy of ATP, perhaps through the Na+ electrochemical gradient generated by the ubiquitous Na+ + K+ -ATPase, while primary active transport directly utilises ATP during the transport cycle. Compounds that cross the apical membrane of the enterocyte, including those which are absorbed passively, may be recognised as substrates for apical active efflux transporters (D). Examples of such transporters included the ATP-binding cassette multidrug resistance proteins, such as P-glycoprotein (P-gp) and MRP2. These efflux transporters will limit drug absorption by transporting compounds back into the intestinal lumen. Alternatively, compounds may be metabolised following uptake into the enterocyte by a variety of enzymes, such as those of the cytochrome P450 family (CYP), particularly CYP3A4/5 in the intestine. Metabolism may be followed by transfer to the circulation (E) or excretion back into the intestinal lumen (F).

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Understanding the various routes and mechanisms for intestinal drug absorption has led to recognition that poor oral bioavailability may linked to a number of factors. These include poor absorption per se or other properties, such as appropriate lipophilicity, which promote absorption by passive mechanisms and simultaneously allow for recognition by other processes. For example, increased intestinal and/or hepatic clearance is linked to increased affinity for intestinal and/or hepatic active efflux transporter systems. During the process of development from lead compound to active drug compounds, recognition and perhaps avoidance of substrates for drug efflux pumps may be considered. Alternatively, novel chemical entities may be natural substrates for absorptive transport systems or such properties may be ‘designed’ in, including the use of pro-drug approaches. Therefore, for the early recognition and assessment of the impact of such properties, it would be desirable to have in vitro and in vivo models, which are relevant to the specific transporter in question. 1.2. Hepatobiliary clearance Compounds that successfully enter the enterocyte, escape intestinal metabolism and exit across the basolateral membrane of the enterocyte, pass through the portal vein directly to the liver; the major site of metabolism in the body. Some compounds may escape immediate exposure to the liver: highly lipophilic compounds may instead be associated with chylomicrons and leave the intestine in the lacteals, so

entering the lymphatics before joining the general circulation after the liver. As in the enterocyte, lipophilic drugs can enter the hepatocyte via passive or facilitated diffusion. In addition, numerous transporters located on the sinusoidal membrane of the hepatocyte can mediate the uptake of lipophilic, amphipathic and polar organic compounds (Fig. 3). Once in the hepatocyte, molecules are exposed to numerous phase I and phase II metabolising enzymes. Following phase II conjugation with co-substrates such as glutathione or UDP-glucuronic acid, these newly formed hydrophilic or amphipathic compounds become organic anions. This increase in polarity, which results in a more water-soluble entity, generally enhances the elimination from the liver. Parent and biotransformed drugs can either be metabolised further or eliminated from the hepatocyte across the sinusoidal membrane, back into blood, where they may be cleared by the kidney into urine. Alternatively, these products may be transported across the canalicular membrane of the hepatocyte into bile, by energy-dependent efflux transport systems, with subsequent excretion in faeces. However, the roles played by various apical (uptake) and basolateral (efflux) transporter mediated processes in the rate-limiting steps in the subsequent metabolism and distributions of drugs are poorly understood at present. Interest in understanding hepatic transport mechanisms has grown for a number of reasons. Firstly, hepatic uptake transporters can be exploited to target the liver. For example, the statin, pravastatin, a cholesterol synthesis inhibiter, is a substrate for the liver specific uptake transporter OATP1B1

Fig. 3. Hepatic drug transporters. Sinusoidal transporters: NTCP (sodium taurocholate co-transporting polypeptide), OATPs (organic anion transporting polypeptides), OATs (organic anion transporters), OCTs (organic cation transporters). Canalicular transporters: BCRP (breast cancer resistant protein), ABCC (ATP-binding cassette subtype C; MRPs), P-gp (P-glycoprotein), BSEP (bile salt export pump), MDR3 (multidrug resistant protein 3), TJ (tight junction).

