0887-2333(95)00080-l Toxic. in Yirro Vol. 9, No. 5, pp. 685-694. 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/95 $9.50 + 0.00 Meeting Report Hepatocyte-based In Vitro Models and their Application in Pharmacotoxicology REPORT OF AN EC DGXII MEETING WITH REPRESENTATIVES OF THE EUROPEAN PHARMACEUTICAL INDUSTRY V. ROGIERS, B. BLAAUBOER*, P. MAURELI_, I. PHILLIPS1 and E. SHEPHARD$ Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium, *Research Institute of Toxicology (RITOX), Utrecht University, PO Box 80.176, 3508 TD Utrecht, The Netherlands, tINSERM U128, CNRS, BP 5051, 34033 Montpellier, France, IDepartment of Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London El 4NS and §Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, UK companies with respect to research priorities and practical needs of immediate importance for the An EC DGXII meeting on hepatocyte-based in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK vitro industry. This initiative was taken as a consequence models and their application in pharmacotoxicology of earlier discussions held at the ECVAM workshop was organized by the Department of Toxicology, on hepatocytes in Ispra (Blaauboer et al., 1994b). Vrije Universiteit Brussels, on 23-24 January 1995 at From that particular workshop it became clear that the Sheraton Hotel, Brussels Airport. The goal of the the pharmaceutical industry already uses primary meeting was to reach a general consensus with reprehepatocytes in a number of ways. However, for well sentatives from the leading European pharmaceutical Introduction Participants Ampara L. (Dunar S.L., E) Backfisch G. (Boehringer Mannheim GMbH, D.) Bayliss M. (Glaxo Research, UK) Berglund C. (Astra-Hassle AB, S) Blaauboer B.J. (RITOX, Utrecht University, NL) Boberg M. (Bayer AG, D) Bouis P. (Ciba-Geigy, CH) Carleer J. (Lilly Development Centre, B) Doehmer J. (Technische Universimt Miinchen, D) Duncan J. (Upjohn Laboratories Europe, UK) Fabre G. (Sanofi Recherche, F) Galli CL. (University of Milan, 1) Garthoff B. (EFPIA % Bayer AG, D) George E. (Glaxo In Vitro Tox Unit, UK) Graham M. (Sanofi Research Division, UK) Gross G. (Hoechst AG, D) Van Era Y. (Notox B.V.. NW Horbach J. (Utrecht University, NL) Guillou F. (Laboratoires Fournier, F) Huempel M. (Schering AG, D) Humphrey M. (Pfizer Central Research, UK) Kervyn S. (Lilley Development Centre, B) Koster H. (Solvay Duphar, NL) Lasserre D. (Rhone-Poulenc Agr, F) Lindros K. (Alko Ltd, SF) Maurel P. (INSERM UI28, CNRS, F) Morley T. (Wellcome Foundation, UK) Mortensen J. (Leo Pharmaceutical Products, DK) Phillips I. (Queen Mary and Westfield College, University of London, UK) Puozzo C. (Pierre Fabre, F) Rogiers V. (Vrije Universiteit Brussel, B) Roguet R. (L’OreaI, F) Roba J. (UCB S.A. Pharma, B) Sauvanet J.-P. (Wyeth-Ayerst, F) Schmidt U. (Bayer AG, D) Semino G. (University of Milan, I) Shenhard E. (Universitv Colleee London. UK) Sm;th D. (P&er Ltd, UK) Thenot J.-P. (Synthelabo Recherche, F) Van Cauteren H. (Janssen Research Foundation, B) Vercruysse A. (Vrije Universiteit Brussel, B) Vermeir M. (Janssen Research Foundation, B) Volz A. (Boehringer Ingelheim, D) Weil A. (RhBne Merieux, F) Weymann J. (Knoll AG, D) Ziogas C. (EC-DGIII) 685 686 V. Rogiers et al. characterized reasons, including phenotypic changes of hepatocyte cultures, lack of standardized conditions and methods and lack of sufficient human cells, their practical use is limited. During the EC DGXII meeting, intensive discussions between representatives of academia and the leading European pharmaceutical companies took place and existing problems and their potential solutions were analysed. These are outlined in this report. General background to the use of bepatocyte-based in vitro models in pharmacotoxicology To ensure the maximum safety of drugs for man, new pharmaceuticals are first tested in uivo using experimental animals, and only at a later stage are they administered to human volunteers. Animaibased in vivo models, however, not only involve large numbers of vertebrates (ATLA, 1994) but are often considered ethically, economically and scientifically inadequate. As the safety demands of new drugs increase, so does the demand for animals for testing purposes. Consequently, the solution to this problem, at least in part, seems to lie in the development of alternative, in vitro models. With respect to the ‘3R’ concept for drug development proposed by Russell and Burch (1959), it should be emphasized that in the near future the use of in vitro models will be limited to the refinement and reduction purposes, rather than for the replacement of in vivo tests on whole animals. However, in vitro models can offer a more scientific approach to understanding the mechanisms of toxic action rather than merely describing toxic events, thus making possible the more efficient and correct planning of in vivo and in vitro experiments (Roberfroid, 1991). In particular, the use of well characterized in vitro models at an early stage of the drug development process is important, since guidance for clinical