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Drug Metabolism Reviews, 39: 159–234, 2007 Copyright © Informa Healthcare ISSN: 0360-2532 print / 1097-9883 online DOI: 10.1080/03602530601093489

PRIMARY HEPATOCYTES: CURRENT UNDERSTANDING OF THE REGULATION OF METABOLIC ENZYMES AND TRANSPORTER PROTEINS, AND PHARMACEUTICAL PRACTICE FOR THE USE OF HEPATOCYTES IN METABOLISM, ENZYME INDUCTION, TRANSPORTER, CLEARANCE, AND HEPATOTOXICITY STUDIES Nicola J. Hewitt Scientific Writing Services, Wingertstrasse, Erzhausen, Germany

María José Gómez Lechón Unidad de Hepatología Experimental, Centro de Investigación Hospital La Fe, Valencia, Spain

J. Brian Houston, David Hallifax, and Hayley S. Brown School of Pharmacy and Pharmaceutical Sciences, University of Manchester, UK

Patrick Maurel INSERM, Montpellier, France; Univ Montpellier, Montpellier, France

J. Gerald Kenna Global Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, Cheshire, UK

Lena Gustavsson and Christina Lohmann Discovery DMPK & BA, AstraZeneca R&D Lund, Sweden

Christian Skonberg Danish University of Pharmaceutical Sciences, Department of Pharmaceutics and Analytical Chemistry, Universitetsparken, Copenhagen

Andre Guillouzo INSERM Université de Rennes, France

Gregor Tuschl Department of Molecular Toxicology, Institute of Toxicology, Merck KGaA, Frankfurterstrasse, Darmstadt, Germany

Albert P. Li The ADMET Group LLC, Rockville, MD

Edward LeCluyse CellzDirect, Hillsboro Street, Pittsboro, NC

Address correspondence to Nicola J. Hewitt; E-mail: [email protected] 159


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Geny M. M. Groothuis Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, University of Groningen, A.Deusinglaan, Groningen, The Netherlands

Jan G. Hengstler Centre of Toxicology, Institute of Legal Medicine and Rudolf-Boehm Institute, Haertelstr, University of Leipzig, Leipzig, Germany This review brings you up-to-date with the hepatocyte research on: 1) in vitro–in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and crosstalk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

INTRODUCTION Drug metabolism and drug transport are major determinants of drug clearance, interindividual pharmacokinetic differences, clinical efficacy, and toxicity of drugs (Gomez-Lechón et al., 2003; Lu, 1998). Inappropriate pharmacokinetics can result in inadequate pharmacodynamic action and/or extremely wide variations in clinical response. Unsatisfactory pharmacokinetic properties have been identified as a major reason for the failure of new chemical entities in drug development in man (Schuster et al., 2005). Moreover, hepatic metabolism of drugs as well as their interactions with hepatic transporters may cause toxicity (either to the liver itself or other organs) and contribute to variability between individuals in susceptibility to such adverse drug reactions. Therefore, key issues in the early phase of drug discovery and development include not only an exhaustive characterization of pharmacological activity, but also investigation of metabolic stability, metabolite profiles, major metabolic routes involved in metabolite formation, the enzymes involved, and the potential for enzyme inhibition or induction. Isolated hepatocytes are now recognized as one of the most relevant and practical models with which the study of drug metabolism and transporter interactions is best performed. When isolated and handled appropriately, they contain a broad complement of metabolizing enzymes and transport proteins, organized in a physiologically relevant context and regulated via cellular processes that occur within the liver in vivo (e.g., nuclear hormone-mediated xenosensors). Microsomes continue to be the first-line screening model for high throughput assays. However, studies undertaken with microsomes are increasingly being replaced or complemented by the use of hepatocytes. The practical and technical difficulties posed by hepatocyte isolation have been largely circumvented by use of cryo-preserved cells. In fact, the number of publications in the last 5 years citing metabolism in hepatocytes have increased by approximately 30%. During the same period, the number of citations of metabolism in microsomes dropped by 25% (source: MedLine). Microsomes are gradually being replaced by hepatocytes because the quality of cryopreserved hepatocytes has improved. As a result of the increasing demand for a more relevant model, the application of hepatocytes to higher throughput assays has been improved by more sophisticated automation instruments and miniaturization methods.


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This review focuses on the use of hepatocytes, as well as hepatocyte-like cell lines for induction, metabolism, clearance, cytotoxicity, and transporter studies. Methodologies used for each of these are outlined and interpretations of results are discussed. In vitro–in vivo correlations are needed to demonstrate the predictive strength of any assay and are discussed using selected case studies as examples. The Basic Hepatocyte Culture—does Matrix Overlay Make a Difference? The establishment of long-term cultures of primary hepatocytes has long been desired and many efforts have been undertaken to achieve this goal (LeCluyse et al., 1996a,b). There is a strong need for robust long-term in vitro screening models, the use of which reduces the number of animals used in drug development. Today, cultures of primary human and animal hepatocytes have been adopted for a variety of pharmacological and toxicological experiments, allowing for the study of chronic effects in vitro. Although in vitro experimental models can never resemble the complexity of a whole organism, their simplicity provides the ability to specifically manipulate and analyze single parameters. Culturing hepatocytes in a sandwich configuration between two layers of gelled extracellular matrix proteins, with collagen I and matrigel being the most commonly used, has dramatically prolonged the longevity of cultures displaying hepatocyte-specific functions (Dunn et al., 1991; Richert et al., 2002). In addition, medium formulation (e.g., the addition/omission of serum or specified hormone mixtures) has a significant influence on the morphological development and cell survival of hepatocytes in culture (Pascussi et al., 2000a,b; Sidhu et al., 2004; Turncliff et al., 2004). Figure 1 depicts the morphological changes occurring over time of hepatocytes cultured in different conditions. Cells are generally seeded in medium containing fetal calf serum since this enhances the surface attachment ability of hepatocytes (Williams et al., 1977). The morphological distinction between hepatocytes seeded onto collagen-coated plates without a collagen gel overlay (conventional monolayers) and those seeded onto collagen gel with a subsequent collagen gel overlay (sandwich) is visible only a few hours after seeding. Conventional monolayer hepatocytes quickly adopt their polygonal shape and establish extensive cell-cell contacts whereas, in sandwich culture this takes markedly longer, being still mostly spherical and singular at this time point (Fig. 1B and C). In general, conventional monolayers appear more flattened than sandwich-cultured cells, a result of the lack of a three-dimensional extracellular matrix environment. All cells display clear cytoplasm and well-delineated plasma membranes. After overnight incubation, sandwich culture hepatocytes form aggregates with a typical cuboidal shape (Fig. 1B and C). In both the sandwich culture models, structures resembling bile canaliculi form during the subsequent 24 h, but to a greater extent in the serum-free cultures (Fig. 1C and D). This is consistent with the findings of Terry and Gallin (1994) who reported that serum is capable of inhibiting the re-establishment of bile canaliculi in hepatocyte cultures. In both serum-free and serum-containing conventional cultures there is a strong perturbation of cell morphology after 72 h, (Fig. 1A, serum-free cultures not shown). Hepatocytes spread out and form fibroblast-like protrusions. The nuclear volume increases and the cytoplasm appears granulated. There are no longer welldelineated plasma membrane borders and bile canaliculi-like structures disappear almost entirely. The morphological stability of conventional cultures can be increased considerably if these cultures are overlaid with collagen gel. However, it must be kept in mind that the time of overlay is crucial for the phenotypic outcome. Since conventional hepatocyte monolayer cultures do not have a steady appearance over time, even in the first days of


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(A) Conventional monolayers with serum 4 hours after plating

(B) Sandwich cultures with serum 4 hours after plating

(C) Sandwich cultures – serum-free 4 hours after plating

24 hours of culture

24 hours of culture

24 hours of culture

72 hours of culture

72 hours of culture

72h of culture

4 day cultures with overlay after 4 hours

Overlay after 72 hours

Figure 1 Morphology of rat hepatocytes cultured for up to 72 h in different conditions. Culture conditions were conventional monolayers cultured on collagen-coated plates with no collagen overlay (A), sandwich cultures on collagen-coated plates with a collagen gel overlay in serum-containing medium (B) and sandwich cultures on collagen-coated plates with a collagen gel overlay in serum-free medium (C).



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culture, they should be overlaid within the first 24 h. Best results are obtained if the monolayer/sandwich hybrid is prepared at the initial medium change, 4 h after seeding. In this configuration, the morphology at the time of overlay can be preserved for more than 1 week. However, if the hepatotypic morphology is lost after several days on collagen-coated plates, it will not be restored after applying the gel overlay; only the state at the time of overlay will be preserved. By overlaying monolayer cultures of primary hepatocytes with collagen gel, it is possible to obtain cultures displaying some features of collagen sandwich cultures over extended periods of time (e.g., bile canaliculi-like structures), although the overall appearance is still different (Fig. 1A). Cells cultured as a collagen sandwich in serum-free medium do not significantly spread out, and polygonal cell formats, clear plasma membrane boundaries and stable bile canaliculi-like networks are still evident after 72 h of culture (Fig. 1C). In contrast, hepatocytes incubated in serum-containing medium noticeably deteriorate and lose cytoplasmic integrity and stability of their canaliculi-like structures (Fig. 1B). This is in agreement with the previously reported beneficial effects of an extracellular matrix overlay on cell viability and the preservation of normal morphology (Dunn et al., 1991; Musat et al., 1993; LeCluyse et al., 1994). Interestingly, hepatocytes seeded on collagen type I gel and overlaid with a layer of matrigel do not demonstrate superior survival in comparison to cells overlaid with another layer of gelled collagen I, exemplified by their lack of stable bile canaliculi-like structures (data not shown). Thus, the use of collagen type I gel as extracellular matrix component for hepatocyte sandwich cultures is appropriate for longterm cultures. The fact that primary hepatocytes, cultured in serum-free collagen sandwich cultures, stay morphologically unchanged for a few weeks and offer the ability to investigate alterations in cellular structures induced by chemical treatment with the use of high content imaging. In addition to the extracellular matrix application and media formulation, cell density also has some influence on the morphology of hepatocytes in culture (Hamilton et al., 2001). If seeded at a very high density (almost 100% confluence), cells in conventional cultures do not tend to spread out significantly, rather they display hepatocyte-like morphology for 1–2 more days, but then start to detach from the surface. Interestingly, the same cell seeding density (cells/cm2) will not result in the same confluency of cells seeded onto collagen film and gel, since more hepatocytes fit into a three-dimensional extracellular matrix gel environment. Since polar differentiation of hepatocytes in culture is only visible in cell aggregates, cell density should always be close to confluency (≥90%). The quality of the cell culture not only depends on the conditions applied after seeding, but also on the initial cell suspension. While for rat hepatocytes, this is usually not a very big issue because, unlike human hepatocytes, hepatocytes from a single rat strain should not vary significantly (Olinga et al., 1998a; Richert et al., 2004). Once the cells are in culture, the addition of the glucocorticoid hormone, dexamethasone, is valuable for the long-term preservation of hepatocyte-specific functions, polygonal hepatocyte morphology, as well as the structural integrity of cytoplasmic membranes, especially bile canaliculi-like structures (Yamada et al., 1980; Laishes and Williams, 1976). Although sandwich culture of hepatocytes under serum-free conditions (after 4 h attachment period) allows conservation of the cells in a differentiated state for several weeks, dexamethasone can considerably improve and prolong the differentiation status of serum-containing cultures. However, culturing hepatocytes with serum-supplemented medium may lead to fibroblast overgrowth after several days, regardless of whether the cells are cultured in a conventional or sandwich configuration.


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METABOLISM In Vitro-In Vivo Correlations Human hepatocytes once isolated, are placed in chemically defined culture conditions where they express typical hepatic biochemical functions and contain the entire hepatic drug-metabolizing enzyme system in an integrated form providing an in vitro model that is a very useful tool for anticipating drug metabolism and drug hepatotoxicity in man (Ferrini et al., 1997; Gómez-Lechón et al., 2003, 2004; Guillouzo et al., 1997). The fact that cultured hepatocytes are kept in an artificial environment differing from that of the liver might result in relevant phenotype changes. Thus, it can be argued that the drug metabolism activities of cultured human hepatocytes may significantly differ from in vivo and this would cast doubt on the value and relevance of in vitro data. Differences in metabolism are frequently reported among cell culture preparations from different donors. This obviously raises the question of whether the variability seen in vitro is an artifactual culture phenotypic change due to the method of preparation or whether this variability is due to or reflects the donor’s phenotype. Several studies show that primary hepatocyte cultures are a good model to qualitatively predict the in vivo metabolic profile of a drug (Bort et al., 1996a,b; Hewittt et al., 2001; Ponsoda et al., 2001; Salonen et al., 2003). Aceclofenac, a well-tolerated antiinflammatory analgesic drug with a well-characterized metabolism, has been studied to directly compare in vitro–in vivo metabolism in the same donors. Aceclofenac undergoes CYP-mediated oxidation in the liver of man to 4′-hydroxy-aceclofenac (Bort et al., 1996a). The drug can also undergo enzymatic hydrolysis by a hepatic esterase to form diclofenac which, in turn, can be 4′-hydroxylated (Bort et al., 1996b). This is a minor pathway representing only 5–10% of the total metabolized drug. Glucuronide conjugates as well as traces of other hydroxylated metabolites are also formed and largely excreted in urine, together with the unchanged drug. The metabolism of aceclofenac was studied in vitro in human hepatocyte cultures prepared from the liver resects of 13 human patients that, after clinical recovery, received a subclinical dose of the drug to examine the metabolism in vivo (drug metabolites in urine were measured). The culture supernatants from incubations of human hepatocytes with aceclofenac were analyzed and the chromatograms were compared to those corresponding urine samples from the same patient after oral administration of the drug (Ponsoda et al., 2001). The study offered the unique possibility of comparing, in the same individuals, the in vitro (cultured hepatocytes) and in vivo metabolism of the drug. There were variations in the metabolite profile and rate of metabolism of aceclofenac observed in different cell preparations, and moreover, they actually reflected the interindividual variability among donors (Ponsoda et al., 2001). The first clear finding was the remarkable similarity between the in vitro and in vivo metabolic profile of the drug for each donor. The rate of 4′-hydroxyaceclofenac formation, which represented about 90% of the hydroxylated metabolites, in vitro correlated well with the excretion in vivo (Fig. 2A). Some variability was found in the degree of in vitro hydrolysis, estimated as the percentage of 4′-hydroxydiclofenac over total 4′-hydroxymetabolites (4′-hydroxydiclofenac plus 4′-hydroxyaceclofenac). However, this variability was also reflected in in vivo samples, which suggested that the variability of the esterase activity observed in vitro mirrored that existing in vivo (Fig. 2B). The rate of metabolism of a drug can easily be determined in vitro by measuring the disappearance over time as well as the formation of specific metabolites. The metabolism of aceclofenac by human hepatocytes in culture was linear for several hours, making it possible to determine the rate of metabolism

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Figure 2 Comparison of metabolism of aceclofenac in vivo and in vitro. Urine was collected during 16 h after drug administration and cultures were incubated for 4 h with 200 μM aceclofenac. CYP2C9 activity (A): Comparison of 4′-hydroxyaceclofenac (4′-OHace) levels in vivo and in vitro, is represented by the % of metabolites formed. Esterase activity (B): Comparison of the ratio of 4′-hydroxydiclofenac and the sum of 4′-hydroxydiclofenac and 4′-hydroxyaceclofenac (4′-OH metabolites). Rate of metabolism (C): a comparison of the levels of 4′-hydroxyaceclofenac in urine and the rate in vitro estimated during 4 h as pmol/min × mg protein. Lineal fit to results between in vivo and in vitro (solid lines), confidence interval (95%, slashed lines) and prediction interval (95%, dotted lines).

for each cell culture preparation. These values were suitable for comparison with the total amount of metabolites excreted in urine following drug intake (Fig. 2C). These results showed, for the first time, conclusive evidence of high in vitro–in vivo similarities existing in drug metabolism. Moreover, the study revealed that the interindividual variations in drug metabolism are also evident in in vitro studies. The results support the use of cultured human hepatocytes to anticipate in vivo metabolic profile of a drug and to explore possible drug-drug interactions. In Vitro CYP Variability as a Reflection of Human Liver CYP Pattern Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to explaining high interindividual metabolic variations. Phenotypic as well as genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes for this variability. Genetic factors, specifically gene polymorphisms, are involved. Among P450 polymorphisms, those affecting CYP2C9, CYP2C19, and CYP2D6 have the highest impact on drug metabolism (Table 1). In particular, CYP2D6 polymorphism requires special mention as it has been estimated that this isoform accounts for the metabolism of 25–30% of drugs used in Table 1 Polymorphic P450 Drug-Metabolising Enzymes. Enzyme CYP1A2 CYP2A6 CYP2C9 CYP2C19 CYP2D6 CYP2E1

Functional allelic variants

Major variant

Allele frequency

Phenotype

13 16 12 16 46 2

CYP1A2*1B CYP2A6* 2 CYP2C9* 3 CYP2C19* 2 CYP2D6* 4 CYP2E1* 3

12 % Japanese 1–3% Caucasians 7–9% Caucasians 13% Caucasians 12–21% Caucasians 500-fold; Fig. 3C), CYP2C9 (10-fold), and CYP2C19 (40-fold), activities that could be partially explained on the basis of genotype distribution. Large differences in CYP activities were also evident in cultured human hepatocytes from different donors and appear to reflect the heterogeneity of CYP expression in human liver among donors. This interindividual variability is observed in total CYP activity as well as in individual CYP activities, such as CYP3A4 (Fig. 3B), CYP2D6 (Fig. 3D),

0

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Figure 3 Variability of major drug-metabolising P450 activities in human liver and human hepatocytes from different donors. P450 activities, determined in microsomes from a human liver bank (A and C) and in 24 h cultured human hepatocytes (B and D) using selective reactions, are plotted and sorted in decreasing order, to show interindividual variability. Genotypes of human P450s were analyzed by PCR in the same livers. CYP3A4 activity was determined by measuring testosterone 6β-hydroxylation and CYP2D6 activity was determined by measuring dextromethorphan O-demethylation. *CYP2D6*1*4; # CYP2D6*4/*4.