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(OATP-C). As the main site of cholesterol synthesis is the liver, it is advantageous that pravastatin is a substrate for OATP1B1. In contrast, similar recognition by OATP1B1 of drugs with target organs outside the liver would be disadvantageous, since liver clearance would be enhanced, with a resultant poorer systemic exposure. Therefore, consideration of the intended target organ for novel chemical entities and recognition by such transport mechanisms is essential for effective drug development programmes. Secondly, potential changes in the expression profiles of both hepatic uptake and efflux transporters, as well as metabolising enzymes, in disease states are at present poorly understood. Similarly, such changes in response to drug treatment require important considerations due to the potential for influencing systemic drug exposure. Thirdly, determining the biliary excretion properties of novel chemical entities (NCE) is a critical issue in the drug discovery and development process. Drug candidates that are extensively excreted into the bile may never achieve adequate concentrations or duration of action in vivo even if their absorption is acceptable. For example, many metabolically stable peptides exhibit short residence times in the systemic circulation and have low bioavailability after oral administration due to rapid and extensive biliary excretion (Ziegler et al., 1996; Chen and Pollack, 1997). Therefore, for any NCE, it is important to elucidate the potential for interaction with such hepatic and efflux systems.

2. Predicting oral bioavailability: in vitro models for oral absorption and hepatobiliary clearance It is clear that for timely and effective drug development, it is important to gain early insight into the potential of lead compounds. This will allow appropriate optimisation of absorption and clearance processes to achieve adequate oral bioavailability and duration resulting in effective systemic concentrations. As large numbers of compounds are to be dealt with, conventional oral bioavailability studies are unrealistic. Such time-consuming and costly procedures should be restricted, as far as practical, to later stages of drug development, once powerful leads have been identified. In contrast, simpler model systems may provide a productive solution for screening and ranking compounds early in the optimisation process. At the simplest level, determination of lipid solubility (log P determination) may provide useful information on the sort of problems that will be encountered during oral absorption. These may be complemented by permeability assays utilising inert membranes (Walter and Gutknecht, 1986; Xiang and Anderson, 1994). A more sophisticated approach is the parallel artificial membrane permeation assay (PAMPA) utilising an inert membrane supporting a lipid bilayer. PAMPA shows definite trends in the ability of molecules to permeate membranes by transcellular passive diffusion (Kansy et al., 1998). Such assays can be carried out in a 96-well filter format, making them amenable to high

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throughput screening and can be modified to investigate the effects of pH, surfactants, other excipients and permeability enhancers (Kansy et al., 1998; Zhu et al., 2002). While PAMPA and even log P determinations have their place, more complex models containing both passive and ‘active’ features of membrane transport are an important second stage. Membrane vesicles, such as those prepared from the brush-border of the small intestine or liver plasma membranes prepared form either hepatocyte canalicular or basolateral membrane domains have proven invaluable in determining features such as substrate specificity, electrogenicity and pH-dependence of transport in the native membrane, including transport proteins (Thwaites et al., 1993; Ishizuka et al., 1997; Yamazaki et al., 1997). Such techniques may be extended to investigate energy-dependent transport processes (Masuda et al., 1997; Niinuma et al., 1999; Ueda et al., 2001). Even more complex systems include the use of isolated mucosal segments separating two compartments in vitro, such as Ussing (or diffusion) chambers. However, the complexity of these more biologically relevant models is also reflected in their inappropriateness for high-throughput screening protocols. An alternative approach is to use simplified in vitro systems which contain a limited number, or often one, identified transport system. Such in vitro systems can help to identify the transporter involved in an absorptive or secretory process and thereby aid the interpretation of more complex models. These in vitro systems include insect cell expression systems, Xenopus oocytes, and transfected mammalian cells, each having their own uses and limitations. For example, Adachi et al. (2001) showed a positive correlation between P-gp-mediated alterations in permeability across MDR1 transfected LLCPK1 cell monolayers and the ratio of compound absorbed across perfused jejunal segments from double-knockout, mdr1a/1b(−/−) mice. These studies demonstrated that the ability of P-gp to alter drug permeability in simple cell monolayers is related quantitatively to its effect on permeability across the intestine. Furthermore, these findings highlighted that there appears to be a close similarity between substrate recognition and transport efficacy for P-gp between the two species. However, in a similar study, Yamazaki et al. (2001) demonstrated that human MDR transfected LLC-PK1 (L-MDR1) and mouse mdr1a transfected LLC-PK1 (L-mdr1a) cells had different P-gp substrate susceptibility for several compounds. This suggests the model may have as strong an influence as the species differences. Investigations on the differences between the transport characteristics of P-gp encoded by human MDR1, mouse mdr1a, and mdr1b genes have revealed that the three P-gp isoforms possess specific and distinguishable functional characteristics (Tang-Wai et al., 1995). In terms of transport, a higher susceptibility in mdr1a than in MDR1 has been reported for several compounds from in vitro experiments with L-MDR and L-mdr1a cells (Schinkel et al., 1996; Yamazaki et al., 2001). Such findings have large implications with regards to accurately predicting human P-gp-mediated interactions