studies can be provided at an early stage, thus expediting the drug development process. In addition, the possibility of using human-derived in vitro models seems of particular importance, since it is known that extrapolation of animal data to humans is sometimes problematic because of both qualitative and quantitative interspecies differences in drug metabolism (Chenery et al., 1987; Le Bigot et al., 1987; Le Bot et al., 1988; Tee e( al., 1987). Since drug metabolism and toxicity are inherently linked, an in vitro model should ideally be relevant for xenobiotic metabolism and thus be able to express key phase I and phase 2 drug-metabolizing enzymes in amounts comparable with the situation in vivo. Consequently, liver-being the major organ with respect to drug metabolism-has been the basis of several different types of preparations used as in vitro Abbreviations: CYP = cytochromes P-450; LRP = liver regulatory protein; PBPK = physiologically based pharmacokinetic. experimental systems in a number of disciplines (Guillouzo and Guguen-Guillouzo, 1992; Skett, 1994). Such in vitro preparations include purified enzymes, subcellular fractions such as microsomes, isolated parenchymal cells (hepatocytes), liverderived cell lines, cell lines that express liver-specific enzyme systems, liver slices and perfused whole organs (Gibson and Skett, 1994). In particular, the use of isolated hepatocytes has attracted much attention over recent decades for several reasons, including an increase in our understanding of the technical aspects of cell isolation and culture and because a homogeneous preparation consisting of a single cell type mainly responsible for drug metabolism can be obtained (Skett, 1994). Thus, the use of hepatocyte cultures combines the convenience of easy handling with ready access to the complex cellular mechanisms of the intact liver in vivo. Among the numerous phase 1 and phase 2 enzyme systems involved in the metabolism of xenobiotics, cytochromes P-450 (CUP) from families CYPl, 2, 3 and 4 play a prominent role (Gonzalez, 1989). The CYP-dependent monooxygenases are expressed mainly in the liver and are able to oxidize an extremely wide range of compounds and, on some occasions, generate toxic metabolites responsible for various pathologies. The expression and function of these enzymes can be affected by a number of factors, including physiological, pathological, genetic and environmental influences. These account for the wide interindividual variability exhibited by human populations in response to drugs and environmental pollutants in terms of metabolism and toxicity. From the above considerations, and from the analysis of numerous clinical reports focusing on adverse drug effects, it is clear that, before a new drug can be administered in vivo to humans with maximum safety, the following questions should be answered: 1. What is the biotransformation pattern of the new drug under physiological conditions? How comparable is this pattern in animal species used for safety evaluation, and in humans? 2. Are there active intermediates formed that could eventually lead to toxic effects (cytotoxic or genotoxic metabolites)? 3. What are the enzymatic systems and isoenzymes involved in the metabolism of the new drug? 4. Is the drug (and its major metabolites) an inducer or inhibitor of drug metabolizing enzymes? What consequences will this have for potential drug interactions? 5. What endogenous and exogenous factors could alter drug metabolism and lead to toxic effects? To answer these essential questions in vitro, shortand long-term cultures of hepatocytes, together with other in vitro systems, could be very useful for the pharmaceutical industry. Short-term culture models, namely conventional monolayer cultures of hepatocytes, can be kept for 687 zyxwvuts Hepatocyte-based zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in vitro models in pharmacotoxicology 2- 3 days and usually maintain phase 1 and phase 2 drug-metabolizing enzymes at an acceptable level in comparison with in viva (Guillouzo, 1986). They are efficient models for establishing qualitatively and quantitatively the biotransformation pattern of a new drug in different species, including human, and are, as such, already frequently being used by the pharmaceutical industry (Blaauboer et al., 1994b). They are also useful in understanding mechanistic toxicity, genotoxicity and biokinetic studies of drugs (Blaauboer et al., 1994a). Long- term in vitro models, of 2-3 wk, expressing key phase 1 and phase 2 drug-metabolizing enzymes close to the in vivo situation, are, however, needed for the in vitro study of long-term events including enzyme induction, some drug interactions, long-term effects of endogenous and exogenous factors on drug metabolism patterns and subchronic/chronic toxicity (Guillouzo et al., 1990; Rogiers and Vercruysse, 1993). At present, such an ideal culture model does not yet exist, although it is clear that more sophisticated culture models of hepatocytes including cultures on specific extracellular