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CYP1A2, CYP2C19, and CYP2C9. This variability is markedly higher than that in other species (Donato et al., 1999). Primary cultured human hepatocytes actively express a full set of P450s, as well as phase 2 drug-metabolizing enzymes, although they show a gradual decrease in CYP expression over time, probably due to the adaptation of cells to the culture environment (LeCluyse, 2001; Rodríguez-Antona et al., 2002; Gómez-Lechón et al., 2004). It is generally accepted that this early decrease in CYP expression applies to all CYPs. However, it can be argued that differences in stability of individual CYPs in culture could result in an artificial culture phenotype that does not reflect the donor phenotype. By comparing CYP3A4 activity in cultured hepatocytes and microsomes from the same liver before cell isolation, reasonably good correlations between both human hepatic models has been demonstrated. However, activities in microsomes were higher than those found in hepatocytes prepared from the same liver sample (Gómez-Lechón et al., 2004). The rates of testosterone hydroxylation at different positions observed in cultured hepatocytes and microsomes obtained from the same livers correlated well with CYP3A4 and CYP2C9 activities and androstenedione formation in the same samples (Gómez-Lechón et al., 2004). These findings suggest that, in spite of losses of CYP activities in cultured cells, metabolic capabilities of the two hepatic models are qualitatively comparable. In summary, it can be reasonably considered that human hepatocytes reflect the heterogeneity of P450 expression in human liver and constitute an invaluable tool for preclinical metabolic and toxicity testing of new chemical entities (Donato et al., 1995; Gomez-Lechón et al., 2004; LeCluyse, 2001). Given the great interindividual variability of CYP patterns in man, the prediction of drug metabolic profiles, drug-drug interactions and toxicity of new drugs in the human population should be investigated in hepatocytes from more than one donor. ENZYME INDUCTION Clinical Relevance A compound may cause an increased activity of one or more enzymes and their induction can have serious consequences: 1) the drug itself, or a coadministered drug, will be cleared more quickly and their pharmacological effect will be reduced; 2) the toxicity of a drug or coadministered drug may be increased due to the production of reactive intermediates via the induced pathway. The clinical implications of enzyme induction should not be ignored since failure to do so has proven to have serious consequences (Lu and Cederbaum, 2006; Shah, 2005). The inhibition of enzymes is easy to measure because the onset of the effect is normally rapid and reproducible in vitro by using recombinant enzymes, human liver microsomes (Favreau et al., 1999; Nomeir et al., 2001) or even hepatocytes (Zhao et al., 2005). It is a direct interaction between enzyme and substrate. In contrast, induction occurs over a period of hours to days or weeks in vivo. Like inhibition, induction results in altered pharmacokinetics or pharmacodynamics (Dilger et al., 2000; Kolars et al., 1991). Large donor variation in the responses to different inducers further complicates the interpretation of induction assays and the prediction of enzyme induction at therapeutic doses. Methods for measuring enzyme induction are more complex because there are a number of mechanisms involved in this process. For example, induction of CYP1A2 by β-naphthoflavone involves de novo synthesis of mRNA, protein synthesis and post-translational modification to a functional enzyme (Hollenberg, 2002), whereas isoniazid induction of CYP2E1 involves the stabilization of the enzyme itself (Fuhr, 2000;


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Koop and Tierney, 1990; Novak and Woodcroft, 2000; Raucy et al., 2004). Our understanding of the mechanisms of enzyme induction is increasing and the use of both primary hepatocytes and cell lines have been integral to this advancement. Human primary hepatocytes are the gold standard for metabolism and induction studies and this is unlikely to change due to the more plentiful supply of fresh hepatocytes and the more widespread and accepted use of cryo-preserved human hepatocytes. Here we describe how hepatic enzymes are regulated and induced and discuss the methods to measure these processes. Relevant Enzymes and Regulation The choice of which enzyme to test for determining the potential induction depends on a number of factors. The FDA suggests that enzymes involved in the metabolism of a compound should be studied for potential induction by that compound in clinical trials. In contrast, enzyme(s) not involved in the metabolism of the compound do not need to be studied in clinical studies (Bjornsson et al., 2003). This is based on the assumption that a compound may induce its own metabolism (autoinduction). As a result, many researchers in the pharmaceutical industry screen new compounds for the contribution of single enzymes to the metabolism of a compound using recombinant microsomes. Enzymes that do not cause over 20% depletion of the parent compound (at low concentrations (μM) over a first-order decay curve) are not tested further in induction studies. However, there are examples of compounds that induce enzymes but are not involved in their metabolism (e.g., omeprazole is metabolized by CYP2C19 (Andersson, 1996) but induces CYP1A2 (Curi-Pedrosa et al., 1994; Daujat et al., 1992). Therefore, in addition to testing those CYPs involved in the metabolism of the compound, other enzymes should be considered. Table 2 summarizes the relative content of CYP isoforms in human liver and the contribution of each CYP to drug metabolism. CYP3A4 is generally considered as the most relevant enzyme to test since it is the most abundant enzyme in the liver and is involved in the metabolism of over 50% of drugs. The CYP2C family also represents a significant proportion of total P450s (2C9, 2C8, 2C19, and 2C18, representing about 20% of the total P450 (Lin and Lu, 1998)) and metabolizes many drugs (Zuber et al., 2002), thus making this enzyme subfamily important to monitor. CYP1A2 is a minor enzyme in the liver and only a small number of drugs (4%) are metabolized by this enzyme (Zuber et al., 2002), however, it is involved in the bioactivation of pro-carcinogens and is therefore considered to be an important enzyme to test (Guengerich et al., 1990). CYP2B6 is emerging as an

Table 2 Relative Abundance of Human CYP Enzymes and their Contribution to Drug Metabolism. Enzyme CYP3A4/5 CYP2D6 CYP2C9 CYP2C8 CYP2C19 CYP1A2 CYP2E1 CYP2B6

Content in liver (% total CYP) (1)

(2)

30 , 29 1.5(2) 4(2) 12(4) 7(4) 0.2(4) 12(1) 13(2) 7(2) 0.2(2)–5(3)

% Of drugs metabolized by enzyme 52(1) 30(1, 2) 11(1) 10(2) n.r. 4(1) 4(1) 6(2) 2(1) 5(2) 25(3)

(1) Zuber et al. (2002); (2) Shimada et al. (1994); (3) Wang et al. (2003); (4) Lin and Lu (1998); n.r. = no reference.



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important enzyme in drug-drug interactions despite a previously reported low abundance in the liver (0.2% of total P450 (Lin and Lu, 1998)). However, once thought to be of minor importance and uninducible in humans (Hollenberg, 2002), CYP2B6 may actually constitute at least 5% of the total P450, contribute to the metabolism of more than 25% of all pharmaceutical drug metabolism (Code et al., 1997; Ekins and Wrighton, 1999; Faucette et al., 2000; Hanna et al., 2000; Hesse et al., 2000; Stresser and Kupfer, 1999) and exhibit high inducibility (Drocourt et al., 2001; Wang et al., 2003; Xie et al., 2000). CYP2E1 is absent in 30% of the sample population (Ekström et al., 1989) and is not considered by the FDA as an important enzyme to test for induction potential (Bjornsson et al., 2003). CYP2D6 has no known inducer and represents only 2% of the total P450 (Lin and Lu, 1998), however, researchers persist to test this enzyme because of its major contribution to drug metabolism and its polymorphism (Zuber et al., 2002). Many drugs are metabolized by direct conjugation with glucuronic acid (Nagar and Remmel, 2006; Soars et al., 2004) but the potential to induce UDP-glucuronosyl S-transferases (UGTs) is seldom studied. There is a large interindividual variability in UGT enzyme activities, possibly due to polymorphisms of the enzyme itself (e.g., UGT2B15*2 (Chung et al., 2005)) or of the regulating nuclear receptor, PXR (Gardner-Stephen et al., 2004). UGT1A1 is induced by fibric acid derivatives in human hepatocytes (although not via PXR), suggesting that these drugs have a potential to cause various pharmacokinetic drug interactions in vivo (Prueksaritanont et al., 2005). However, Soars et al. (2004) found that although UGT isoforms could be statistically significantly increased, the induction varied greatly between donors and was not as large as that observed for CYP1A2 or CYP3A4. They concluded that the clinical relevance of the induction responses obtained in their study was unclear. Selective induction of SULT has been demonstrated in human hepatocytes, the consequence of such induction can lead to failure of the therapeutic efficacy of the compound (Li et al., 1999a). XENOSENSORS The aryl hydrocarbon receptor (Ahr), pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are integral to the regulation and induction of the main P450s. These receptors control the expression of CYP1A (Ahr), CYP2, and CYP3A (PXR and CAR) families, as well as UGTs and glutathione-S-transferases and the transporters MDR1 and MRP2 (Bock et al., 1990; Kullak-Ublick and Becker, 2003). It is important to understand the regulation of these enzymes, transporters, and receptors from a clinical point of view because they play a major role in drug detoxification and drug-drug interactions (McInnes and Brodie, 1988). Ahr and CAR are located in the cytoplasm and after ligand-binding are translocated to the nucleus. CAR is thought to be present in the cytosol in its inactive form, bound to endogenous steroids but in the presence of phenobarbitone (PB) or “PB-like” inducers, it is dephosphorylated to trigger its translocation to the nucleus (Kawana et al., 2003; Squires et al., 2004). The Ah receptor is present as a complex with Hsp90 (a heat shock protein) but dissociates with the Hsp protein and translocates to the nucleus once an inducer binds to it (Hollenberg, 2002). PXR may also be found in the cytoplasm (Kawana et al., 2003; Squires et al., 2004) but is primarily located in the nucleus. Once activated, the receptors form heterodimers with other factors, such as Arnt (Ahr nuclear translocator) and retinoid X receptor (RXR for both PXR and CAR) and then bind to the target xenobiotic response elements (XRE) located in both the proximal and distal P450 gene promoters, resulting in the transcription of the respective CYP isoform (Goodwin et al., 1999; Lehmann et al., 1998; Moore et al., 2000; Sueyoshi et al.,


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1999). In turn, PXR and CAR expression is mediated by the glucocorticoid receptor (GR) in human hepatocytes (Pascussi et al., 2000a,b). Inducers of PXR-mediated CYP3A4 include natural glucocorticoids and synthetic steroids as well as a wide spectrum of xenobiotics (Drocourt et al., 2001; Goodwin et al., 2002; Kliewer et al., 2002; Waxman, 1999). Calcium channel modulators such as nifedipine, as well as being CYP3A4 inducers, also induce CYP2C9, CYP2C8, CYP2C19, and CYP2B6 (Drocourt et al., 2001) suggesting that there is co-regulation of these five enzymes via PXR and/or CAR (as well as GR, which regulates both CAR and PXR) (Gerbal-Chaloin et al., 2001; Smirlis et al., 2001; Xie et al., 2000). The regulation and the mechanisms of induction of CYPs have been extensively reviewed (Hollenberg, 2002; Lin and Lu, 1998; Tang et al., 2005). Cross-Talk between Xenosensors: Another Argument Why Primary Human Hepatocytes are Essential One of the reasons for the differences in drug metabolism and signal transduction between primary hepatocytes and hepatoma cell lines is downregulation of several xenosensors. PXR and CAR are strongly downregulated or absent in cell lines, such as HepG2 or Hepa-1c1c7 (Pascussi et al., 2001). This is a likely reason why CYP3A4 is not expressed (or strongly reduced) in hepatoma cell lines. In contrast, Ahr is still expressed in HepG2 and Hepa-1c1c7 cells; therefore, CYP1A enzymes are still inducible in these hepatoma cell lines. In view of these well-documented differences, one might predict differences in xenobiotic metabolism between primary hepatocytes and hepatoma cell lines. However, Gerbal-Chaloin et al. (2006) have demonstrated that the complexity of crosstalk between PXR/CYP3A4 and Ahr should not be underestimated. In their studies omeprazole sulfide, a metabolite of the gastric proton pump inhibitor omeprazole, alone had no effect on CYP1A1 expression in Hepa-1c1c7 (Fig. 4A). However, the TCDD-mediated induction of CYP1A1 mRNA was strongly suppressed by omeprazole sulfide (Fig. 4A). Similar results were obtained in HepG2 cells. In sharp contrast to the hepatoma cells, the omeprazole sulfide induced CYP1A1 mRNA in primary hepatocytes (Fig. 4B) and did not significantly reduce the TCDD-induced induction of CYP1A1 mRNA expression (Fig. 4B). As a result of a series of elegant studies, Gerbal-Chaloin et al. (2006) put forward a hypothesis explaining the discrepancy between primary hepatocytes and hepatoma cell lines (Fig. 5). Omeprazole sulfide is an antagonist of Ahr by stabilizing its inactive form. Therefore, conversion of the agonist-dependent Ahr to a DNA-binding form is inhibited. In contrast, omeprazole is a hydrocarbon-like inducer of CYP1A1/2 (Diaz et al., 1990). In primary human hepatocytes, CYP3A4 rapidly converts omeprazole sulfide to omeprazole (Fig. 5), leading to activation of Ahr and expression of its target genes, such as CYP1A1/2. In contrast, omeprazole sulfide is not metabolized in hepatoma cell lines, due to lack of CYP3A4. Therefore, omeprazole sulfide inhibits Ahr, thereby decreasing TCDD-induced expression of CYP1A1 mRNA. This scenario also explains the influence of CYP3A4 modifiers. The CYP3A4 inducer, rifampin, not only increases CYP3A4 activity it plays a role in the activation of Ahr-controlled genes in the presence of omeprazole sulfide. The reverse is true for CYP3A4 inhibitors, such as ketoconazole, which antagonize Ahr-mediated effects. It should be considered that CYP3A4 expression decreases in long-term cultures of human hepatocytes in the absence of inducers, such that the cells behave similarly to hepatoma cells, showing no CYP1A1 mRNA induction by omeprazole sulfide, but an inhibition of Ahr-dependent effects (Fig. 4C). This underlines the importance of culturing human hepatocytes in the presence of appropriate cocktails of inducers to maintain a



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A

Hepa-1 cells

B

C

Freshly plated

15 Days culture

Primary human hepatocytes Figure 4 Different effects of omeprazole sulfide (OMS) in the human hepatoma cell line Hepa-1c1c7 and in primary human hepatocytes. In Hepa-1c1c7 cells (A) OMS alone has no effect on CYP1A1 mRNA expression but suppresses TCDD induced induction of CYP1A1 mRNA. In freshly plated primary human hepatocytes (B) OMS induces CYP1A1 mRNA expression but does not significantly antagonize induction of CYP1A1 mRNA by TCDD. After 15 days in culture in the absence of inducers (C) primary human hepatocytes behave similar to hepatoma cells, showing no CYP1A1 induction by OMS but a decrease in CYP1A1 induction by TCDD (from: Gerbal-Chaloin et al., 2006).

metabolic competence in these cells (Pichard-Garcia et al., 2002). In conclusion, the mere use of hepatoma cell lines can be misleading, since differences in expression of drug metabolizing enzymes may compromise the complex cross-talk between xenobiotics, metabolites and xenosensors. A Model for Physiological and Stress-induced Cross-talk of Xenosensors The differential effects of low and high glucocorticoid concentrations on the drug metabolizing enzymes and nuclear receptors have been well documented. Pretreatment of hepatocytes with low concentrations of dexamethasone (for example, 30 nM), does not cause increased expression of CYP3A or CYP2B per se but serves as a permissive factor allowing a stronger induction by enzyme inducers (Pascussi et al., 2001,2003; Ringel et al., 2002). In contrast, high dexamethasone concentrations of more than 1 μM lead to increased levels of CYP3A and CYP2B without additional xenobiotics. Figure 6 summarizes a proposed model, based on a large number of studies, explaining the different roles

N. J. HEWITT ET AL.

Ketoconazole

OMS

CYP3A4

PXR

Rifampin RXR


172

OM

AhR

AhR XRE ARNT

Transcription of CYP1A1 and other AhR Target genes Figure 5 The cross-talk between PXR/CYP3A4 and Ahr explains the differences between human hepatoma cell lines and primary human hepatocytes. OMS is an antagonist of the Ahr. In contrast, omeprazole (OM) is a hydrocarbon-like inducer of CYP1A1/2. CYP3A4 converts OMS to OM. In primary human hepatocytes OMS is metabolized to OM, leading to activation of the Ahr and expression of CYP1A1. However, hepatoma cell lines do not express CYP3A4 and consequently OMS remains unmetabolized. Therefore, OMS inhibits the Ahr, thereby decreasing expression of CYP1A1. This scenario also explains the influence of the CYP3A4 modifiers rifampin and ketoconazole (from: Gerbal-Chaloin et al., 2006).

of sub- (A) and supramicromolar (B) concentrations of glucocorticoids. Hydrocortisol, the major glucocorticoid in blood, exhibits a circadian fluctuation with concentrations ranging between 0.1 and 0.45 μM. In this concentration range, hydrocortisone increases expression of PXR, CAR, and RXR (Fig. 6). Depending on the presence of basal concentrations of xenobiotic ligands, PXR, CAR, and RXR may serve as positive transcription regulators of CYP3A, CYP2B, MDR1, and MRP2 (Fig. 6A). This model is supported by the observation that the circadian variation of hepatic CYP3A4 in man correlates with cortisol in its circadian fluctuation (Ohno et al., 2000; Pascussi et al., 2003). In addition, physiological concentrations of 0.1–0.45 μM hydrocortisone have been shown to be sufficient for activation of the GR. Besides transcriptional activation of the xenosensors PXR, CAR, and RXR, glucocorticoides induce expression of CYP2C9 by interacting with a glucocorticoid response element in the 5′-flanking region of CYP2C9 (Gerbal-Chaloin et al., 2002). Supramicromolar concentrations of hydrocortisone may occur in vivo as a consequence of severe stress, for instance after intoxication. In fact, glucocorticoids are direct ligands activating PXR but only at supramicromolar concentrations. Under these conditions



A

173

Glucocorticoid

NUCLEUS

GR

HSP

GR

CYP2C9

PXR

GR

GRE

(CAR, RXR)

GRE Xenobiotic ligands

CYP3A4 CYP2B6 MDR1 MRP2

PXR XRE

Physiological levels

B

Glucocorticoid

HSP NUCLEUS

GR

CYP2C9

GR

GR

GRE

Direct ligand only at supramicromolar concentrations

PXR

(CAR, RXR)

GRE

PXR XRE

CYP3A4 CYP2B6 MDR1 MRP2 Induced levels

Increased detoxification and excretion

Figure 6 Different responses at physiological and stress-induced glucocorticoid concentrations. Physiological concentrations (0.1–0.45 μM hydrocortisone) (A). In this concentration range, hydrocortisone increases expression of PXR, CAR and RXR after binding of the activated glucocorticoid receptor (GR) to the glucocorticoid responsive element (GRE). At supramicromolar concentrations (B) glucocorticoids are direct ligands activating PXR. Under these conditions hydrocortisone induces expression of CYP3A4, CYP2B6, MDR1 and MRP2 independent from other xenobiotic ligands (from: Pascussi et al., 2003).