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from in vitro mouse mdr1a-based assays. For example an overestimation in the contribution of P-gp-mediated transport in limiting absorption, in biliary excretion and into the brains of humans may occur. Furthermore, the potential for creating “false-positive” predictions surrounding drug–drug interactions involving of P-gp-mediated transport is also a concern. Hepatocytes isolated from human liver are a particularly attractive system to study hepatobiliary clearance. Metabolism may be preserved and thus modelled accurately. In contrast, cell polarity is usually lost, such that modelling transport may be difficult. Modification of methodology can produce hepatocyte couplets, or three-dimensional structures in collagen matrixes, which enable the complexity of hepatocyte transport to be explored. Isolated hepatocytes are a powerful model, yet limited by availability, costs in preparation due to labour-intensive methods, and due to the individuality of each hepatocyte preparation, lack of standardisation both between and within laboratories. Finally, inter-donor variability has also to be addressed when using isolated hepatocytes (Modriansky et al., 2000). Cell lines have the potential to illustrate much of the complexity of the processes involved in oral bioavailability, while allowing for routine procedures, which may at least partially reflect the needs of timely information. The complexity should reflect the variety of membrane transport systems, cytosolic metabolism and polarised, apical or basolateral, expression of transporters found in vivo. While the intestinal model system of Caco-2 cells are almost universally accepted as a powerful model of the intestine, no similar system is available for the liver. The liver-derived cell line HepG2 is considered as an alternative. In this review, we focus our consideration on human cell lines as model systems. The choice of human cells has the advantage of eliminating species differences, when considering development of drugs for use in man

3. Modelling intestinal absorption As indicated above, the Caco-2 cell system is used extensively in research and in drug development as a model of the human intestine. While other human cell lines are available, which may be suitable models of colonic crypt- (T84) or ileo-caecal (HCT-8) colonocyte-type phenotypes, Caco-2 cells illustrate a largely small intestinal absorptive enterocyte phenotype. 3.1. Caco-2 cells First isolated by Fogh et al. (1979), the Caco-2 line originates from a human colorectal carcinoma. Since its isolation a large body of information on intestinal drug absorption has been obtained using this model (Artursson and Karlsson, 1991; Collett et al., 1996; Yee, 1997). One of the main advantages of this cell line is that once confluent,