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hydrocortisone induces expression of CYP3A4, CYP2B6, MDR1, and MRP2 independent from other xenobiotic ligands (Fig. 6B). The resulting levels are much higher compared to physiological nonstress conditions. Thus, stress-associated glucocorticoids work synergistically to upregulate genes involved in detoxification and elimination of xenobiotics which may be crucial during life-threatening intoxication.

Adenoviral Transduction of Transcription Factors: Insight into the Regulation of CYPs More insight into the regulation of CYP enzymes has been gained by the use of replication-defective adenoviruses to deliver transgenes into hepatocytes. This is a very efficient method of introducing a foreign gene into human hepatocytes in culture (80% of cells are transduced after 1 h) without affecting urea and plasma protein synthesis or xenobiotic biotransformation activities (1A2, 2A1, 2B6, 3A4/5). Although glycolysis and gluconeogenesis in transduced hepatocytes were moderately altered (by only 20%), the expression of inducible genes (acute-phase plasma proteins, CYPs) was not impaired in CYP3A4 transduced human hepatocytes upon stimulation with interleukin (IL-6) or methylcholanthrene (Castell et al., 1997a). Inflammation and more specifically, cytokine release, causes downregulation of CYPs. CYP3A4 is downregulated by IL-6 (the principal regulator of the hepatic acute-phase response) and this requires activation of the glycoprotein receptor gp130 (Jover et al., 2002). By overexpressing dominant-negative STAT3 and C/EBPβ-LIP (CCAAT-enhancer binding protein β) using adenoviral vectors in hepatocytes, it was found that IL-6 downregulates CYP3A4 through translational induction of C/EBPβ-LIP, which competes with and antagonizes constitutive C/EBP transactivators. An adenoviral vector was used to study the role of hepatocyte nuclear factor 4 (HNF4) in the regulation of CYPs in human hepatocytes (Jover et al., 2001). HNF4 is a member of the nuclear receptor superfamily that activates specific CYP promoters from several species. It is also the key transcription factor regulating responses to xenobiotics through activation of the PXR gene during fetal liver development (Kamiya et al., 2003). Transduction of human hepatocytes with the recombinant adenovirus containing an HNF4 antisense RNA caused a time-dependent increase in the HNF4 antisense transcript, followed by a concomitant decrease in apolipoprotein C III mRNA (a target gene of HNF4). CYP gene expression analysis of human hepatocytes transfected with HNF4 antisense RNA indicated that HNF4 is involved in the regulation of CYP3A4, CYP3A5, CYP2A6, CYP2B6, CYP2C9, and CYP2D6 (but not CYP2E1). HNF-3γ is another hepatocyte nuclear factor important for the maintenance of specific liver functions. Gene reporter experiments with proximal promoters showed that HNF-3γ transactivated CYP2C8, CYP2C9, and CYP2C19 but not CYP2C18. Overexpression of HNF-3γ in hepatoma cells induced CYP2C9, CYP2C18, and CYP2C19 mRNA but did not activate CYP2C8 (Bort et al., 2004). Adenovirus transduction has also been used to try to differentiate hepatocyte-like cell lines by overexpressing molecules such as p21 (a potent cyclin-dependent kinase inhibitor involved in terminal cellular differentiation). Cells that had been infected with p21 showed differentiated hepatic phenotypes in morphology and increased CYP3A4 and CYP2C9 protein expression (Kobayashi et al., 2002a; Kunieda et al., 2002). Studies incorporating the use of adenoviral vectors as efficient transfection tools undoubtedly add knowledge to the field of CYP regulation and phenotype differentiation.



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Effects of Culture Time and Format on CYP Expression In order to measure induction effects of compounds, it is important to recognize that changes in CYP expression occurs even in the absence of inducers. Cultures of primary hepatocytes show significant alterations in CYP expression over time but also in relation to each other. In particular, in the rat, cytochrome P450 genes CYP2C and CYP4A1 are considerably decreased over time in the serum-containing conventional monolayer cultures after 3 days (Tschul and Mueller, 2006). CYP1A1 levels are strongly increased in the serum-free conventional cultures and to a lesser extent in sandwich cultures. After 72 h in culture, CYP mRNAs are more abundant in the serum-free cultures than in serumcontaining cultures. Indeed, there is evidence that the presence of dexamethasone (Sidhu et al., 1995) and a three dimensional extracellular matrix environment (Sidhu et al., 1993) helps maintain constant levels of cytochrome P450 expression. The low abundance of CYP mRNAs at 72 h indicates a limited metabolic capacity in serum-containing collagen conventional cultures over extended periods of time, making more suitable for short-term applications (Grant et al., 1985). In general, variations in gene expression over time were least pronounced in the serum-free collagen sandwich cultures. Therefore, this culture seems most adequate for the illustration of gene expression alterations triggered by compounds. Species-Specific Model Inducers Animal induction studies are useful to interpret initial in vivo animal PK studies but they should not be used to predict induction responses in humans. The reason for this is the marked differences in CYP isoforms, their substrate specificities and the optimal CYP inducers (Table 3). A well-known example of this is CYP3A4 in human hepatocytes, which is potently induced by rifampin and moderately induced by dexamethasone (Lu and Li, 2001; Maurel, 1996). In contrast, dexamethasone and pregnenalone-16α-carbonitril (PCN) are potent inducers of rat CYP3A1/2 whereas rifampin is a relatively poor inducer of this enzyme (Lu and Li, 2001; Maurel, 1996). There are also species differences in omeprazole induction of CYP1A2, which is induced in human but not in rat, mouse or rabbit hepatocytes (Diaz et al., 1990; Lu and Li, 2001; McDonnell et al., 1992). The rat is most noted for its sex-specific differences in CYP activities, which is in direct contrast to human CYPs (Parkinson et al., 2004). CYP2A2, CYP2C11, CYP2C13, and CYP3A2 are 10 times higher in male than in female rat livers—attributed to the pulsatile secretion of growth hormones specific only to male rats. Female rats continuously secrete growth hormones that cause them to have female-specific enzymes such as CYP2C12 and steroid 5α-reductase (Parkinson, 2001; Waxman et al., 1991a). CYP2B and CYP3A2 induction by PB and PCN, respectively, was higher in female than in male rat hepatocytes but only because the basal levels of these enzymes were much lower than those in male rat hepatocytes (Parkinson, 2001; Parkinson et al., 2004; Waxman et al., 1991b). FDA Regulations and Pharmaceutical Practice The FDA publishes guidelines for the conduct and interpretation of induction studies. However, according to a survey of 30 researchers of the pharmaceutical industry (Hewitt, accepted for publication in Chemico-Biological Interactions), few strictly adhere to these suggestions. The survey was carried out to get an insight into pharmaceutical

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CYP Inducer Substrate CYP Inducer Substrate

CYP Inducer Substrate

1A

2C

2A6 PB, RIF Coumarin 2E1 Isoniazid, PB, RIF

Chlorzoxazone

CYP Inducer Substrate CYP Inducer

Substrate

2A

Chlorzoxazone, p-nitrophenol

N/A 2E1 Ethanol, Acetonepyrazine

Chlorzoxazone p-nitrophenol

Absent PB N/A 2E1 Ethanol

Burforolol

Dextromethorphan

p-nitrophenol

2E1 (not isoniazid)

Dextromethorphan, Bufurolol

2D15

Lauric acid 2D9–13

2C21(minor CYP) PB Tolbutamide

1A1/2 OM, BNF 3-MC Phenacetin, ER 2B11 PB BR, diazapam

Absent

Absent

Dog 3A12 RIF Testosterone, Midazolam

4A10/12/14

2C29 Phenytoin Tolbutamide

1A1/2 PB Phenacetin ER, MR 2B9/10/13/19 PB BR, PR

3A11 PB Testosterone, Nifedipine

Mouse

4A1/2/3/8 Clofibrate (4A1) Lauric acid 2D1/2

2C11, CYP2C6 Dex Diclofenac, Testosterone (16α- and 2α-hydroxide)

1A1/2 BNF, 3-MC, PB Phenacetin, ER 2B1/2 PB BR, PR, testosterone

3A1/2 DEX, PB Testosterone, Midazolam

Rat

Coumarin 2E1 3-MC, Polychlorinated biphenyl Chlorzoxazone

2A

Dextromethorpham

2D17

2C, 2C20 Atorvastatin Tolbutamide (2C20) S-mephenytoin (2C). Testosterone (ASD formation) Absent

1A1/2 BNF Caffeine, ER, Phenacetin 2B17 PB Testosterone (16α-OHT)

3A8 PB, RIF Testosterone, Midazolam

Monkey

RIF=rifampin, PB=phenobarbitone, Dex=dexamethasone, BNF=β-naphthoflavone, 3-MC=3-methylcholanthrene, ER=ethoxyresorufin, MR=methoxyresorufin, BR=benzyloxyresorufin, PR=pentoxyresorufin, OM=omeprazole. Main reference for P450 in different species: Nelson et al. (1993). Specific references for CYPs: Kobayashi et al. (2002b); Shou et al. (2003); Zuber et al. (2002); Roussel et al. (1998); Kuroha et al. (2002); Mankowski et al. (1999); Paulson et al. (1999); Burke et al. (1985); Harauchi and Hirata (1994); Arlotto et al. (1991). References for substrates and inducers of human P450s: http://www.theberries.ns.ca/Archives/cyp450.html; http://www.dcmsonline.org/jax-medicone/1998journals/ august98/hivdrugstables.htm; http://References for animal CYP induction References for animal CYP induction: Galisteo et al. (1999); Posti et al. (1999); Emoto et al. (2000).

2E

2D

CYP Inducer Substrate CYP Inducer Substrate

4A

4A9/11 Clofibrate Lauric acid 2D6 No known inducer Dextromethorphan

3A4/5 RIF Testosterone, Midazolam, Nifedipine 1A1/2 OM, BNF, 3-MC Phenacetin, ER 2B6/7 PB S-mephenytoin (N-demethylation) Bupropion, BR, PR 2C8/9/18/19 RIF (2C9, 2C19), PB (2C19) Paclitaxal (2C8), Tolbutamide, Diclofenac (2C9), S-Mephenytoin (hydroxylation, 2C19)

CYP Inducer Substrate

3A

2B

Human

CYP

Table 3 A Summary of the CYPs in Various Species – Isoforms, Substrates and Inducers.




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practices of induction studies and was prompted by a project aimed at consolidating experimental conditions. The results were somewhat surprising considering that all companies have to satisfy the FDA regulations. It may be that companies conduct these studies according to the class of compound, knowing that they might exhibit certain properties predisposing them to induction potential but filtering these compounds out when they reach the drug development stage. Sandwich cultures are the preferred culture format of the FDA (to maintain the viability and morphology, not necessarily to increase the induction response) but only 30% of those questioned used sandwich cultures. One researcher noted that the bioavailability of polypeptide test compounds was compromised by the collagen overlay, thus making conventional monolayers with no overlay more appropriate in their studies. The sandwich culture format allows hepatocytes to remain relatively differentiated compared with cultures with no overlay. Investigating CYP induction in serum-free collagen sandwich cultures of primary hepatocytes may confer a more stable expression of CYPs over longer times, resulting in more reproducible results (Runge et al., 2000). An advantage of long-term hepatocyte cultures is that they allow researchers to determine whether induction is reversible once treatment is stopped and the culture continued. Based on this theory, one suggestion is to use serum-free collagen sandwich cultures to examine CYP induction of several consecutive test compounds in the same cultures with recovery periods between treatments to return CYP expression back to basal levels. This would be a step towards more efficient use of cultures in preclinical drug development. It has been shown that sandwich cultures express P-gp transporter proteins, polarized in the bile cannuliculi (Liu et al., 1999). The presence of P-gp proteins, as well as uptake transporters such as OATP and OCT (discussed later in this review) determines the intracellular concentration of an inducer and therefore determines its induction potency. For example, rifampin is taken up into hepatocytes by OATP 1B1, which has been shown to exhibit a number of variants (Tirona et al., 2001). If cells express the variant OATP 1B1, then the induction by rifampin may potentially be weakened. The effects of transporter variants highlights the need to determine how culture conditions influence the expression and function of uptake and efflux proteins and how this subsequently affects the induction response of compounds that are substrates for these transporters. Human hepatocyte cultures are normally allowed 48 h to establish good cell-cell contact, a critical characteristic for differentiation (Corlu et al., 1991; Guguen-Guillouzo et al., 1983) and induction potential (Maurel, 1996). The induction response is much lower if the cells are not allowed time to recover (Maurel, 1996). The majority of survey participants allowed a 24–48 h recovery period; however, one researcher allowed only 3 h for the cells to recover but still reported good induction responses. The induction period is suggested by the FDA to be at least 72 h (Bjornssen et al., 2003) in order to detect increases in enzyme activities and to take into account the half-life of 20–40 h of the CYP proteins (Maurel, 1996). Most researchers in the survey and others (Kostrubsky et al., 1999; Maurel, 1996; Raucy et al., 2004) incubated compounds for only 48 h, whereas others preferred longer periods (Madan et al., 2003; Meunier et al., 2000; Robertson et al., 2000). All participants used the recommended positive controls (Bjornsson et al., 2003), however, they set the validation criteria at different thresholds, some expecting more than 2-fold and other more than 3-fold induction for rifampin induction. Moreover, the threshold at which a test compound was considered to be positive varied from 1.5- to 5-fold above control. Most used the FDA recommendation at the time of the survey of 2-fold or 40 % of the positive control (Bjornssen et al., 2003). Although there were close similarities, out of 30 participants, no two participants carried out and interpreted their induction studies in


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exactly the same way. Once a compound is found to be positive in an in vitro hepatocyte induction assay, few pharmaceutical companies will terminate the development the compound (only 17 % in the survey). Most take the advice of the FDA and request extra samples to be included in the first clinical studies to check for possible drug-drug interactions (Bjornsson et al., 2003). Many will also take other parameters of the drug into consideration, such as bioavailability, protein-binding, therapeutic plasma concentration, co-therapies, and so on, rather than make one criterion “kill or keep” decision. Strategies of Interpreting In Vitro Induction The interpretation of the in vivo relevance in man of in vitro induction data poses a number of problems since many compounds that have been tested for their induction potential do not advance to clinical studies (Smith, 2000). Retrospective studies suggest that induction observed in vivo can be reproduced in vitro and there are numerous reports of good correlations (Drocourt et al., 2001; Faucette et al., 2004; Hariparsad et al., 2004; Kato et al., 2005; Komoroski et al., 2004; Kostrubsky et al., 1998; Li et al., 1997; PichardGarcia et al., 2000; Raucy et al., 2004; Ring et al., 2005; Robertson et al., 2000; Roymans et al., 2005; Sahi et al., 2003), however, ambiguity remains as to how to accurately predict the potency of potential inducers. Induction is dose-dependent, however, many test only three concentrations of test compound (a possible therapeutic concentration, 10-fold therapeutic concentration, and the highest nontoxic concentration). Care must be taken when using high concentrations, since the induction effect has been shown to disappear at high concentrations (Maurel, 1996), possibly due to cytotoxicity. Now that higher throughput multiwell assays are being developed, the possibility of testing a range of test compound concentrations means that EC50 values can be determined for induction responses. One other suggestion is a safety factor to reflect the difference between the highest concentration which caused no induction (NOEL) and the therapeutic concentration. Others use an EC50 value to extrapolate potency data; however, this may not take into account the slope of the dose response curve thus the NOEL may serve as a better indicator of induction potential. In this way, lead optimization assays can be used to give a quantitative risk assessment and a ranking of compounds into strong, moderate, and noninducers. The use of Fresh and Cryo-preserved Hepatocytes for Induction Studies There are many reports of successful comparisons of drug metabolism in fresh and cryo-preserved human hepatocytes (Hewitt et al., 2001; Li et al., 1999b; McGinnity et al., 2004; Soars et al., 2002) but the ability to cryo-preserve hepatocytes which also attach in culture has been somewhat of a holy grail over the last 20 years. The plateability of cryopreserved hepatocytes cannot be attributed to a single parameter but undoubtedly the better the quality of the hepatocytes cryo-preserved, the higher the probability of them being plateable after thawing. Indeed, some have shown that attachment of both cryo-preserved rat and human hepatocytes can be improved by preincubating the fresh hepatocytes with fructose and/or alpha-lipoic acid prior to freezing (Terry et al., 2006). Likewise, Silva et al. (1999) simply preincubated fresh cells in glucose-containing Krebs-Henseleit buffer for 30 min in a 5% CO2 and 95% O2 atmosphere prior to cryo-preserving to improve the attachment efficiency of both rat and human cryo-preserved hepatocytes. It is likely that other factors also influence the recovery of hepatocytes, such as the transport medium used to transfer the liver to the laboratory, the time between organ procurement and the