Caco-2 cells differentiate both structurally and functionally into cells resembling mature enterocytes (Pinto et al., 1983). Caco-2 cells have also been shown to express several transport systems for sugars (Mesonero et al., 1994), amino acids (Hidalgo and Borchardt, 1990a), bile acids (Hidalgo and Borchardt, 1990b), and xenobiotics, including P-gp (Hunter et al., 1993; Taipalensuu et al., 2001) and MRPs (Walgren et al., 2000; Taipalensuu et al., 2001; Prime-Chapman et al., 2004). Therefore, Caco-2 cells can be used to investigate both passive and active transport processes. Indeed, there is wide use by pharmaceutical companies of Caco-2 cells to screen NCE to determine absorptive potential, reflecting the good correlation between Caco-2 permeability and drug absorption in humans in vivo (Cogburn et al., 1991; Artursson and Karlsson, 1991; Yee, 1997; Polli and Ginski, 1998). Typical experimental procedures using Caco-2 cells for this purpose involve determining apparent permeabilities (Papp ) of a compound in the absorptive (Papp,A–B; Fig. 2) and secretory (Papp,B–A ; Fig. 2) directions at a given donor concentration under sink conditions. The ratio of Papp,BA and Papp,AB (efflux ratio) has been used to gauge the effects of P-gp-mediated efflux activity and to predict attenuation of oral absorption due to this activity. However, it has been highlighted that such an efflux ratio may not quantify how P-gp-mediated efflux activity attenuates absorption in vivo (Benet et al., 1990; Schinkel, 1998). This has resulted in Troutman and Thakker (2003) proposing new experimental parameters, the absorptive quotient (AQ) and the secretory quotient (SQ) which provides a direct readout of the attenuation of absorptive transport and enhancement of secretary transport, respectively, caused by P-gp-mediated efflux in a polarised epithelial. Although Caco-2 cells are a useful system for investigating intestinal absorption, their application as a biliary model requires further evaluation. Since the early findings by Hunter et al. (1993) showing that there is functional expression of P-gp at the apical membrane of Caco-2 cells, a major aim in the field has been to determine what other ABC transporters are expressed in these cells (Hunter et al., 1993). Taipalensuu et al. (2001) investigated the mRNA expression of a number of known efflux proteins in both human jejunum and Caco-2 cell monolayers using quantitative real time PCR. P-gp, MDR3, MRP 1–6, BCRP and the lung resistant-related protein are all expressed in both human jejunum and Caco-2 cells. The order of mRNA expression was BCRP ∼ MRP2 > MDR1 ∼ MRP3 ∼ MRP6 ∼ MRP5 ∼ MRP1 > MRP4 > MDR3. Indeed, on exclusion of the BCRP data, the transcript levels of the other transporters correlated well between jejunum and Caco-2 cells (r2 = 0.9), hence, Caco-2 cells capture the expression pattern of these transporters, with the exception of BCRP, of the healthy human jejunum (Taipalensuu et al., 2001). This is in line with studies using Northern blotting and semi quantitative RT-PCR, showing that MRP2 transcripts are the most abundant MRP isoform expressed in Caco-2 cells, where MRP1 and 5 are the least abundant (Hirohashi et al., 2000; Prime-Chapman et al., 2004). However, this is