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start of collagenase digestion, the condition of the liver (fatty livers require longer collagenase digestion times) and the isolation time itself. The effect of age, ethnicity, gender, steatosis, and transport buffers on fresh hepatocyte viability, attachment efficiency, and enzyme activities has been comprehensively reviewed (Gomez-Lechon et al., 2004; Parkinson et al., 2004). The viability and attachment efficiency of fresh hepatocytes was higher when they were isolated from biopsies of liver than from whole liver perfusions. Terry et al. (2005) reported a difference in the attachment efficiency of cryo-preserved hepatocytes originating from whole liver and from healthy sections of liver where the other part was used for transplantation however, the post-thaw viabilities of both were very low (36% and 47%, respectively). Li et al., (1992) were rather more successful with isolating fresh hepatocytes and reported over 80% viability of hepatocytes isolated from all liver samples (including healthy and diseased livers). Although the initial viability is not a direct indication of the attachment potential, high initial viabilities are required if they are to show any attachment in culture. Cryo-preserved hepatocytes with high viabilities and good metabolic capacities have been reported (Chesne and Guillouzo, 1988; Hewitt et al., 2001; Kafert-Kasting et al., 2006; Li et al.; 1999b; Silva et al., 1999; Skett et al., 1999). Early reports demonstrated a limited attachment efficiency of human hepatocytes after thawing (Chesné and Guillouzo, 1988; Lawrence and Benford, 1991). In these studies, cells were cryo-preserved in 16–20% DMSO and thawed in ice-cold glucosecontaining medium to offset the osmotic effect of diluting DMSO. Another common method to reduce the osmotic effect of diluting DMSO was to slowly add small volumes of medium over a period of 10 min to allow DMSO to equilibrate between the medium and the intracellular environment (de Loecker et al., 1993; Li et al., 1999b). Some have used culture plates coated with human liver extracellular matrix to improve the attachment of thawed cells (Moshage et al., 1988), while others improved attachment by co-culturing hepatocytes with rat liver epithelial cells (Fautrel et al., 1997) or removed dead cells by Percoll purification (Garcia et al., 2003; Skett et al., 1999). Kafert-Kasting et al. (2006) tried to overcome poor attachment efficiencies by seeding double the normal number of fresh cells per well onto a collagen gel and cryo-preserving the entire culture plate. The cultures were cryo-preserved in 10% DMSO, stored in vapor phase nitrogen and then thawed in a 37°C incubator. This is a method that was previously used by Koebe et al. (1996, 1999) to cryo-preserve porcine hepatocytes but they used a double gel sandwich culture. The advantage of the single gel method is that it allows for any dead cells to detach from the plate after thawing, leaving only the functional cells. A second layer of collagen can be added subsequently. Despite the efforts to optimize attachment, it was reported in 1997 (Ruegg et al., 1997) that only one in nine human hepatocyte preparations survived in culture after cryo-preservation. The most important improvement in the attachment efficiency of cryo-preserved human hepatocytes came when the method of thawing was switched to a “quick thaw” – cells were thawed rapidly in a water bath and then poured into warmed medium (kept at 37°C). After centrifugation, and importantly without the need for Percoll purification, these cells formed confluent monolayers, viable for at least 5 days (Roymans et al., 2005). At least in the experience of Albert Li (personal communication), cryo-preserved human hepatocytes that were previously nonplateable became plateable by using the warm-thaw method (personal communication). This suggests that at least for some batches of hepatocytes, the hepatocytes were of good quality and the cryo-preservation was optimal but the method thawing was equally important. Silva et al. (1999) had previously reported that the attachment of cryo-preserved human hepatocytes was improved by preincubating the cells to increase the ATP levels in the


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cells but in their studies, they also thawed the cells in warmed medium. The combination of not one but both of these conditions may well have increased the success of these experiments. Although enzyme activities of freshly thawed cryo-preserved human hepatocytes are similar to that in fresh hepatocytes (Li et al., 1999b), there are reports that some batches of cryo-preserved hepatocyte cultures tend to have lower activities than those reported in corresponding fresh hepatocytes (Kafert-Kasting et al., 2006; Roymans et al., 2004, 2005). Despite this, all batches of cells that have attached were responsive to inducers at similar fold-inductions to that seen for fresh cells. Moreover, the induction was evident as mRNA induction, protein increases, and functional enzyme activities (Garcia et al., 2003; Kafert-Kasting et al., 2006; Roymans et al., 2004, 2005; Silva et al., 1999). Despite the advances in the production of cryo-preserved hepatocytes for induction studies, strong debate remains as to the validity of using these cells. Up until October 2004, the FDA recommended that only fresh cells could be used for induction studies because there was not sufficient evidence that cryo-preserved hepatocytes would attach and form healthy confluent monolayers needed to achieve an induction response (Bjornssen et al., 2003; Maurel et al., 1994). However, as a result of a number of reports, the FDA issued a draft in 2004 suggesting that cryo-preserved hepatocytes could be used if they were shown to attach sufficiently in culture. As with fresh hepatocytes, cryo-preserved hepatocytes should be shown to be responsive to positive control compounds and the fold induction should be similar to that seen in fresh cells. Figure 7 shows a comparison of induction of mRNA and enzyme activities of CYP1A2 (A) and CYP3A4 (B) in fresh and cryo-preserved hepatocytes (from different donors). The ranges of induction seen in cryopreserved hepatocyte cultures were comparable with fresh hepatocytes. A major advantage of cryo-preserved hepatocytes is that the experiment can be repeated using the same conditions with the result that the data is reproducible. Figure 8 shows the magnitude of induction of CYP3A4 varies considerably between different donors (A) but the induction response using cryo-preserved hepatocytes from the same donor is reproducible from one experiment to another (B). Roymans et al. (2005) studied the induction response of cryo-preserved hepatocytes to prototypical inducers of CYP1A, CYP2C9, CYP219, CYP2B6, and CYP3A4. They found that there was a donor-to-donor variation, also observed with fresh hepatocytes, but hepatocytes from each donor were able to respond to the selective inducers. Interestingly, in an earlier report by Roymans et al. (2004), the relative potency of three glitazone drugs, troglitazone, rosiglitazone, and plioglitazone, in inducing CYP1A2 and CY3A4 in cryo-preserved human hepatocytes reflected that already seen in fresh hepatocyte studies. Omeprazole and lanzoprazole were demonstrated to be equipotent in inducing CYP1A2 and moderate inducers of CYP3A4 in cryo-preserved hepatocytes, in accordance with that observed in fresh cells (Curi-Pedrosa et al., 1994). Roymans et al. (2005) extended their studies to other P450s and found that CYP2C9, CYP2B6, and CYP2E1 in cryo-preserved hepatocytes were inducible. The induction of CYP2E1 is not via mRNA formation and subsequent protein synthesis but through increased translational efficiency and stabilization of the protein involving ubiquitinylation and proteasomal degradation (Fuhr, 2000; Koop and Tierney, 1990; Novak and Woodcroft, 2000). Induction of CYP2E1 by omperazole and ethanol was evident as an increase in chlorzoxasone 6-hydroxylation and an increase in the CYP2E1 protein detected by western blotting, whereas CYP2E1 mRNA was unaltered. Thus, cryopreserved hepatocytes were able to reproduce mechanisms known to occur in fresh hepatocytes, at least with respect to the major enzymes involved in drug metabolism. Work

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continues to increase the success rate for isolating and cryo-preserving human hepatocytes that can be used for longer-term studies such as induction. Conclusions on the Use of Hepatocytes for Induction Studies The methods of determining enzyme induction, whether at the mRNA or protein enzyme levels, have been constantly refined over the last decade, resulting in extensive knowledge of the regulation and alteration of phases 1 and 2 metabolic enzymes. The use

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of alternative cell systems has increased our understanding of enzyme regulation and the importance of the balance of factors (such as nuclear receptors and coexpressed enzymes) involved in this process. Human hepatocytes are clearly the most relevant model for assessing enzyme induction, since they are much more differentiated than cell lines. The advances in cryo-preservation techniques have allowed for multiple studies on human hepatocytes from the same donor, making these assays more robust. The relevance of enzyme induction and its interpretation may well be more complex than simply measuring an alteration enzyme function per se, and consideration of EC50s may be more appropriate when making “go–no go” decisions in the drug development process. TRANSPORTER-MEDIATED UPTAKE AND EFFLUX Relevant Transporters and Regulation It has long been recognized that compounds can only pass membranes by passive diffusion if they are lipophilic. Hydrophilic, and in particular, charged (ionized) compounds can only pass a cell membrane with the help of a transporter. This transport process is carrier-mediated and may be facilitated by driving forces such as electrochemical gradients, or by an active, energy-dependent process. Both facilitated and active carriermediated processes are temperature-dependent and saturable but only active transport is ATP-dependent. It is now known that a large number of drugs and their metabolites enter or exit cells via transporters. Around 1980, it was generally thought that there were three categories of transporters: one for negatively charged, one for positively charged, and one for neutral compounds. This knowledge was based on functional and inhibition studies in perfused livers and isolated hepatocytes. However, with the emergence of molecular biology techniques, there has been a burst of available data to identify transporters involved in uptake and excretion of drugs. We now know that several dozen transporters are involved in drug uptake or excretion and that they are present in virtually every organ in the body. Endogenous substrates have been identified for most of these transporters. They also



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exhibit multispecific affinity for exogenous compounds, such as drugs and toxins. Although their functions have been most extensively studied in the liver, there are increasing data available on transporters present in the intestine, kidney, brain, and other organs (van Montfoort et al., 2003). As with metabolic enzymes, transporters can be categorized into families and subfamilies, based on amino acid homology. More transporters continue to be identified but the most important families thus far identified are 1) the solute carrier SLC family, comprising among others such as Na+-taurocholate co-transporting polypeptides (NTCP), organic anion-transporting polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs); and 2) the ATP-binding cassette (ABC) transporter family, including the multi drug resistance proteins (MDR), bile salt export pump (BSEP) (both belong to the ABCB family), breast cancer resistance protein (BCRP; belonging to the ABCG or White family) and the multi drug resistance associated proteins (MRP’s), belonging to the ABCC family. Like metabolizing enzymes, transporter families have several different members (isoforms) which vary considerably between species. The isoforms exhibit species variations in expression as well as substrate specificity. Figure 9 is a schematic representation of the most relevant transporters in human hepatocytes. The SLC members are expressed on the basolateral side of the hepatocyte. The OATPs have broad substrate specificities and are responsible for uptake of large amphipathic organic anionic drugs, uncharged compounds, and large amphipathic cationic drugs. The OATs and OCTs are involved in hepatic uptake of small organic anions and cations, respectively. The OATPs, OATs, and OCTs are essentially bidirectional transporters and although they usually function as uptake carrier, they may also serve as sinusoidal excretion carrier, dependent on conditions such as substrate concentration gradients and other driving forces. NTCP is mainly responsible for sodium-dependent uptake of conjugated bile salts and in rat but not in human, also of sulfated steroids and thyroid hormones. As this transport is dependent on the Na+ gradient, this transport is active and unidirectional. MDR1 (also referred to as P-glycoprotein or P-gp) is localized at the apical membrane of the hepatocyte and is involved in biliary excretion of a large variety of structurally unrelated compounds, among others bulky hydrophobic cationic compounds, but also OATP2B1

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steroid hormones and uncharged compounds. MDR1 expression is very low under basal conditions but is rapidly upregulated under stress conditions or by the inducing effects of drugs. In rat and mouse, there are two isoforms, mrd1a and mdr1b, which have different substrate specificities and regulation. MDR3 is involved in phospholipid excretion whereas BSEP is responsible for the excretion of bile salts into the bile. The members of the MRP subfamily transport mainly bulky amphipathic organic anions. MRP2 is localized in the apical membrane and excretes mainly anionic conjugates, among others bilirubin glucuronides, leukotriene C4 and glutathione. The other members of the MRP (MRP 1,3,4,6) family are located on the basolateral membrane and are responsible for the removal of conjugates formed in the liver via the urine. They mediate the excretion of organic anions into the sinusoidal bloodstream as well as the basolateral excretion of glutathione and leukotriene C4. MRP1 and 3 are expressed in low levels under normal conditions, but are upregulated under conditions in which MRP2 is downregulated (for instance in cholestatic conditions). They have similar substrate specificities with the exception of glutathione conjugates, which are poor substrates for MRP3, and bile salts, which are poor substrates for MRP1. BCRP (or ABCG2) is a half transporter and it is suggested to function as homodimer. It is involved in the biliary excretion of a variety of anticancer drugs and may also transport endogenous compounds such as porphyrins (Doyle et al., 2003). As transporters may influence significantly the pharmacokinetics of a drug, it is important to study the involvement of drug transporters when the pharmacokinetic behavior of a new drug is studied. Transporters mediate nonmetabolic clearance of compounds into bile. Moreover, transporters determine the intracellular concentration of drugs and their metabolites and thereby the exposure of the organ to the drug itself and its metabolites. As metabolites may be toxic, the toxicity of drugs can also be related to transporter function. Comprehensive knowledge of transporters may help to understand species and interindividual differences, as well as predict drug-drug and food-drug interactions. Furthermore, various disease states may influence the expression of the transporters and thereby influence the pharmacokinetics of a drug. It is, thus, important to increase our knowledge of organ specificity and cellular localization of transporters, their substrates and inhibitor specificity, species differences, and changes in expression due to disease and compounds, and so on. The expression levels of transporters are regulated in coordination with metabolizing enzymes so it is not surprising that they share common nuclear factors. For instance, several drugs that are known to induce metabolic enzymes via the CAR-mediated pathway also induce MRP2 and MRP3. Bile acids that influence the expression of CYP7A1 and CYP8B1 also influence the expression of BSEP, OATP 1B3, and MRP2 in rat but not ntcp in human (Jung et al. manuscript in preparation). A large number of drugs concomitantly induce the expression of CYP3A4 (via the PXR pathway) and MDR1, MRP2,3,4, and OATP1A2. Recently, Elferink et al. (2004) showed that lipopolysaccharide-induced downregulation of NTCP in human liver was correlated with TNFα and IL-1β levels. The downregulation of mrp2 mRNA expression in rat liver upon lipopolysaccharide treatment was not observed in human liver, although protein expression of MRP2 was decreased, indicating species differences in the regulation of this transporter (Elferink et al., 2004). In addition to this common regulatory pathway, there is another level of interaction between metabolism and transport. Many transporters and metabolizing enzymes exhibit affinity for the same substrates. In addition, many transporters transport metabolites, therefore, it is easy to envisage that metabolism and transport also have a high level of interplay, and that inhibition of transporters may influence the amount of drug metabolized



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and vice versa (Shitara et al., 2003). The most studied example is the interaction between P-gp and CYP3A4 (Fig. 10). Promoter studies have helped to understand the regulation of transporters by identifying responsive elements and analyzing the effects of transcription factors. To investigate whether these in vitro findings are operative in the human liver cell, expression levels of transporters may be analyzed in human tissues obtained from healthy and diseased patients. In vitro studies with human isolated hepatocytes or liver slices are very useful to analyze the effects and mechanisms of regulatory factors on both RNA and protein expression and on the function of the expressed transporters. This is important because regulation does not only take place at the mRNA expression level but also on the levels of protein synthesis, breakdown, localization, and (de)activation.

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Hepatocyte Models for Transporter Studies Numerous ex-vivo or in vitro models are available for exploring the contribution of transporters to drug metabolism and excretion, including liver perfusion (Ruiz et al., 2006), liver slices (Elferink et al., 2004; Olinga et al., 2001), primary hepatocyte cultures (either as a conventional monolayer with no overlay or as a sandwich culture (Liu et al., 1999; Rose et al., 2006)), primary hepatocyte suspensions (Reichard et al., 2003; Sandker et al., 1994) and transfected cell lines expressing defined transporters (Biermann et al., 2006; Sahi, 2005). The identification of which transporters are involved in the uptake and efflux of a particular drug requires a model that maintains physiological expression of most, if not all transporters. Hepatocytes in liver slices retain transporters in a polarized localization, they have an intact architecture with both parenchymal and nonparenchymal cells, representative of the in vivo environment and they are easy to prepare. Using this model, clear species differences have been identified in the expression and regulation of transporters (Elferink et al., 2004) and the substrate specificities (Olinga et al., 2001; Terdoslavich et al. (manuscript in preparation)). The main disadvantage of liver slices is that the supply of intact human tissue is prohibitive; therefore, many researchers prefer to use isolated human hepatocytes to investigate transporters. Initial experiments aimed to optimize methods using fresh rat hepatocytes and subsequent assays were successfully extended to fresh and cryo-preserved human hepatocytes (Liu et al., 1999; Olinga et al., 2001; Sandker et al., 1994; Yamashiro et al., 2006). Sugiyama’s group has extensive experience of using cryo-preserved human hepatocytes for transporter studies. One such report compared the uptake properties of taurocholate and estradiol 17β-D-glucuronide in freshly isolated and cryo-preserved human hepatocytes. Uptake of taurocholate was sodium-dependent in both fresh and cryo-preserved hepatocytes. Moreover, the Km values for these compounds in cryo-preserved human hepatocytes were within the range of those previously reported for NTCP (taurocholate), OATP 1B1, and OATP 1B3 (estradiol 17β-D-glucuronide). Cryo-preserved human hepatocytes have also been used to determine the relative importance of specific uptake transporters in drug uptake clearance. One such example is pitavastatin, a novel potent 3-hydroxymethylglutaryl-CoA reductase inhibitor (Hirano et al., 2004). The uptake clearance of pitavastatin was compared with that of reference compounds (estrone-3-sulfate, a selective substrate for OATP 1B1 and cholecystokinin octapeptide for OATP 1B3) in transporter-expressing HEK293 cells and cryo-preserved human hepatocytes. Using this method, the observed uptake clearance of pitavastatin in human hepatocytes could be almost completely accounted for by OATP 1B1 and OATP 1B3 and about 90% of the total hepatic clearance could be accounted for by OATP 1B1. Thus, cryo-preserved human hepatocytes retain sufficient transporter functions to be used as a practical alternative to fresh hepatocytes to study the mechanism of hepatic uptake of drugs. Uptake Studies in Hepatocytes Uptake of drugs across the sinusoidal membrane is the first step in hepatobiliary clearance and for some polar drugs with low passive permeability (e.g., pravastatin) this mechanism has been proposed to be the rate determining step (Treiber et al., 2004). The uptake CLint combined with CLint for efflux and metabolism are cellular processes that make up the total clearance (Liu and Pang, 2005). A commonly used method to determine the contribution of transporter-mediated uptake to total uptake of a drug is to perform uptake studies at 37°C and 4°C. Preferably, this should be combined with a concentration dependence study to look for saturation of uptake at higher drug concentrations.