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in contrast to the findings of Delie and Rubas who suggested that mRNA levels in Caco-2 cells do not correlate well with human jejunum, while Nakamura reported that MDR1 and MRP1 levels correlate to those in normal colorectal tissue and colorectal adenocarcinoma (Delie and Rubas, 1997; Nakamura et al., 2002). In comparisons to the liver, the expression of MRP1, 3, 4, 5 and MDR1 mRNA are all higher in Caco-2 cells. MRP1 was absent from the liver in this study, where MRP 3, 4, and 5 are expressed 10-fold higher, and MDR1, two-fold higher in Caco-2 cells than the liver (Borlak and Zwadlo, 2003). hOCT1, hOCT2 and hOCT3 mRNA have all been shown to be expressed in Caco-2 cells (Zhang et al., 1999; Bleasby et al., 2000; Martel et al., 2001). Like P-gp, MRP2 is also located at the apical membrane of Caco-2 cells (Hunter et al., 1993; Walgren et al., 2000; Prime-Chapman et al., 2004). MRP2 also appears to reside in intracellular vesicular pools, where MRP1 and MRP3 appear to be expressed in the cytoplasm and at the lateral membrane, respectively, while no staining of MRP5 could be detected using the anti-MRP 5 antibody, M5 I-I (Prime-Chapman et al., 2004). The localisation of MRP 4, 5, 6, 7, 8 and 9 have still to be determined in Caco-2 cells. There is a large body of evidence supporting functional expression of P-gp and MRP2, the transporters involved in biliary excretion, at the apical membrane of Caco-2 cells. For example, numerous P-gp substrates such as digoxin, erythromycin, fexofenadine and fluoroquinoline antibiotics to name but a few, are secreted across Caco-2 cells, suggesting that P-gp may play a role in limiting absorption in vivo (Cavet et al., 1996; Takano et al., 1998; Lowes and Simmons, 2002). Indeed, in man, 11% of an intravenous dose of digoxin was secreted into the intestinal lumen, double that observed in the presence of quinidine, an inhibitor of P-gp (Drescher et al., 2003). Similarly, the intestinal excretion of digoxin in mdr1a/1b knockout mice was reduced eight-fold, from 16 to 2% of the administered dose when compared to wild type animals (Schinkel et al., 1997). A net secretion of digoxin was also observed across Ussing chamber mounted mouse intestine and was completely abolished in the presence of either quinidine or verapamil (Stephens et al., 2002). Paclitaxel, an anti-cancer drug, and P-gp substrate has also been shown to be transported in an asymmetric manner across Caco-2 cells with an efflux ratio of 184. This ratio was abolished by the P-gp specific inhibitor PSC-833 (4 ␮M), due to an increase and decrease in the A–B and B–A fluxes of paclitaxel, respectively. This resulted in a >100-fold increase in cellular accumulation of paclitaxel (Crowe, 2002). In agreement with this, the net secretion of paclitaxel across mouse intestinal tissue decreased 12- and 6-fold in the presence of the P-gp inhibitors, quinidine and verapamil, respectively (Stephens et al., 2002). Therefore it is clear that Caco-2 cells can be used to investigate P-gp-mediated interactions in the intestine, however, it is unclear if such observations can be related to other tissues, which also express P-gp at the apical membrane, i.e. interaction at the canalicular membranes of hepatocytes.

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Calcein, a MRP substrate, is also predominantly effluxed across the apical membrane of Caco-2 cells by MRP2 (Prime-Chapman et al., 2004). Parallel observations have been made in vivo by Fujita and co-workers who determined that calcein is effluxed from rat jejunum (Fujita et al., 1997). Calcein excretion is also observed across the intestine of mdr1a(−/−) mice, this secretion can be abolished by MK571, a MRP specific antibody (Stephens et al., 2002). This again confirms that observations involving transport in Caco-2 cells can reflect the situation in vivo, however again it is unknown whether such observations involving intestinal MRP2 can be used to study interactions at the biliary level. Caco-2 cells also express phase I and phase II metabolising enzymes such as CYP1A1 and glutathione Stransferase (Sergent-Engelen et al., 1993; Borlak and Zwadlo, 2003). Indeed Caco-2 cells endogenously express CYP1A1, CYP1A2, CYP2C8-19, CYP2E1, CYP3A5 and CYP2D6 mRNA, at 12-, 28-, 78-, 71-, 4- and 3-fold less than is expressed in human liver (Borlak and Zwadlo, 2003). In Caco-2 cells, the levels of CYP3A4, the main drugmetabolising enzyme of the intestine and liver, are low in comparison to normal tissue (Gan et al., 1996; Borlak and Zwadlo, 2003). To overcome this limitation, a number of groups have induced or transfected CYP3A4 into Caco-2 cells to investigate the relationship between P-gp and CYP3A4 in co-ordinating drug metabolism and excretion (Schmiedlin-Ren et al., 1997; Cummins et al., 2001, 2003; Benet and Cummins, 2001; Borlak and Zwadlo, 2003; Chan et al., 2004). Schmiedlin-Ren et al. (1997) showed successful induction of CYP3A4 at the levels of transcription and translation in Caco-2 parental cells by treating them with 1,25(OH)2 D3 . In a study by Cummings using CYP3A4 transfected Caco-2 cells, CYP3A4 protein levels were induced 40-fold with sodium butyrate and TPA, this 40-fold increase in protein was comparable with an observed increase in metabolism of midazolam, a specific CYP3A4 substrate (Cummins et al., 2001). In the same model the metabolism of K77 and sirolimus, substrates of both P-GP and CYP3A4, decreased when P-gp was selectively inhibited with GF120918, highlighting that P-gp increases the exposure of drugs to CYP3A4 in the intestine by repeated cycling of drug via diffusion and active efflux (Benet and Cummins, 2001). This drug metabolism alliance observed in vitro was mirrored in vivo using a rat single pass intestinal perfusion model, where K77 metabolism decreased on selective inhibition of P-gp (Cummins et al., 2003), highlighting the validity of observation made in Caco-2 cells in context to understanding metabolism/transporter interactions in vivo. P-gp-mediated observations in Caco-2 cells have been shown to predict the drug pharmacokinetics of orally administered drugs in wild type and mdr1a(−/−) mice. Collett et al. (2004) measured the ratio of A–B transport in the presence and absence of P-gp inhibitor, GF120918, of 10 compounds across Caco-2 cells. These data were then correlated with ratios of oral plasma levels in mdr1a(−/−) or mdr1a/1b(−/−) and wild type, calculated from literature