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Transporter-mediated uptake is highly temperature-dependent and for compounds with low passive diffusion, very low uptake is observed at 4°C (Fig. 11). However, passive diffusion is also temperature-dependent to some degree; therefore, it may be difficult to distinguish the contribution of carrier-mediated transport and passive diffusion for compounds with a high passive permeability. Concentration dependence experiments will give a fuller picture of the contribution of transporter-mediated vs. passive permeability and moreover, Km and Vmax calculations will aid the understanding of the affinity and capacity of the

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uptake. One important limitation of the concentration dependence experiment is that the uptake of low solubility compounds will be limited by the compound available in solution.The concentration-dependent uptake of two compounds into rat hepatocytes is illustrated in Fig. 12. Compound A, a low log D compound, shows a clear transporter-dependent uptake with

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very little uptake at 4°C and a saturation at high compound concentrations. The Km for the uptake was 27 μM and calculation of CLint from Km and Vmax resulted in a predicted hepatic clearance that was in good agreement with the clearance observed in vivo in rat. The uptake profile of the second compound B, which has a log D of 2, is typical when passive diffusion is the major mechanism for uptake. Another approach to evaluate transporter-dependent uptake of compounds that allows a higher throughput, is to measure the rate of uptake in the presence of transporter inhibitors (Fig. 13). Preferably, inhibitors that are relatively broad are useful in order to

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identify compounds that have a transporter-dependent uptake for example, cyclosporine A for OATPs, quinidine for OCTs, and glibenclamide for OATPs and OATs (Arndt et al., 2001; Sahi, 2005; Shitara et al., 2002; Uwai et al., 2000). This approach could be used as a first step to identify which group of transporter-mediated uptake plays an important role in compound uptake. The second step in the compound evaluation may then be to perform concentration dependence studies at 37°C and 4°C to get a better understanding for the uptake kinetics. Uptake of compounds can be directly monitored by incubating suspensions or cultured hepatocytes (either short-term (2 h) or longer term (4 days) with transporter substrates and measuring the amount of compound in lysed cells after a set period (either by radiodetection or LC MS/MS). The uptake in hepatocyte suspensions can be stopped by rapid centrifugation of the cells through a silicon cushion to prevent loss of the compound back into the medium (Hirano et al., 2004). For plated hepatocytes, cells are rapidly washed with ice-cold buffer and lysed. Another novel approach to measure real-time uptake is to culture cells onto scintillating microplates (96-well), which detect only labeled compound which has been taken up into the cells (dubbed a scintillation proximity assay, SPA). SPA has been used extensively for radioimmunoassay, ligand-receptor binding assays and enzyme assays (Wu and Liu, 2005) but relatively seldom for transporter assays. In transporter assays, the cells act as a barrier between the scintillation layer and the medium, therefore, radiolabeled test compound in the medium would only produce a minor signal. However, following cellular uptake, the compound would come into close proximity with the scintillation surface and (if radiolabeled) would produce a signal. To support this theory, experiments have been undertaken using [14C]-taurocholate, [14C]-tetraethylammonium, and [3H]-estradiol-17βglucuronide. All the three test compounds were found to be taken up into fresh rat hepatocytes in a time- and concentration-dependent manner, which was also saturable (Lohmann et al., manuscript in preparation). The uptake kinetics obtained using SPA was comparable to those using conventional uptake methods, indicating that this assay could provide a higher throughput, reliable and rapid method to study compound uptake. [14C]-mannitol was used as a negative control because this compound does not enter cells and uptake of this compound in vivo is paracellular (between cells). Using the SPA, uptake of [3H]-estradiol17β-glucuronide was selectively inhibited by pravastatin, suggesting the involvement of OATPs. The SPA technique can also be adapted to explore the interaction of nonradiolabeled compounds with transporters, by evaluating whether the compound exerts an inhibitory effect on transport of a radiolabeled substrate. Efflux Studies in Hepatocytes When primary hepatocytes are cultured on a collagen plate without an overlay of collagen or matrigel, they form flat monolayers, prone to dedifferentiate and lose their polarization and thus expression of relevant efflux transporters and metabolic enzymes. However, short-term cultures (i.e., 2 h after plating) have proven useful for uptake studies (Fig. 1C). Cell suspensions can also be used for accumulation and efflux studies (Alvarez et al., 1997; Scala et al., 1997). In these studies, the accumulation of a fluorescent or radiolabeled compound is determined in the absence and presence of efflux transporter inhibitors. If efflux is inhibited, then compound accumulation is increased. If hepatocytes are cultured in sandwich format, they re-polarize and establish functional bile canaliculi (Fig. 1). The time for the expression of biliary efflux transporters varies between transporters and



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between species (up to 5 days for rat hepatocytes and up to 10 days for human hepatocytes). Hepatocytes cultured in sandwich culture configuration also express tight junctions, which surround and seal canalicular pockets between hepatocytes. Any excreted compound remains in the canalicular compartments such that accumulated compound in the monolayer represents both cytosolic and canalicular compartments. The tight junctions are dependent on calcium and can be opened by removing calcium from the medium (Liu et al., 1999). Therefore, the accumulation into the monolayers incubated in calcium-free buffer represents the cytosolic compartment only. Studies undertaken with hepatocytes in sandwich culture in the presence or absence of calcium can be used to assess the relative contributions of biliary and sinusoidal transporters to drug excretion (Liu et al., 1999). In addition, confocal microscopy can be used to investigate canalicular excretion of drugs directly, although this does require that the drug be suitable for cell based imaging analysis (e.g., inherently fluorescent or fluorescently tagged). Uptake and Efflux Studies in Other Models One of the complicating factors of the studies on transporters is the widespread lack of specific substrates and inhibitors. This means that it is difficult to determine the contribution of individual transporters to the uptake and excretion of test compounds. Some compounds do show some selectivity such that competitive inhibition can be studied. A more direct method of determining if a compound is a substrate for an individual transporter is to use transfected cell lines or oocytes (Fig. 14; Meijer and van Montfoort, 2002). One major advantage with transfected cells is that the uptake into the transporter-transfected cells can be compared to the uptake into the corresponding empty vector- or nontransfected cells resulting in a quantification of transporter-mediated uptake vs. passive

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diffusion (Fig. 14A). HEK293 cells can be stably transfected with a single transporter to study uptake of compounds (Biermann et al., 2006; Sahi, 2005). As with uptake transporters, efflux transporters may also be transfected into different cell types for functional studies to identify if compounds are substrates or inhibitors of drug transporters. For efflux transporters it is more common to use polarized cells such as MDCKII cells for transfection and to measure the vectorial transport of the compound between the apical and basolateral compartments (Fig. 14B). One example is MDCK-MDR1 cells that are commonly used within drug industry to screen for MDR1 substrates and inhibitors (Zhang and Benet, 1998). To have a better understanding of the interplay between a specific uptake and efflux transporters, MDCK cells, transfected with both an uptake and efflux protein, can be cultured on Transwells to reflect the unidirectional transport of a compound from one compartment to another. Cui et al. (2001) demonstrated the expression and localization of basolateral OATP 1B3 and apical MRP2 in double-transfected MDCK cells grown on Transwell membrane inserts (Fig. 14C). The rate of sulfobromophthalein (BSP, a substrate for both transport proteins) basolateral to apical absorption was at least six times faster by double-transfected MDCK-MRP2/OATP 1B3 cells than by single-transfected cells (either OATP 1B3 or MRP2 cells). This effect is likely to be due to the fact that BSP inhibits its own uptake if allowed to accumulate, or simply to the fact that diffusion BSP across a cell membrane lacking respective transporters is very slow. However, in the double-transfected model, BSP accumulation is prevented by MRP2 which removes it from the cells into the apical compartment. There have been a number of other transfected cell models for selected transporters, including oocytes (Meijer and van Montfoort, 2002) and baculovirus-Sf9 cells. The latter is based on a transporter protein mutation resulting in additional phosphorylation sites to increase the ATPase activity (Szabo et al., 1997). The transporters are expressed on the cell surface, from which membrane vesicle fractions are prepared. If a compound is a substrate for the transporter, then ATP is converted to ADP and inorganic phosphate, which can be detected spectrophotometrically. Vesicles (inside out) are used to determine the rate of efflux of the test compound, or the inhibition of efflux of a known radiolabeled substrate. An advantage of this expression system is that compounds do not need to be radiolabeled, which is often the case in early compound screening.

Pharmaceutical Practice There is now an increasing evidence that drug transporters in the liver play a key role in the pharmacokinetics of some compounds (Shitara et al., 2006). The FDA has started to include drug transporter studies in their guidelines and focus on the risk of drugdrug interactions and toxicity. However, understanding in vitro–in vivo correlations in the clinic is complicated by the fact that drugs are often substrates for both drug metabolizing enzymes and transporters, and specific in vivo probes for drug transporters are lacking. Importantly, clinical data have demonstrated the implications of drug transporter polymorphisms for the pharmacokinetics of drugs (Kim et al., 2001; Nishizato et al., 2003). Although drug transporters are still an emerging area in which an increased knowledge is needed to develop optimal and well validated tools, the pharmaceutical industry is clearly aware of the potential implications of transporters in clinical end points, such as clearance, bioavailability, and drug-drug interactions, and is looking to incorporate transporter studies early on in the discovery phase (Kim, 2006).



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Conclusions on Model Systems for Transporter Studies in Drug Discovery and Development In the development of new drugs, primary hepatocytes may be used to study uptake (suspension or plated) and efflux (sandwich culture) of drug transporter studies. Together with transporter-transfected cell lines or membrane vesicles, with which the particular transporters that are involved may be identified, hepatocytes provide a powerful tool to understand the pharmacokinetics of drug transporter substrates and the contribution of transporter-mediated processes to hepatobiliary clearance. The potential for drug-drug interactions may also be studied in the same systems. Further understanding of species differences and in vitro–in vivo correlation regarding transporters is required to acquire the same degree of validation that exists for drug metabolism models. This is needed before the pharmaceutical industry will be able to apply transporter models as predictive tools to drive chemistry of compounds in a more consistent way. PREDICTION OF METABOLIC DRUG CLEARANCE Are Hepatocytes Better than Microsomes? The pharmacokinetics of drugs is important to establish in the early phases of development and therefore, there has been an increase in the use of in vitro techniques to improve the throughput of these assays. It is estimated that about 60% of marketed compounds are cleared by hepatic CYP-mediated metabolism (McGinnity et al., 2004) making the liver the main focus for these studies. It must be kept in mind that compounds may also be metabolized by extra-hepatic organs, especially drugs undergoing extensive conjugation (Obach, 1999; Soars et al., 2002). Different in vitro systems such as hepatocytes, liver slices, microsomes, and recombinant microsomes have been compared and found to be equally effective in determining the metabolism and clearance of exclusively CYPmetabolized compounds (Andersson et al., 2001; Houston and Carlile, 1997; Olinga et al., 1998b; Salonen et al., 2003). Other reports on liver slices are not as positive and have suggested that these are a poor model due to the inaccessibility of the compounds to the core of the slices (Houston and Carlile, 1997). Human hepatic microsomes are traditionally used for in vitro based prediction of metabolic clearance and drug-drug interaction potential. However, there are obvious criticisms of this particular in vitro system which result from two main effects. Firstly, the loss of structural integrity, as the preparation of microsomes is a destructive technique. This results in an in vitro incubation matrix where there is a greater potential for nonspecific binding than in the intact cell. Also the removal of the outer plasma membrane results in the loss of any transporter protein systems which may be important for either the uptake or efflux of the drug and/or metabolites. Secondly, microsomes have a bias in the complement of enzymes available for metabolism. It is common practice to optimize for cytochrome P450 reactions by adding appropriate cofactors (NADPH), hence other microsomal reactions, for example, UGT, do not operate. Also there are other drug metabolizing enzymes, such as those in the cytoplasm, that are removed in the microsomal preparation. This disruption in the normal (in vivo) sequential/parallel metabolism of a compound may result in a number of consequences that can confound the clearance prediction. There may be product accumulation due to loss of sequential metabolism and this may result in a possible feedback inhibition effect (Jones et al., 2005). Neither of these two types of criticism are applicable to hepatocytes, therefore, they have a clear advantage over subcellular fractions (Bayliss et al., 1999; Begue et al., 1983; Lave et al., 1997).


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Hepatocyte Cultures or Suspensions? The clearance (CLint) of a number of compounds was compared in freshly isolated rat hepatocyte suspensions and cultures (Griffin and Houston, 2005). The in vitro CLint of only two of seven compounds was comparable with in vivo, although the overall rank order of compounds was the same in both models. The measured in vitro CLint for highturnover compounds was lower for conventional monolayer cultures compared with suspensions but conventional cultures gave a higher estimation of in vitro CLint for the low-turnover compound S-warfarin compared with suspensions. Thus, hepatocyte cultures may be more suitable for predicting the CLint of low clearance compounds (below 0.1 μl/ min/106cells). Blanchard et al. (2004) also compared the clearance of compounds in suspensions and cultures in both rat and human hepatocytes (Blanchard et al., 2005). They found that the CLint of the high clearance compound, naloxone, was slightly higher in hepatocyte suspensions than in cultures and suggested this was due to the continuous mixing of the suspensions. In contrast, the clearance of two other (medium and low clearance) compounds was comparable in both systems. Can Cryo-preserved Hepatocytes be Used Instead of Fresh Hepatocytes? Fresh human tissue is not available to many laboratories; therefore, the question of whether cryo-preserved (commercially available) human hepatocytes are valuable for prediction of clearance is important. There is evidence from a number of studies (Blanchard et al., 2006; Lau et al., 2002; McGinnity et al., 2004; Shibata et al., 2002; Soars et al., 2002) to support the routine use of cryo-preserved human hepatocytes. In general, kinetic profiles are similar in cryo-preserved and fresh hepatocytes and, in some cases, microsomes. For example, when Vmax values are scaled up per g liver, there is a good relationship between cryo-preserved hepatocytes values and microsomes. However Km values tend to be lower in cryo-preserved hepatocytes than in microsomes and as a result the particular clearance value obtained for a drug using hepatocytes may be slightly higher or similar to microsomes (Brown et al., 2006; Hallifax et al., 2005). Cryo-preserved hepatocytes also display the CYP3A4 atypical kinetic phenomena that have been well characterized in microsomes, namely autoactivation, heteroactivation with testosterone and substrate inhibition (Brown et al., 2006; Hallifax et al., 2005; Witherow and Houston 1999). They also show a clear advantage in the prediction of clearance by UGT enzymes. This has recently been exemplified by a study on AZT (Engtrakul et al., 2005) where it was found that hepatocytes gave a good prediction of in vivo clearance whereas microsomes, despite extensive work to optimize conditions, would only provide a clearance value one-tenth of the hepatocyte value (Table 4). The effect of incubating cryo-preserved hepatocytes from three different donors with serum on compound clearance was recently reported by Blanchard et al. (2006). The CLint of the compounds decreased in presence of serum but made the values more reproducible across the donors. The CLint was donor-dependent, as observed with fresh hepatocytes from different donors. Shibata et al. (2002) suggested that hepatocytes should be pooled to give an “average donor”, however, an advantage of single donor incubations is that clearance may be linked to enzyme characteristics, as was demonstrated with four of the six compounds tested by Blanchard et al. (2006). Another advantage is that information about the variation in the patient population can be anticipated. The two drugs that were not linked to enzyme characteristics were antipyrine and nalxone. The lack of

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Table 4 Observed and Predicted Intrinsic Clearances from Hepatic Microsomes and Hepatocytes from Rat. All Data Expressed per g Liver Using Appropriate Scaling Factors to Allow Direct Comparison Between Systems. Intrinsic clearance(ml/min/g liver) Drug

Microsomal

AZT Propranolol Digoxin Saquinavir

Hepatocyte

In vivo

Reference

15 198 711 50

20 245 600 95

Engtrokut et al., 2005 Hallifax and Houston, 2006 Lam and Benet, 2004 Parker and Houston, 2006

1.7 193 161 350

correlation was attributed to the fact that antipyrine is metabolized by many CYPs, thus making a correlation with specific enzymes difficult. Naloxone is glucuronidated by UG2B7 (Coffman et al., 2001; Wahlstrom et al., 1989) and was compared to hydroxycoumarin glucuronidation, which is conjugated by UGT1A6 (Lampe et al., 1999) so a correlation would not be expected. Single donor incubations also allow for calculations for extremes of characteristics. CYP2C9 deficient donors may clear compounds differently from the “average” donor. Hepatocytes pooled from several donors are now commercially available. These reflect the “average donor” in the same way as pooled microsomes, their advantage is that they are an intact whole cell system. So far, the reproducibility of the enzyme activities and clearance of phases 1 and 2 substrates between different batches is excellent (Eneroth et al., 2006). A good comparison with fresh cells is also reported for these cells (Koganti et al., 2005). Figure 15 demonstrates the prediction of in vivo clearance of 41 drugs by cryopreserved human hepatocytes. In this data set (Brown et al., 2006) are substrates for CYPs and UGTs taken from four independent studies, some of these studies involved kinetic information from metabolite formation and the others from drug depletion. It is clear that there is a good correlation between predicted and observed values and this correlation 1000 Hepatocyte CLint (ml/min/kg)



100

10

1

0.1 0.1

1

10

100

1000

10000

In vivo CLint (ml/min/kg) Figure 15 Comparison between observed human in vivo CLint and CLint predicted using cryo-preserved hepatocytes for 41 substrates. Open symbols represent predictions from drug depletion studies by Soars et al., (2002), Shibata et al., (2002) and Lau et al., (2002); closed symbols from metabolite formation studies by Hallifax et al., (2005) and Brown et al., (2006), , respectively. The solid line represents the line of unity, whereas the dashed line represents a 5.6-fold under-prediction.