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data on these compounds. A significant correlation (r2 = 0.8, P < 0.01) was observed between the ratio of A–B transport in the presence and absence of GF120918 and the in vivo knockout/wild type oral plasma ratios. Therefore, the ability of Caco-2 cells to model a multi-component system, in which the combined effects of P-gp-mediated transport at several different barriers (e.g. intestine, liver) influence the plasma concentration of the drug suggest that the basic characteristics of transport are similar at each site. This supports the hypothesis that Caco-2 cells may be a valid model for P-gp-mediated transport for multiple tissues. This is in line with data generated in Caco-2 and the human hepatoma cell line, HepG2 in our own laboratory. A significant positive correlation was observed, all be it from a small data set, between the IC50 values of verapamil, erythromycin, quinidine and propranolol for the inhibition of rhodamine123 transport across and from Caco-2 and HepG2 cells, respectively.

poor yields. Ultimately, membrane vesicles are an artificial experimental system and using CMV alone cannot predict the contribution of any transporter in the biliary excretion of any given drug. Therefore, other systems that are more physiological must be used; such systems include cell cultures. Cell cultures represent a more advanced model than CMVs that may prove to be closer to the whole animal situation. Cell cultures in most cases are easier to manipulate, represent a more complete, cost-effective system, which has the potential to be more representative of the in vivo system. However, no truly representative in vitro cell culture model for predicting biliary interactions has yet been identified. HepG2 and WIF-B cells have been successfully employed in transport studies (Sormunen et al., 1993; Zaal et al., 1994; Roelofsen et al., 1997; Nies et al., 1998; Bravo, 1998). WIFB cells are a complex model, illustrating dual rat and human characteristics (Shanks et al., 1994). Routine culture is not trivial compared with more established lines. Thus, we focus on the potential of the HepG2 cell line.

4. Modelling hepatobiliary drug transport 4.1. HepG2 cells When screening compounds for their potential to undergo biliary clearance, the choice of model often represents a compromise between the level of throughput achievable and the predictive potential of the model. For example, whole animal intravenous bile duct cannulations (IVBDC) and isolated perfused liver studies reflect the true physiological state of the liver i.e. the liver has retained structure, functional hepatocytes and produces bile. However, such techniques are costly in terms of time, money and animal consumption, whilst being complex with regards to studying individual hepatic transporters, thus offsetting the benefits of such a model for high throughput screening. Freshly isolated hepatocytes are less complex than whole animal and whole organ studies and if isolated from human liver avoid species issues regarding hepatobiliary disposition (Tee et al., 1985). Although hepatocytes maintain liver specific functions which are lacking from other in vivo systems, such as canalicular membrane vesicles (CMVs) and non-mammalian expression systems, the loss of cell polarity during relatively short times in culture limits this system for studying biliary excretion. The development of methodology for the separation of canalicular and sinusoidal membranes of the hepatocyte has enabled the uptake and export mechanisms of these cells to be studied (Meier et al., 1984) as well as producing a valuable tool for screening compounds for the potential to undergo biliary clearance. Such experiments have the advantage that intracellular events such as metabolism, binding to intracellular proteins and organelles will not affect transport characteristics. Furthermore, due to the ease in which intracellular and extracellular conditions can be manipulated, ATP-dependence along with ion requirements for transport can be investigated. However, isolation of purified CMV, in the proper orientation is not trivial, due to multiple spinning and fractionation steps together with the issues of purity and

Among the human hepatic cell lines, HepG2 cells were derived from a human liver tumour and has been characterised to retain many xenobiotic-metabolising activities, along with several hepatocyte specific characteristics such as bile acid and albumin synthesis which are normally lost by hepatocytes in culture (Dierickx, 1989; Knowles et al., 1980; Javitt, 1990). Also, when grown on a monolayer a certain fraction of HepG2 cells become polarised, with microvilli lined vacuoles between adjacent cells, which resemble bile canaliculi. These vacuoles contain apical markers such as actin, fodrin, and villin, and are sealed by tight junctions and desmosomes (Chiu et al., 1990; Sormunen et al., 1993). HepG2 cells have been useful in predicting the metabolism and cytotoxicity of chemicals in human liver (Dai and Cederbaum, 1995; Wu and Cederbaum, 1996; Yoshitomi et al., 2001). However, HepG2’s show about 10% of the P450dependent monoxygenase activity of freshly isolated human hepatocytes, although the cell line does retain many phase II systems such as UDP-glucuronyltransferases, sulphate transferases and glutathione-S-transferases; indeed, the latter system is expressed at comparable levels to human adult hepatocytes (Doostdar et al., 1988; Duthie et al., 1988; Yoshitomi et al., 2001). This decrease in metabolic activity with regards to the phase I metabolism can be overcome by transfecting HepG2 cells with the appropriate enzymes. For example, the cytotoxicity of both ethanol and acetaminophen has been examined in HepG2 cells stably transfected with CYP2E1 (Dai and Cederbaum, 1995; Wu and Cederbaum, 1996). More recently CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 isoforms have been stably transfected into HepG2 cells, which provide valuable tools for the studying