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extends over 4 to 5 orders of magnitude; on average however, there is a 5-fold underprediction. The 5-fold bias in cryo-preserved hepatocyte predictions should be contrasted with the 9-fold bias observed with human liver microsomes (Ito and Houston, 2005). It is of interest that a similar difference in bias has been demonstrated for these two in vitro systems using rat tissue. Rat liver microsomes show a 2-fold bias in predictions whereas fresh rat hepatocytes show a minimal bias of 20% (Ito and Houston, 2004). Thus, the general conclusion is that cryo-preserved human hepatocytes provide an in vitro prediction of hepatic clearance numerically closer to the in vivo value than that obtained from hepatic microsomes. Hepatocellularity One of the main factors affecting the prediction of clearance is the scaling of in vitro data to in vivo values. Most researchers use the well-stirred model for scaling up CLint (expressed as μl/min/million cells) to hepatic clearance in vivo (expressed as ml/min/kg body weight) (Blanchard et al., 2006; Houston, 1994; Iwatsubo et al., 1997; Obach, 1999). There is some debate as to the hepatocellularity of human and animal livers (Bayliss et al., 1999; Houston, 1994; Wilson et al., 2003). Almost all predictions for human clearance are made using 120 million cells/g liver weight (Andersson et al., 2001; Bachmann et al., 2003; Bayliss et al., 1999; Davies and Morris 1993; Fisher et al., 2002; Iwatsubo et al., 1997; Lave et al., 1997; McGinnity et al., 2004; Naritomi et al., 2003; Salonen et al., 2003; Soars et al., 2002). Reports by Wilson et al. (2003) suggest that this may be lower, although the liver samples used in these studies were from liver tumor patients, which are known to exhibit hepatomegaly (Lam et al., 2004) and such may have lower hepatocellularities than nondiseased tissue. Nevertheless, the point made by Wilson et al. (2003) emphasizes the need to accurately measure true hepatocellularity in healthy liver tissue. Of notable exception to this problem of hepatocellularity is when slices are used. The scaling can be easily calculated on the basis of the slice wet weight or protein content. Competing and Rate Limiting Processes in Hepatocytes Kinetic studies in hepatocytes, as opposed to microsomes, involve additional complexities concerned with the drug concentration gradients between the inside of the cell and the extracellular medium and the process of hepatocellular uptake. In addition to diffusion through the outer membrane, transporter proteins can pump drugs in and out of the cell (Chandra and Brewer 2004; van Montfoort et al., 2003). Once in the cell, intracellular binding can occur in a similar manner to that encountered in microsomes. The consequences of a high hepatocellular uptake, which can be quantified by a cell-to-media concentration ratio (Kp), can confound the interpretation of clearance from cellular systems. For the vast majority of drugs, intracellular binding occurs (Austin et al., 2005) and it is necessary to correct for this in order to determine the true drug concentration in the medium, which in turn will be in equilibrium with the intracellular drug concentration available to the enzyme (see Eq. 1).

CE = fu⋅inc⋅Cmedium

(1)

where CE and Cmedium are the drug concentrations available to the enzyme and in the extracellular medium, respectively and fuinc is the fraction of unbound drug in the incubation.

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The unbound intracellular concentration may differ from that in the external medium due to transporter-mediated uptake; the consequences of this are not necessarily intuitive (Wu and Benet, 2005). This effect is only true when active transport is involved. Most uptake transporters facilitate passive transport and the unbound concentration inside and outside the cells should be the same when equilibrium is reached. Also the uptake process may be the rate-limiting step for the clearance of a compound. These different consequences can be illustrated by considering four different drugs, namely imipramine, propranolol, digoxin, and saquinavir. In these examples, rat hepatocytes were used rather than human cells to demonstrate various phenomena. Kp values of several hundred are observed for both imipramine and propranolol, and probably many other lipophilic basic drugs (Hallifax and Houston, 2006). The actual value achieved depends upon the drug concentration and as the drug concentrations are increased there is a decrease in the Kp value. This can be described by a two-site model with a high infinity, low capacity saturable component and a linear component that is not saturated over the concentration range studies. The saturable component can be removed either by incubating at low temperature (4°C) or by treatment with a membrane permeabilizer (saponin); also, competition from other lipophilic basic drugs can be demonstrated for this process. However, despite the very high Kp achieved for these drugs, this does not appear to rate-limit the hepatic clearance as has been demonstrated quite clearly with propranolol (Hallifax and Houston, 2006). Clearance predictions based on unbound (incubation medium) drug concentrations are in good agreement with in vivo observed values, and indeed with predictions from hepatic microsomes (Table 4). The relationship between fuinc, Kp and the volume ratio (VR) of the cell to the total incubation (Jones et al., 2004) is summarized in Eq. 2.

fuinc =

1 1 + K p ⋅ VR

(2)

The impact of intracellular binding for three different hepatocellularities is illustrated in Fig. 16. It can be seen that only when Kp values exceed several hundred does this result in a significant fraction unbound (fuinc < 0.5). This is due to the impact of the volume ratio value, (e.g., 0.003 at a hepatocellularity of 106 cells/ml). 1 Fraction unbound



0.8 0.6 0.4 0.2 0 1

10

100

1000

10000

Cell/media concentration ratio Figure 16 Relationship between fraction of unbound drug concentration in the hepatocyte incubation (fuinc) and the cell to medium concentration ratio (Kp) according to Equation 2. Three different hepatocellularities are shown (with corresponding volume ratios) – solid line 0.5 × 106 (0.0015), dashed line 1 × 106 (0.003) and dotted line 2 × 106 (0.006) cells/ml.


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Digoxin is a clear example of a hepatocyte clearance determination that is far more useful than a microsomal clearance. Lam and Benet (2004) have shown a good agreement between hepatocyte predicted clearance and in vivo clearance whereas for microsomes this prediction is far less (Table 4). These studies in the rat involve the CYP3A metabolism in addition to the action of the OATP 1B1 and P-gp transporter proteins. Saquinavir is another good example of the consequences of transporter proteins (Parker and Houston, 2006). In this case, the uptake of drug is rate-limiting metabolic clearance. Clearance predictions from microsomes are far in excess of hepatocytes and over-predict by 5-fold in vivo observed values (Table 4). Permeability of saquinavir is similar to the hepatocyte clearance indicating a rate limitation. A similar situation occurs with at least two other HIV protease inhibitors, ritonavir, and nelfinavir (Parker and Houston, 2006). Conclusions on the Utility of Hepatocytes to Predict Clearance Not only are hepatocytes theoretically better than microsomes for prediction of metabolic clearance, this can be demonstrated experimentally. At the very least, hepatocytes can fully substitute for microsomes but they can also delineate more hepatic events such as non-P450 reactions (UGT, SULT NAT, and so on) and the consequences of transporter proteins. However, kinetic studies in hepatocytes can be complicated to interpret and it is likely that any routine studies will require additional, specific, follow up studies in order to fully characterize hepatic events and ensure correct conclusions are drawn. HEPATOTOXICITY Scientific Principles Within the pharmaceutical industry, drug induced liver injury (DILI) is a frequent finding during preclinical safety testing and can also occur in man at all clinical phases. The consequences are project attrition or project delay throughout discovery and development, as well as failure to gain approval for new drug registrations, cautionary labeling, and post-marketing withdrawal of approved drugs (Kaplowitz, 2005; Walgren et al, 2005). In addition, DILI due to licensed drugs is an important cause of adverse drug reactions in man. More than 600 current drugs have the potential to cause liver injury in man and DILI is increasingly recognized as a major cause of serious illness (including acute fulminant hepatic failure or chronic persistent liver damage that ultimately may require transplantation) (Ostapowicz et al., 2002). One problem posed by DILI is the incidence of significant liver dysfunction in man caused by individual drugs is generally low (of the order of 1:1,000 or less). Furthermore, many of the drugs that cause idiosyncratic DILI in man do not produce overt signals of significant liver damage during preclinical safety testing in experimental animals. Consequently, conventional in vivo safety studies in animals are of limited value for prediction and management of DILI in man. To deal with these challenges, it is important to understand the molecular and cellular processes of DILI which underlie susceptibility. This understanding can be used to influence drug design and selection with the least possible potential to initiate liver injury and to manage these candidates through clinical development. DILI is not a single type of toxicity but rather many different pathologies and patterns of presentation that arise via diverse and complex mechanisms which remain relatively poorly understood (Lee, 2003). The complexity of DILI tends to reflect the



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complexity of liver function and liver homeostasis, as well as the crucial role played by this organ in “whole body homeostasis” and host defense (against chemical insult plus invading microorganisms). It is clear that development and progression of significant liver dysfunction is a multistep process, which involves initial chemical insult followed by biological response (Fig. 17; Lee, 2003; Kaplowitz, 2005). It can be hypothesized that the balance between protective responses and responses that result in propagation and amplification of tissue injury and also individual susceptibility factors, determines whether or not toxicity arises (Fig. 17; Kaplowitz, 2005). In man, the most frequent patterns of DILI are hepatocellular (affecting hepatocytes), cholestatic (affecting the biliary system), and mixed hepatocellular/cholestatic (Lee, 2003). This indicates that it is especially important to focus on processes involved initiation and/or progression of injury affecting hepatocytes and also to consider bile formation and bile flow. Another important feature of DILI is the key role of drug metabolism and disposition. Most drugs are metabolized within the liver, a process well known to play a key role in drug elimination. It converts relatively nonpolar compounds to more polar metabolites that are either excreted directly into bile or via the kidneys into urine. Frequently the metabolites are nontoxic; hence drug metabolism and excretion are generally considered a detoxication process, although some metabolites may mediate toxicity (Fig. 18). Chemically reactive metabolites are of particular interest because they play a key role in DILI caused by numerous compounds (Walgren et al., 2005). These include drugs that cause dose-dependent and reproducible liver injury due to generation of toxic metabolites and drugs that cause idiosyncratic DILI in man. Covalent binding of reactive metabolites to proteins frequently plays an important role in the mechanism of liver injury. This may mediate cell toxicity directly (e.g. paracetamol) or may trigger adaptive immune responses in susceptible patients that are targeted against metabolite-modified liver protein neoantigens and cause immune-mediated liver injury (e.g., halothane and dihydralazine (Park et al., 2005). It has been estimated that roughly two-thirds of drugs that cause idiosyncratic DILI in man form reactive metabolites, suggesting that this could be an important risk factor. Another factor is CYP induction, which can potentiate the formation of toxic metabolites (Li et al., 2002). Interaction of drugs with transporters can also influence or cause DILI. Many drugs are actively transported into cells by uptake transporters, which are highly expressed in hepatocytes. It is likely that this process plays an important role in DILI, although this has yet to be explored experimentally. Inhibition of transporters responsible for efflux of bile salts from hepatocytes into bile has been proposed as an important mechanism of cholestatic DILI (Stieger et al., 2000). For these drugs, the liver injury could occur because of accumulation of high levels of cytotoxic bile salts within hepatocytes. However, it is also possible that cell toxicity caused by intracellular accumulation of drugs or drug metabolites also plays a contributory role.

Use of Hepatocytes in Toxicity Testing Isolated hepatocytes are well suited to assessment of hepatocellular liver injury. When isolated and handled appropriately they express a broad range of metabolizing enzyme activities, hepatic transporters, and other differentiated functions (Dambach et al., 2005; Farkas and Tannenbaum, 2005; Guillouzo, 1998, O’Brien et al., 2004, Gómezlechón et al., 2004,). Important chemical insults that can be explored include:

200

Outcome

(how the individual responds)

Biological res pons e

C he m i c a l insult

Variability

– E n v i r o nm e n t a l

diet, disease status etc)

– Genetic – Epigenetic (age, sex,

metabolism and excretion)

Hepatotoxicity

Figure 17 Steps involved in development of DILI and role of individual susceptibility.

Balance determines outcome

Between and within species

Cell toxicity Cel l prolifer ation Inflammatory cell infiltration Immune activation (innate, adaptive) Collagen deposition, tissue remodelling

Propagation and amplification – – – – –

Pr oc es se s af fecti ng toxic i ty Disposition (uptake, distribution,

C h e mic a l d et o x ic a t io n Stress response responses Cell repair Tissue repair

Resolution, adaptation

– – – –

P r o tec ti o n a nd re s o l u t io n

Reactive m e tabolites S e c on d a r y p har ma c ol og y Sec o n dar y bioc he mis try – e n z y me in h ib iti o n – transporter interactions CYP450 induction Et c … . .




DRUG

201

Excreted metabolites Detoxication (GSH conjugation etc.)

“Toxic” metabolite

Non-covalent interaction with cellular macromolecules

Covalent binding to protein

Amplifying events (innate immune system?)

Adaptive immune response

Direct or idiosyncratic hepatotoxicity

Immune-mediated hepatotoxicity

Figure 18 Role of metabolism in DILI.

1. Formation of chemically reactive metabolites that can trigger oxidative stress or interact covalently with proteins and other macromolecules. 2. Interference with biochemical systems, such as lipid degradation (causing steatosis) and phospholipid degradation (causing phospholipidosis). 3. Impairment of mitochondrial function (e.g., impairment of respiration). 4. Inhibition of biliary transporter activity (causing intracellular accumulation of toxic bile salts and possibly also intracellular accumulation of toxic drug metabolites). An initial priority is to ascertain hepatic-specific or basal toxic effects and to establish whether toxicity is associated with the metabolism of a given compound. A number of parameters can be measured to assess cell viability, mainly achieved in pharmaceutical practice by quantifying both cellular ATP content (an index of metabolic capacity) and enzyme leakage from cells into the culture medium (reflecting plasma membrane leakage). Release of cytosolic lactate dehydrogenase is assessed commonly, although other enzymes can also be determined (e.g., glutamate dehydrogenase, a mitochondrial enzyme). Representative ATP and LDH data obtained with cultured mouse hepatocytes exposed in vitro to cocaine are shown in Fig. 19. Cytotoxic end points represent a first approach to assess hepatotoxicity and may be adequate if the purpose of the study is simply to rank test compounds for ability to cause hepatocellular injury. However, evaluation of hepatocyte cytotoxicity alone will not provide information on the type of toxicity or possible mechanisms and will not address xenobiotics that impair cell function without causing cell death. Impairment of cellular function may not

N. J. HEWITT ET AL. (A) Release of LDH

40

LDH (nmol/min/well)

24 hr 30 4 hr 20 10 0 0

2

4

6

8

10

Concentration (mM)

(B) Depletion of ATP

12

ATP (nmol/mg protein)


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8

4

4 hr 24 hr

0 0

2

4

6

8

10

Concentration (mM) Figure 19 Toxicity of cocaine to cultured mouse hepatocytes. Mouse hepatocytes were cultured as conventional monolayers on collagen-coated plates. After 24 h in culture, cells were incubated with cocaine in Williams E medium, at concentrations ranging form 0–10mM, for 4 h or 24 h. Cell supernatants were removed and assayed for LDH activity (A), and cellular ATP content (B).

be critical for the hepatocyte itself but can be of toxicological significance for the whole organism. It is an undesirable liability, since impaired hepatocellular function may predispose an individual to synergistic liver injury caused by other agents ingested concomitantly, such as other drugs, dietary components, or herbal remedies. By examining effects on hepatocytespecific metabolism, it is possible to determine whether relevant hepatic specific functions become altered by the presence of a xenobiotic. Several metabolic parameters, representative of the most characteristic liver functions, should be evaluated (gluconeogenesis, glycogen metabolism, ureogenesis, plasma protein synthesis, synthesis of very low density lipoproteins, and so on). In general, metabolic parameters are more sensitive to the toxic effect of hepatotoxins than cytotoxic end point indicators. Concentrations to which cells are exposed in such cell metabolism studies should not cause perceptible cell death (Castell et al., 1997b). To characterize the type of liver toxicity and to explore mechanisms, various parameters can be investigated. These include cell morphology, mitochondrial membrane potential (using various redox sensitive dyes), accumulation of lipid or phospholipids, induction



203

of apoptosis, glutathione status, lipid peroxidation, levels of various intermediary metabolites, transporter activity, expression and localization of individual proteins, covalent binding to proteins, and gene expression at the mRNA level (by quantitative PCR and using global gene arrays, described later). The choice of parameters will be dictated by the purpose of the experiment. If ranking of compounds for their ability to cause hepatocellular injury is sufficient, cell cytotoxicity studies may be adequate. Most studies described in the literature have been undertaken using hepatocyte suspensions or cultures. A particular advantage of suspensions over cultures is that metabolic capability is well preserved allowing for estimating and ranking the ability of compounds to cause marked cell necrosis mediated by metabolism (O’Brien and Siraki, 2005). However, the data obtained can be confounded by the short timescale available before cells lose viability (no more than 6 h). Hepatocyte suspensions are not suitable for investigation of toxicity of compounds that are turned over slowly or for analysis of parameters requiring good preservation of cell morphology. In practice, toxicity to hepatocytes caused by drugs tends to be a relatively slow process that takes many hours, for example cocaine, which is illustrated in Fig. 19. Therefore, hepatocyte toxicity is explored most appropriately using hepatocyte cultures, not suspensions. Sandwich culture configuration can improve morphology and viability of human hepatocytes in culture, although the effect is less marked than for rat hepatocytes. The sandwich culture configuration does not interfere with cytotoxicity assessments and similar IC50 values for cell cytotoxicity have been described for a range of test compounds evaluated using rat hepatocytes in conventional monolayer vs. sandwich culture (Hopwood et al., 2004). A particular advantage of the sandwich culture configuration was that the reproducibility of data was improved, possibly due to higher basal cell viability throughout the experiment. Use of sandwich cultures also results in excellent morphology and so is well suited to analysis by microscopy (even to the level of electron microscopy), as well as studies of toxicologically relevant interactions between drugs and biliary transporter activity (which have been discussed elsewhere [LeCluyse et al., 1994; Liu et al., 1999]. The use of hepatocytes provides an opportunity to study toxicity alongside metabolism and disposition and thereby to develop an integrated understanding of the relationship between these different parameters. Currently, this information is of most value for comparison between related compounds, to develop a rank order of toxicity potential and also for investigation of underlying mechanisms plus possible species differences. It is not possible to use data from in vitro toxicity studies on hepatocytes to accurately assess the risk that an individual compound will cause DILI in vivo, in preclinical species or in man. This is because hepatocyte studies focus on chemical insult and other steps involved in initiation of liver injury but do not address subsequent events that may lead to protection or amplification and also do not consider possible individual susceptibility factors (Fig. 17). More precise risk assessment will require a marked improvement in our understanding of all the steps involved in development of DILI. Case studies in Use of Hepatocytes to Study liver Toxicity: MDMA and Troglitazone Most drugs are metabolized to inactive or less active metabolites in the liver (Arand et al., 2003; Hengstler et al., 2003). In contrast, there are examples in which drugs are converted to reactive and more toxic metabolites than the parent compound. Primary hepatocytes are a powerful tool for identification of both, substances that are activated as well


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as inactivated by hepatic metabolism. Increased susceptibility to 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) results from extensive hepatic metabolism and increased concentrations of the metabolites (Carmo et al., 2006). MDMA has been recognized as one of the most significant examples of drug abuse over the past decades, especially among young adults. MDMA is a ring-substituted amphetamine derivative and is a potent releaser and/or reuptake inhibitor of presynaptic serotonin, dopamine and norepinephrine. MDMA abuse is due to the intense feelings of euphoria, friendliness, comfort, intimacy, pleasure, empathy, and hyperactivity induced by the drug. Misuse of MDMA can induce severe acute toxic effects including hepatotoxicity. However, despite its widespread use, the number of MDMA-related deaths and severe intoxications is relatively low (Green, 2004), therefore, MDMA is commonly misrepresented among its users as being safe. It is known that there is large interindividual variation in the propensity to the adverse effects of MDMA (Carmo et al., 2006). Some individuals have died after ingestion of a single tablet, while others have survived after high doses of MDMA, suffering only mild sympathetic symptoms such as tachycardia and hypertension (de Letter et al., 2004; O’Donohoe et al., 1998). Large differences in MDMA-induced toxicity have also been observed in primary hepatocytes from different donors. The toxicity of MDMA was clearly increased in cells expressing CYP2D6*1 compared to cells devoid of CYP-dependent enzymatic activity (Carmo et al., 2006). Likewise, formation of the oxidative MDMA metabolite N-Methyl-α-MeDA was greatly enhanced in CYP2D6*1-transfected cells. In contrast to CYP2D6, CYP3A4 did not enhance MDMA toxicity. The increase in the cytotoxic effects of MDMA was, therefore, attributed to the CYP2D6-mediated production of the oxidative metabolite. This was supported by cytotoxicity studies demonstrating that Nmethyl-α-MeDA itself was more than 100-fold more toxic than the parent compound (Carmo et al., 2006). Thus, CYP2D6 ultrarapid metabolizers ingesting Ecstasy may be more susceptible to hepatotoxicity. Troglitazone deserves mention purely because its mechanism of toxicity remains uncertain even after extensive investigations. Troglitazone was the first member of the thiazolidinediones for treatment of type II diabetes. It was withdrawn from the market because of liver toxicity and fulminant hepatic failure in several patients (Gitlin et al., 1998; Neuschwander-Tetri et al., 1998). In humans, troglitazone is predominantly conjugated with sulfate (70% of all metabolites (Loi et al., 1999a,b) and only 3% of orally administered troglitazone is excreted unchanged in the urine. The debate continues as to whether this compound is toxic per se or whether it is bioactivated, although there are good data to support both. Arguments for troglitazone being the toxic entity stem from early studies in which troglitazone cytotoxicty in cultured human hepatocytes correlated with increased unmetabolized troglitazone (Kostrubsky et al., 2000). Inhibition of troglitazone sulfation with 2,6-dichloro-4-nitrophenol increased the toxicity to human hepatocytes. It was suggested that a limited sulfation capacity in susceptible patients was a possible mechanism for troglitazone-induced hepatotoxicity (Kostrubsky et al., 2000). Porcine hepatocytes, which lack SULTs, were resistant to troglitazone possibly due to their high glucuronidation capacity, thus consuming troglitazone as rapidly as SULTs in resistant human hepatocytes. Further evidence for trogliazone being a direct toxicant came from Masubuchi et al. (2006). They showed that troglitazone, but not the relatively nonhepatoxic thiazolidinediones, rosiglitazone, and pioglitazone, induced mitochondrial swelling and decreased mitochondrial membrane potential and Ca2+ accumulation. This is in accordance with others who reported structural and functional abnormality of mitochondria in hepatoma cell lines and isolated hepatocytes treated with troglitazone



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(Haskins et al., 2001; Narayanan et al., 2003; Shishido et al., 2003; Tirmenstein et al., 2002). Another mechanism of cell death is apoptosis, which was reported to be specific to troglitazone but not its sulfate, glutathione or quinine metabolites (or to chemically similar pioglitazone and rosiglitazone) using HepG2 cells (Yamamoto et al., 2001). The evidence supporting bioactivation is also compelling. Hewitt et al. (2002) showed that the cytotoxicity of troglitazone in cryo-preserved human hepatocytes from 27 different donors was correlated to CYP3A4 activities, together with the ratio of SULT and UGT activities. The higher the CYP3A metabolism and sulfation and the lower the glucuronidation, the more cytotoxic troglitazone was in that donor. There was no direct correlation between cytotoxicity and CYP3A4 only or the SULT:UGT ratio only—the three enzymes together had to be combined to make the correlation. This suggests there is a balance of effects whereby troglitazone is bioactivated to the sulfate and quinine (which is found to have some toxic effects, albeit lower than troglitazone itself (Yamamoto et al., 2001)) but detoxified by glucuronidation, as noted using pig hepatocytes which predominantly glucuronidate troglitazone (Kostrubsky et al., 2000). Another pathway thought to be important in the overall cytotoxicity of troglitazone is conjugation with reduced glutathione (GSH). Cryo-preserved human hepatocytes form two GSH-conjugates (Prabhu et al., 2002). Low GSH conjugation correlated to higher sensitivity of these cells to troglitazone toxicity. Bioactivation pathways may result in direct and indirect actions. Troglitazone covalently binds to microsomal protein after activation by NADPH-dependent CYP metabolism. The covalent binding, predominantly to CYP3A4, was completely inhibited by ketoconazole and GSH (He et al., 2004). How this binding to CYP3A4 ultimately results in cytoxicity is not known. Troglitazone sulfate may also exert an indirect toxic effect by compromising hepatocellular function, rather than causing cell necrosis or apoptosis. Nozawa et al. (2004) demonstrated that troglitazone reduced the bile flow within 1 h in isolated perfused rat livers, an effect that was sex-specific and related to the extent of troglitazone sulfate formed. Male rat livers, with 5-fold higher troglitazone sulfate levels than females, were more sensitive to cholestasis than female livers. Using isolated canalicular rat liver plasma membrane preparations, it was shown that troglitazone sulfate potently inhibited the canalicular BSEP with an IC50 of 0.4–0.6 μM, which is lower than the reported plasma concentrations of troglitazone (3.6–6.3 μM (Loi et al., 1997). Troglitazone itself was ten times less potent than its sulfate. This suggests that intrahepatic cholestasis in rats was a result of bioactivation of troglitazone to the sulfate and subsequent inhibition of Bsep. The debate continues as to the precise mechanism of liver toxicity of troglitazone. Perhaps the very nature of its idiosyncratic toxicity is a reflection of how difficult this type of toxicity is to reproduce in vitro or in vivo. Hepatoxicity of Phase 2 Metabolites Attention with regards to bioactivation has been paid mostly to the phase 1 metabolites of drugs, most likely because of the plethora of possible reactive metabolites. However, some phase 2 metabolites deserve mention, that is, acyl-glucuronides and acyl-CoA thioesters. These conjugates of carboxylic acid drugs have been studied intensely since several NSAIDs, have been associated with hepatotoxicity (Bakke et al., 1995; Zimmerman, 1994) most of which are carboxylic acids, but share few other structural similarities. In addition, a number of these NSAIDs have been withdrawn post-marketing due to hepatotoxicity or allergic reactions, e.g., ibufenac, zomepirac, benoxaprofen, and bromfenac


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(Lee, 2003). Several studies have shown that both acyl-glucuronides and acyl-CoAs are able to trans-acylate GSH and proteins (Benet et al., 1993; Bolze et al., 2002; Grillo et al., 2002, 2003; Sidenius et al., 2004) forming thioester-, ester- or amide-bound covalent adducts. Hence, it is very likely that these metabolites may play a role in the observed adverse reactions. The notion that acyl-glucuronides could react chemically with proteins most likely originated from the observation that glucuronides of bilirubin and a number of carboxylic acid compounds formed adducts with albumin in vitro (McDonagh et al., 1984; van Breeman et al., 1985). Studies by Smith et al. (1986) subsequently showed that the acylglucuronides (both the β-1-O-glucuronide and its isomers) of zomepirac were able to trans-acylate serum albumin in vitro. There was a good correlation between the in vivo AUC of the glucuronides in human volunteers and the amount of irreversible plasma protein adduct formed (Smith et al., 1986). Kretz-Rommel and Boelsterli (1993) showed that the in vitro covalent binding of diclofenac to hepatocellular proteins (in cultured hepatocytes) was dependent on diclofenac-GlcA (D-GlcA) formation, and inhibition of D-GlcA formation by either borneol or TPHU (7,7,7-triphenylheptyl-UDP) decreased covalent binding by up to 87% (Kretz-Rommel et al., 1993). However, the D-GlcA-dependent covalent binding could not be correlated to acute cytotoxicity in the cultured hepatocytes, underlining the need to understand better the mechanisms involved in metabolism-mediated cytotoxicity. Further studies by Hargus et al. (1994, 1995) showed that in vivo and in vitro formation of a 110 kDa protein adduct (found to be DPP IV) was also dependent on diclofenac-GlcA formation and could be ameliorated by co-incubation with β-glucuronidase, which hydrolyzes β-1-O-glucuronides. Using TR-rats, deficient in MRP2, Seitz et al. showed that when D-GlcA transport into the bile was absent, no formation of what was believed to be the same DDP IV-adduct was seen (Seitz et al., 1998). Recently, interest has turned to acyl-CoAs as chemically reactive metabolites, although speculations on sources of reactive metabolites other than acyl-glucuronides already arose in the 1980s (Drew and Knights, 1985; Hertz and Bar-Tana, 1998; Stogniew and Fenselau, 1982). However, the number of metabolic pathways in which acyl-CoAs participate makes it interesting to investigate the disposition of xenobiotic acyl-CoA thioesters. For example, several studies have shown that xenobiotic carboxylic acids, e.g., fenbufen and ibuprofen, can be incorporated into complex lipids, thus prolonging the time the drug stays in the body and potentially causing adverse effects through inhibition of lipid metabolizing enzymes, and so on (Dodds, 1995). Another pathway of lipid metabolism, which may be affected by xenobiotic acyl-CoAs, is the β-oxidation of fatty acids. Formation of xenobiotic acyl-CoAs may interfere with this pathway by depleting either CoA or carnitine, or by inhibiting some of the enzymes involved in the β-oxidation pathway (Fromenty, 1995), causing ATP-production from fatty acids to be inhibited. Both hypoglycin and valproic acid have been shown to form CoA-thioesters in addition to inhibiting β-oxidation (Bjorge and Baillie, 1985; Silva et al., 2001, 2004; Wenz et al., 1981). The hypoglycin metabolite, methylenecyclopropylacetate-CoA, is known to inhibit β-oxidation of fatty acids by suicide-inhibition of multiple acyl-CoA dehydrogenase enzymes in mitochondria (Lieu et al., 1997; Wenz et al., 1981). In the case of valproic acid, however, it may be that a diene ((E)-Δ2,4-valproate) is formed through CoA-thioester formation and subsequent β-oxidation and that this metabolite—or a subsequent βoxidated metabolite, 3-oxo-4-ene-valproic acid—can react with intracellular proteins to inhibit β-oxidation. Alternatively, or in addition, these metabolites may act by depleting intramitochondrial GSH or by shifting the mitochondrial redox-state through inhibition of



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glutathione reductase (Kassahun et al., 1994; Tang et al., 1995). This is supported by studies showing that the α-fluorinated analog of valproic acid, which does not form the CoA-thioester (Grillo et al., 2001), does not lead to steatosis in vivo in rats or depletion of mitochondrial GSH, as well as inhibition of glutathione reductase (Tang et al., 1995). As for acyl-glucuronides, studies concerning acyl-CoA thioester-formation and the cellular effects can best be studied in cultured hepatocytes, which contain a complete cellular network and processes necessary for the formation of these metabolites and their reactions with subcellular entities, i.e., mitochondria and proteins. Specifically, the use of inhibitors of the metabolic processes is easily studied in hepatocytes, with regard to both acute and long-term cytotoxic markers. In this respect, studies on proteomics and metabonomics using hepatocytes may prove to be an interesting model for in vitro work. Genomics and Proteomics One of the key approaches used in recent years to enhance our understanding of mechanisms underlying DILI is evaluation of changes in gene expression at the transcript level (i.e., mRNA expression). The ease with which thousands of genes can be measured has led most of the scientists to register changes of as many genes as possible, with the hope that an in-depth biostatistical analysis would reveal which of those genes were clearly linked to toxic events. These techniques are being used to construct databases that aim to predict the type of toxicity caused by individual test compounds in vivo, based on analysis of patterns of change in gene expression (Hengstler et al., 2006). Encouragingly, gene array analysis has allowed the differentiation between different subtypes of hepatotoxicity at the level of gene expression. Specific mRNA expression patterns for hepatocellular necrosis, inflammation, hepatitis, fibrosis, microvesicular lipidosis, and bile duct hyperplasia have been identified from in vivo studies (Huang et al., 2004; Waring et al., 2001). A further interesting outcome is the identification of patterns of gene expression deregulation specific for carcinogens. Recently, Ellinger-Ziegelbauer et al. (2005) presented gene expression data on rats (in vivo) exposed to four nongenotoxic hepatocarcinogens (methapyrilene, diethylstilbestrol, Wy-14643, and piperonylbutoxide) and four genotoxic carcinogens (2-nitrofluorene, dimethylnitrosamine, NNK, and aflatoxin B1) for up to 14 days and identified substance-specific alterations in mRNA expression patterns. For instance, nongenotoxic substances predominantly deregulated genes related to signal transduction pathways in cell cycle progression and responses to oxidative DNA damage. In contrast, the genotoxic carcinogens induced predominantly genes involved in DNA damage response, apoptosis and survival signaling. Investigations of patterns of pathwayassociated genes allowed a correct assignment of tested substances to the groups of “genotoxic” or “nongenotoxic” rat carcinogens (Ellinger-Ziegelbauer et al., 2005). However, analysis of a single gene or pathway usually will be insufficient to assign a specific mechanism of carcinogenicity. Basal gene expression in human liver tissue and freshly isolated hepatocytes is reported to be distinctively different from one culture to another, regardless of the culture conditions applied (Richert et al., 2006). Therefore, one needs to take into account gene expression changes taking place in hepatocyte cultures over time without any treatment. As well as gene expression changes over time, there are differences in mRNA levels between the specific cell cultures at a given time-point (Tuschl and Mueller, 2006). Figure 20 compares the temporal gene expression changes after 72 and 24 h of 22 hepatotoxicity-related genes in DMSO-treated primary rat hepatocyte conventional and sandwich cultures in

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991-fold

20

10

Txn2

Sult1c2

SOD2

OAT1

COX2

ACOX

MDR1

HSP70

HNF4a

GSTa1

FASN

GADD45a

FABP2

CYP4A1

CYP2C

CYP1A1

cyclinG1

cyclinD1

ApoE

AlkP

- 10

AFP

0 MRP3

Fold regulation


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- 20

-116-fold

-57-fold

- 30

Figure 20 Gene expression analysis of primary rat hepatocytes cultured as conventional monolayers or sandwich cultures with or without serum. The different culture conditions were: () conventional monolayers with serum, ( ) conventional monolayers without serum, () sandwich cultures with serum and ( ) sandwich cultures without serum. One microgram of total RNA was transcribed to cDNA. cDNA (5 ng) per sample was loaded for real-time PCR analysis with TaqMan Low Density Arrays. Shown are values of fold regulation for the temporal gene expression changes occurring at 72 h compared to the values at 24 h (set as 1) in the respective 0.5% DMSO-treated cell cultures. Messenger RNA levels were determined using quantitative real-time PCR. Values were calculated by the efficiency-corrected comparative CT method with 18S rRNA serving as calibrator. Bars illustrate mean values from quadruplicate measurements of a single PCR run with standard deviation. Elevated levels of gene expression are indicated by positive figures > +1, downregulation is indicated by negative figures < –1. Positive as well as the negative y-axis are scaled logarithmically.

serum-containing and serum-free media. There are significant differences in the observed expression patterns for the four cell culture models tested. There is a marked downregulation of most of the assayed genes in the serum-containing monolayer cultures whereas, in the three other cultures, almost all genes increased in abundance at 72 h. The observed increase in mdr1 expression over time in primary hepatocytes is also reported by Fardel et al. (1993) and Chieli et al. (1994). The increase in MDR1 expression over time is less pronounced in sandwich cultures, which is in agreement with Lee (2002). Cyclins D1 and G1 were increased over time, especially in serum-containing cultures, indicating a disturbed cell cycle regulation. Another noticeable feature was the increase in the heat shock factor, HSP70, over time in the serum-containing cultures (Fig. 20), which was still present in conventional monolayer cultures but had disappeared in the collagen sandwich cultures by 72 h. GADD45α expression, although altered only weakly, displayed high levels in serum-containing conventional cultures. Since GADD45α is associated with stress signaling, this indicates that the level of cellular stress was highest in the serum-containing conventional monolayer culture, making them less appropriate for the detection of toxic effects. DMSO was used in these studies because it is a very commonly used solvent. Su and Waxman (2004) reported that the addition of 2% (v/v) DMSO did not affect CYP1A1 mRNA levels but had great influence on the expression patterns of other CYPs and several liver transcription factors. DMSO was capable of restoring normal CYP2B1, CYP3A1,