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interaction involving these phase I enzymes (Yoshitomi et al., 2001). With regards to transporter expression RT-PCR analysis has revealed that OATP1A2 (OATP-A), OATP1B1, OATP3A1 (OATP-D), OATP4A1 (OATP-E), OATP1B3 (OATP-8), P-gp, MRP1, MRP2, MRP3 and MRP6 are expressed at the mRNA level in HepG2 cells (Lee and Piquette-Miller, 2001; Jung et al., 2001; Cantz et al., 2000). This is in contrast to findings by Koike and co-workers who found no MRP1 mRNA expression in HepG2 cells (Koike et al., 1997). Na+ -taurocholate co-transporting polypeptide (NTCP) is not expressed in HepG2’s as taurocholate uptake is Na+ -independent, however the uptake and efflux of taurocholate is sensitive to intracellular GSH levels (Lee and Piquette-Miller, 2001). P-gp, MRP1, MRP2 and MRP3 proteins are expressed at the apical, lateral, apical and basolateral membranes of HepG2 cells, respectively (Cantz et al., 2000; Roelofsen et al., 1997). The apical transporters are located in apical vacuoles and appear to be active with regards to P-gp and MRP2 activity, as known substrates of these transporters, such as rhodamine-labeled phosphatidylethanolamine, rhodamine123, glutathione-methylfluorescein (GS-MF), as well as fluo 3 have been shown to accumulate in these microvilli lined vacuoles (Sormunen et al., 1993; Zaal et al., 1994; Roelofsen et al., 1997). The bile salt export pump (BSEP/ABCB11) also appears to be functional at the apical membrane of HepG2 cells, as a fluorescent derivative of the bile salt cholyglycine is secreted into the apical vacuole of HepG2 cells (Keppler and Konig, 2000). MRP2 protein distribution is regulated by protein kinase C in HepG2 cells as treatment with the phorbal ester PMA, a protein kinase C agonist, results in a rapid relocation of apical localised MRP2 to the basolateral membrane, with a loss of pseudocanaliculi, and reduced secretion of GS-MF within 4 h. This redistribution of MRP2 is mirrored in PMA induced cholestasis rat liver (Kubitz et al., 2001). MRP2 mRNA and protein levels are inducible by rifampicin, tBHQ, arsenite and clotrimazole through the activation of the pregnane X receptor in HepG2 cells (Kauffmann et al., 2002). A twofold increase in MRP2 mRNA was observed in HepG2 cells when treated with rifampicin (10 ␮M), as was the case in primary human hepatocytes (Kast et al., 2002). The farnesoid X-activated receptor (FXR) and the constitutive androstane receptor (CAR) are also involved in MRP2 regulation in HepG2 cells as CDCA (100 ␮M), a FXR antagonist, and phenobarbitone (1 mM), a CAR antagonist, induce MRP2 mRNA by two-fold in both HepG2 cells and PXR null mice hepatocytes, respectively (Kast et al., 2002). MRP3 is also inducible by phenobarbitone (three-fold), 2-AAF (1.5-fold), cisplatin (1.5-fold) and rifampicin (1.5-fold), where MRP5 is induced by 2-AAF (five-fold), cisplatin (1.5-fold) and rifampicin (2.5fold) in HepG2 cells (Kiuchi et al., 1998; Schrenk et al., 2001). Therefore, HepG2 cells express and regulate numerous hepatic transporters observed in vivo therefore making HepG2 cells a good candidate for studying P-gp and MRP2 mediated interactions.

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5. What do the models mimic? It is only stating the obvious, that models are not the real situation; it cannot be emphasised enough that models are just that. Each model is a compromise between simplicity and the situation it is trying to model. Cultured cell models of intestinal or liver function offer an intermediate level of complexity, with multiple transport systems, which may reflect the organ in vivo. In this respect, what may be more important is that the model reflects the extent and nature of the transport (and metabolising) systems observed in the organ in situ, rather than necessarily reflecting any other organospecific phenotype. In this respect, intestinal Caco-2 cells may be a useful model of certain features of hepatobiliary clearance, notwithstanding their intestinal derivation. What is key is that the user understands the model and its limitations. In terms of drug discovery and development, it will still be essential to utilise animal models to demonstrate clearly the propensity for oral absorption and the limitations exposed by hepatobiliary clearance mechanisms. However, utilisation of models such as the Caco-2 and HepG2 cells could allow significant reductions in the need for such in vivo models, by allowing earlier identification, and hence elimination, of drug candidates with unfavourable absorptive characteristics. Furthermore, with the emergence of ‘animal on a chip’ technology which in theory consists of a silicon wafer which holds cells from a rat’s brain, heart and liver in different trenches linked by tiny fluid chambers (Khamsi, 2005), Caco-2 and HepG2 cells may be candidates for the production of ‘man on a chip’ technology. It is unlikely, given the appropriate emphasis on consumer safety, protection and efficacy of treatments, that these cell model systems will allow in vivo (animal) models to be readily dispensed with. However, careful application of these cell models at the early stages of development will not only reduce wastage and expense in drug development, but also allow for more strategic (reduced) numbers of in vivo animal experiments.

Acknowledgements We thank Dr. Anne Cooper, AstraZeneca R&D Charnwood, for helpful discussions. RAF was supported by a BBSRC-CASE studentship in collaboration with AstraZeneca Charnwood.

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