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CYP4A1, HNF4, and CEBPα (a cyclic AMP response element-binding protein) levels; it also decreased initially elevated HNF6 expression and maintained constant levels of HNF3α, HNF3β, and CEBPβ (Su and Waxman, 2004). This property of DMSO is used for the hepatocyte differentiation of the hepatoma derived cell line HepaRG (Aninat et al., 2006; Gripon et al., 2002; Guillouzo and Chesne, 1996). This cell line is described in more detail later in this review. Other organic solvents also affect the metabolic enzyme expression of hepatocytes, therefore the concentration used for dissolving test agents must be assigned with caution (Chauret et al., 1998; Easterbrook et al., 2001; Hickman et al., 1998). The examples presented above illustrate the potential of genomic technologies to provide novel insight into key mechanisms of toxicity. A major problem encountered when studying the influence of test substances on gene expression patterns in cultured hepatocytes is the relatively high variation in basal mRNA expression. Cultured rodent hepatocytes in particular exhibit a large number of gene up- and downregulations, simply as a result of culture conditions. When undertaking cytotoxicity studies, it is essential to understand how different parameters (other than the test compounds) affect the gene expression. Culture conditions for primary hepatocytes have been optimized to guarantee acceptable levels of drug metabolizing enzymes and responsiveness to enzyme inducers (Carmo et al., 2005; Ringel et al., 2002, 2005). A disadvantage of in vitro genomic studies is that gene expression profiles in rat hepatocytes in vitro and in vivo may differ markedly. Therefore, further research is required to establish cell culture conditions that result in in vitro responses at the transcriptome level that more closely resembles the in vivo situation. Nevertheless, promising results have already been obtained in vitro. For instance, aflatoxin B1, dimethylnitrosamine, acetylaminofluorene, and paracetamol all induced characteristic induction of specific transcription factors (E2F1 and Id1) in cultured human hepatocytes (Harris et al., 2004). Compound-associated specific alterations of mRNA expression patterns have also been observed using cultured rat hepatocytes and liver slices (Boess et al., 2003; de Longueville et al., 2003). Proteomics holds the promise for global analysis of changes in the quantities and post-translational modifications of the proteome. Proteomic analyses are most frequently conducted using 2D gel electrophoresis for protein separation, and mass spectrometry for identification of proteins. Other recently developed technologies include surface enhanced laser desorption ionization (SELDI), antibody microarrays, and various types of liquid chromatography tandem mass spectrometry techniques (LC MS/MS) (Ferguson and Smith, 2003). The first successful applications have been identification of potential markers of toxicity using a proteomics approach alone (Fella et al., 2005), or using proteomics in combination with other ‘omics-technologies (Ruepp et al., 2002). In conclusion, gene or protein expression profiling in cultured hepatocytes has provided valuable new data that may in the future improve our ability to predict toxicological end points. However, further research is needed to solve problems such as basal alterations in gene expression and to provide a better correlation with the in vivo situation. ALTERNATIVE CELL SYSTEMS TO PRIMARY HUMAN HEPATOCYTES AND THEIR LIMITATIONS Hepatocyte-Like Cell Lines Whether originated from tumors or obtained by oncogenic immortalization, human hepatoma cell lines lack a substantial set of liver-specific functions, especially


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many CYP-related enzyme activities. Even, HepG2 cells do not express the major metabolizing enzyme activities; they only exhibit some enzyme activities depending on the culture conditions and the source of the cells (Hewitt and Hewitt, 2004). HepG2 cells stably integrated with specific P450 isoforms have been shown to be responsive to CYP inducers (Allen et al., 2001; Yoshitomi et al., 2001) but are of limited value for induction assays due to their lack of liver-specific functions (Dai et al., 1993; Waxman et al., 1991). Reexpression of CYPs has also been obtained by transfection of plasmid constructs expressing liver-specific transcription factors such as c/EBP-alpha but similarly they do not mimic regulation of gene expression observed in normal hepatocytes (Jover et al.; 1998). Recent advances in hepatocyte-like cell lines have been obtained by the use of HepaRG cells derived from a human hepatocellular carcinoma (Aninat et al., 2006; Gripon et al., 2002). These cells exhibit a pseudodiploid karyotype with two main abnormalities: a surnumerary and remodeled chromosome 7 and a translocation with a loss of the fragment 12p leading to a monosomy 12p (Gripon et al., 2002). When passaged at low density, HepaRG cells show unique features: they revert to undifferentiated elongated cells, actively divide and after having reached confluence they differentiate to form colonies of hepatocyte-like cells with one or two nuclei and bile canaliculi and are surrounded by biliary epithelial-like-cells. Maximum differentiation is obtained by maintaining confluent cells for two weeks in the presence of hydrocortisone and 2% DMSO. Differentiated HepaRG cells express mRNA encoding various nuclear receptors (Ahr, PXR, CAR, and PPARa), the major P450s (CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4) and phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1) in similar levels as in 3–5 days human primary hepatocyte cultures (Aninat et al., 2006). CYP activities in differentiated HepaRG cells are also comparable to those usually found in primary human hepatocyte cultures and are responsive to prototypical inducers. Transcript levels, basal enzyme activities and induction rates of a number of P450s are further increased by the addition of 2% DMSO. An exception is CYP3A4 which is expressed at much higher levels upon DMSO-induced differentiation and is not increased further after a 72 h treatment with rifampicin (Table 5). One of the reasons for huge differences in the expression of the major CYPs between HepaRG and HepG2 cells is the expression of the main nuclear factors PXR and CAR in the former. A high expression of CAR has never been reported in another human hepatoma cell line. Other liver-specific markers, including aldolase B that is found only in adult hepatocytes, are expressed in differentiated HepaRG cells. Presently, all liver-specific functions investigated in these cells have been detected. However, it must be borne in mind that, as observed in primary human hepatocytes, their levels are differently modulated by experimental culture conditions. When seeded at high density, HepaRG cells remain differentiated and can be used after 1–3 days (Aninat et al., 2006). HepaRG cells have also been shown to be more sensitive than HepG2 cells to bioactivated toxins such as aflatoxin B1, supporting their more appropriate use for early cytotoxicity screening as well as induction studies (Aninat et al., 2006). Moreover, HepaRG cells should represent a surrogate to primary human hepatocytes for the study of genotoxic compounds. Stem Cell Derived Hepatocyte-like Cells Numerous articles have reported the generation of hepatocyte-like cells from different types of extrahepatic stem or precursor cells (Beerheide et al., 2002; Hengstler et al.,



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Table 5 mRNA Levels and Specific Activity of CYP3A4 in HepaRG Cells. (A) CYP3A4 mRNA Levels Days of culture Human hepatocytes

HepaRG cells

HepG2 cells

0 1 3 5 5 8 15 30 30 (+DMSO) Confluency

mRNA levels 100 22 6 30 0 0 4.5 8.1 176 0

(B) CYP3A4 Activity (Testosterone 6 b-Hydroxylation) Days of culture 15 30 (−DMSO) 30 (+ DMSO)

Untreated 10.9 + 4.7 (n = 5) 39.7 + 15 (n = 3) 576 + 3&& (n = 3)

+ Rifampicin (50 μM) 129.3 + 22*** (n = 6) 212.9 + 48** (n = 3) 806 + 104&&& (n = 3)

A – Comparative levels of CYP3A4 mRNA in primary human hepatocytes, HepaRG cells and HepG2 cells measured by RT-qPCR. The results are expressed as percentage compared to the mean value obtained with a pool of three different freshly isolated human hepatocyte populations (Day 0) arbitrarily set at 100 %. Cultured primary hepatocytes corresponded to one cell population and were maintained in the absence of DMSO for 1, 3 or 5 days. HepaRG cells were seeded at low density (2.6 × 104 cells/cm2) and cultured for either 30 days in the absence of DMSO or between days 15 and 30 in the presence of 2% DMSO (+ DMSO). HepG2 cells were used at confluence. B – CYP3A4 activity estimated by determination of testosterone 6 β−hydroxylation. Enzyme activity was measured in HepaRG cells seeded at low density after 15 and 30 days. Some cultures were exposed to 2% DMSO between days 15 and 30 (+ DMSO). Pretreatment with 50 μM rifampicin or vehicle (control) was performed for 72 h. Results are expressed as pmol/mg protein/min. Student’s t test was used for statistical analyses between untreated and rifampicin-treated cells (**p < 0.01, ***p < 0.001) and between DMSO-non exposed (–DMSO) and DMSO-exposed (+DMSO) cells (&&p < 0.01, &&&p< 0.001). Adapted from Aninat et al. (2006).

2005; Nussler et al., 2006). At a first glance, this seems to open exciting new opportunities for all fields of research where primary human hepatocytes are applied. However, after a careful evaluation of published studies, all presently available stem cell derived “hepatocyte-like cells” are not yet sufficient to justify their introduction into routine pharmaceutical or toxicological assays. The group of verfaillie reported that “multipotent adult progenitor cells (MAPCs) differentiate into functional hepatocyte-like cells” (Schwartz et al., 2002) and that they “had phenobarbital-inducible cytochrome P450” (Jiang et al., 2002). With more than 1000 citations, these are perhaps the most cited articles in this field. Schwartz et al. (2002) isolated CD44–, CD45– HLA class I– and II– as well as c-kitadherently growing cells from human, rat (Sprague-Dawley) and mouse (C57Bl/6) bone marrow, termed hMAPCs, rMAPCs, or mMAPCs, respectively. These cells were cultured on 1% matrigel in a DMEM-based medium containing 10 ng/ml FGF-4 and 20 ng/ml HGF and subsequently used to study the induction of pentoxyresorufin-O-dealkylase activity (PROD) by phenobarbital. After 4 days with 1 mM phenobarbital, PROD activity


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was increased by 1.3–1.4-fold. Schwartz et al. therefore concluded that MAPCs “had phenobarbital inducible cytochrome P450.” However, this conclusion is equivocal when one considers that PROD activity in conventional cultures of primary hepatocytes is induced more than 10-fold by phenobarbital (Hengstler et al., 2000). Therefore, the author’s conclusion that the MAPC form “functional hepatocyte-like cells” should be treated with caution. Jang et al. (2004) reported that haematopoietic stem cells “become liver cells when co-cultured with damaged liver separated by a barrier”. The authors used a relatively complex procedure for the purification of haematopoietic stem cells and exposed these cells to damaged liver tissue of mice using Transwell plates (which provide the barrier). After just 48 h, albumin and CK18 became detectable in 2–3% of the stem cells. Several liver transcription factors and cytoplasmic proteins expressed during the differentiation of liver (αFP, GATA4, HNF4, HNF3β, HNF1α, and C/EBPα) and in mature hepatocytes (CK18, albumin, fibrinogen, transferrin) were analyzed in the haematopoietic stem cell derived cell population. The expression of all markers increased over time, with the exception of αFP which initially increased and later decreased, indicating possible maturation (Jang et al., 2004). One limitation of this model is the relatively small percentage of cells in the stem cell derived cell population expressing the specific markers. The percentage ranged between as little as 2% of the cells expressing albumin and transferrin and 17% of cells expressing HNF3β. Unfortunately, the heterogeneity of the cell population hinders characterization of enzyme activities and application as an alternative to cultured primary hepatocytes. Experiments aimed at differentiating different human extra-hepatic stem cells to hepatocytes have been performed by a relatively large number of groups (Avital et al., 2001; Eberhardt et al., 2006; Fiegel et al., 2003; Lavon et al., 2004; Lee et al., 2004; von Mach et al., 2004; Zulewski et al., 2001). However, quantitative analysis of enzyme activities and direct comparison of the stem cell derived cell types with primary hepatocytes is not yet available. Recently, three studies have been performed to reprogram human peripheral blood monocytes and differentiate them to hepatocytes (Ruhnke et al., 2005a,b; Zhao et al., 2003). The method involves culturing human blood monocytes with β-mercaptoethanol, M-CSF and IL-3 for 6 days, followed by a 14 day incubation with FGF-4 (Ruhnke et al., 2005a and b). The resulting cell type (termed NeoHep cell) were hexagon-shaped with diameters between 54 and 112 μm, which is larger than cultured primary human hepatocytes. The NeoHeps expressed mRNA for albumin, CYP3A4, CYP2B6, CYP2C9, coagulation factor VII and asialoglycoprotein receptors 1 and 2. Albumin, α1-antitrypsin, CYP2D6 and CYP2C9 proteins were also detected by immunostaining. Enzyme activities of NeoHep cells have been compared to those of cultured primary human hepatocytes. Although some activities of xenobiotic metabolizing enzymes were detectable, CYP3A4 activities were much lower in NeoHep cells compared to primary hepatocytes (Ruhnke et al., 2005a), probably due to a lack of expression of PXR. In conclusion, promising results have been obtained with extrahepatic stem cells since some previously silent hepatocyte markers become expressed during differentiation. However, a human stem-cell derived cell type equivalent to primary hepatocytes does not yet exist. The “Virtual Hepatocyte” Cellular behavior and cell fate scenarios are the result of the coordinated activation and deactivation of multiple signaling pathways. Following genomic sequencing—one of



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the challenges systems biologists are now facing—is to quantitatively understand the complex interactions which cause the purposeful behavior of mammalian cells through mathematical modeling. Goals are, for example, being able to explain the cross-talk of signaling pathways through mathematical models, as well as understanding the process by which interactions between transcription factors and their target sequences result in complex cellular responses. A proper understanding of mammalian cell behavior requires the merging of disciplines and the collaboration of life scientists, mathematicians, physicists, chemists, engineers, and computer scientists. Metabolism, detoxification, regeneration, and differentiation of hepatocytes are typical examples of cell functions regulated by highly dynamic multiple signaling networks. Critical for the success of mathematical modeling of these processes is high quality quantitative data. Recently, primary mouse hepatocytes isolated and cultured under strictly standardized conditions have been shown to fulfill the necessary criteria to permit analysis by a systems biology approach (Klingmüller et al., 2006). All signal transduction pathways in hepatocytes known to be functional in vivo have been shown to be active also in cultured hepatocytes. These include the dosedependent, reproducible and dynamic activation of the JAK-STAT, SMAD, PI3K-Akt, ras-ERK, Wnt/β-catenin,and the FasL-induced signaling pathways (Klingmüller et al., 2006). An example of this is the IL-6-induced time-dependent activation (phosphorylation) of STAT3 in primary mouse hepatocytes (Wang et al., 1999). Presently, a network of scientists (HepatoSys: http://www.systembiologie.de/en/index.html) has initiated a research program aimed at mathematical modeling of detoxification, metabolism, regeneration, and endocytosis in primary mouse and human hepatocytes. However, the “in silico hepatocyte”, predicting specific responses of hepatocytes in vivo, still has a long way to go! Body Culture! A multicell Model This review centers on the use of hepatocytes for determining a number of end points but a single cell system is only representative of one organ, in this case the liver. A major drawback of most in vitro systems is that they do not reflect multiple organ interactions which occur in the whole body. A novel cell culture system, the “integrated discrete multiorgan cell culture” system (IdMOC) is based on the “wells within a well” concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). To “connect” the different cell types, a volume of medium is added to the containing well to flood all inner wells. So far, this system has been successfully used to determine organ-specific toxicity. The wells are flooded with either vehicle control or test compound and, after incubation, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate end points (such as ATP content). Using this technology, the toxicity of tamoxifen (an anticancer agent with known human toxicity) was determined in primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells), and tumor cells (MCF-7 human breast adenocarcinoma cells; Li et al., 2004). The IdMOC model demonstrated clear quantitative differences in the anticancer effects of tamoxifen (i.e., cytotoxicity towards tumor cells) vs. its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels). MCF-7 cells were most sensitive to tamoxifen toxicity and hepatocytes least sensitive. Cytotoxic anticancer agents in general are cytotoxic to most dividing cells, and the relative resistance of hepatocytes towards tamoxifen


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compared to the other cell types is probably a result of its high metabolic capacity as well as it being a nondividing cell type. It is noteworthy that low dose tamoxifen treatment has been associated with neurotoxicity, especially optic neuritis (Noureddin et al., 1999), which was consistent with the high sensitivity of astrocytes to tamoxifen toxicity in the IdMOC model. The interconnection of the multiple cell types in the IdMOC model by a common medium has two advantages: 1) it ensures that all cell types are treated under virtually identical conditions, thereby avoiding differences in response due to experimental artefacts; 2) it allows metabolites from one cell type to interact with a different cell type. For example, metabolites generated by hepatocytes can diffuse and interact with nonhepatic cells. The physical separation of the cell types (each in an individual well) mimics the in vivo condition, where organs are physically separated from each other. The IdMOC system is being characterized further for its application in drug development, with emphasis on drug metabolism, drug distribution as well as drug toxicity. CONCLUSIONS Primary hepatocytes continue to be the most relevant in vitro model for advancing our knowledge of liver functions, the mechanisms underlying the regulation and druginduced changes of metabolic enzyme expression and hepatocellular integrity. The value of hepatocytes has been enhanced further in recent years by technical improvements in cell handling conditions (most notably cryo-preservation and cell culture, especially sandwich culture), in the methods available to explore effects of xenobiotics on critical functions (transporter assays, ‘omic technologies, toxicity end points and so on), and in our understanding of the key role played by xenosensors. Microsomes, once favored for their ease of preparation and use, are now considered even further away from the in vivo situation. Emerging knowledge of uptake and efflux transporter proteins have shown that even simple phase 1 metabolism in microsomes can be significantly influenced because the homogenous mixing of enzymes and test compound biases metabolism towards a reaction that may not occur in an intact cell. The use of adenoviruses and transfected cell lines has increased our knowledge of cellular mechanisms but these cannot yet replace primary hepatocytes for prediction of in vivo drug-drug interactions or hepatotoxicity. Likewise, “hepatocyte-like” cells and in silico modeling, although are promising, have a long way to go before they can be considered an alternative to primary hepatocytes. The pharmaceutical perspective is to generate sensible data which can be used to make “go, no-go” decisions; the ultimate aim is to make the models used as predictable of the in vivo situation as possible. With the increasing supply of good quality fresh and cryo-preserved human hepatocytes, researchers are further on in their quest to reach this goal.

ACKNOWLEDGMENTS This review was written as a result of the 1st Medicon Valley Hepatocyte User Form (MV-HUF) Symposium which was held in Copenhagen on January 26/27th 2006. The authors would like to thank the 2005/6 organizing committee of the MV-HUF: Lassina Badolo, Gerda Marie Rist, Lykke Jacobson, Anette Gemal, Wendy Kappers, Birgitte Andersen, Johnny Arnsdorf Hansen, and Maria Ekblad. We would like to thank Becky McGee and Scott Lloyd from In Vitro Technologies, Maryland, US, for the data presented in Figs. 7 and 8.



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