matrices, co-cultures with rat liver epithelial cells of primitive biliary origin or cell lines, and three-dimensional cultures in hydrated collagen I gel, display properties that are promising (e.g. maintenance of drug-metabolizing enzymes) (Guillouzo et al., 1990; Rogiers, 1993). Analysis of problems encountered Phenotypic changes in long- term cultures Long-term hepatocyte cultures, currently in use in academia and the pharmaceutical industry, undergo phenotypic changes and consequently do not accurately reflect the situation with regard to foreign compound metabolism in vivo. When cultured under conventional conditions for 1 wk or more, hepatocytes of different species suffer a rapid decline in many liver-specific functions (Rogiers and Vercruysse, 1993). Of particular concern is the reduction in the concentrations of several of the cytochromes P-450 that are of central importance in the metabolism of foreign compounds and the loss of the ability to increase the expression of these proteins in response to some inducers. This decrease in abundance is not uniform for each member of the P-450 superfamily and hence the complement of P-450s present in long-term cultured hepatocytes differs both qualitatively and quantitatively from that of the adult liver in viva. Long-term hepatocyte cultures suffer from the same problem with respect to phase 2 drug-metabolizing enzymes (Vandenberghe et al., 1988, 1989 and 1990) and probably also with respect to non-cytochrome P-450-dependent phase 1 metabolism. In the case of the latter proteins, our knowledge is very limited. A number of drugs are enzyme inhibitors. The consequences of enzyme inhibition are generally of equal importance to those caused by enzyme induc- tion, resulting in modifications of therapeutic effects, side-effects and toxicity of these drugs and of other xenobiotics. As is the case for inducers, inhibitors exhibit some degree of enzyme specificity. In many of the hepatocyte culture systems currently in use, inducers as well as inhibitors sometimes may not be detected because of the reduced expression of drugmetabolizing enzymes and the inability of the cells to respond adequately to such compounds. The development of a long-term hepatocyte model for induction studies in vitro and for subchronic/chronic toxicity experiments may be considered as a key issue in modern drug development. Need for additional characterization and validation of culture models Whatever the in vitro model used for drug development or risk assessment, irrespective of its species of origin, the value of the results obtained will be critically dependent on the state of the characterization and standardization of the model. The lack of comprehensive sets of probes, specific for a range of key phase 1 and phase 2 drug-metabolizing enzymes at the functional (substrate), protein (antibody) and mRNA (cDNA) levels is a serious limitation to the development and application of hepatocyte cultures in pharmacotoxicology. This conclusion was also reached in the ECVAM report on hepatocytes (Blaauboer et al., 1994b). In particular, there is a lack of probes for enzymes involved in non-cytochrome P-450-dependent phase 1 metabolism and those involved in phase 2 biotransformation. In this respect, efforts should be focused not only on the expression of members of the CYP l-4 families and of other components of the CYPmediated monooxygenases, but also on epoxide hydrolases, flavin-containing monooxygenases and phase 2 enzymes such as UDP-glucuronyltransferases and glutathione S-transferases in various species. Specific substrates, antibodies and cDNA probes are needed for these key enzymes. In this respect, the order of importance of different species for the pharmaceutical industry is as follows: human, dog, monkey, rat, mouse and rabbit. Pig should receive more attention in the near future, since it could represent an alternative species in pharmacotoxicology. The probes must be available in quantities sufficient to meet the needs of academia and the pharmaceutical industry. Modern techniques such as raising specific antibodies by phage-display (McCafferty et al., 1990), and the construction of specific cDNA probes by reverse transcription/polymerase chain reaction (PCR) (Nanji et al., 1994) may be useful in this respect. Interspecies variability and need for human cells In addition to the difficulties associated with extrapolating in vitro data to the situation in vivo, problems associated with interspecies extrapolations are of real concern since both qualitative and 688 V. Rogiers ef zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM al. quantitative species differences exist in the biotransformation of drugs and in the intrinsic toxicity of these compounds and their metabolites (Chenery et al., 1987). There is no doubt that the use of humanderived hepatocyte cultures in pharmacotoxicology is preferable to the use of cultures derived from other species (Fentem, 1994; Guillouzo et al., 1995; Rogiers, 1993). However, it is clear that serious limitations exist concerning the use of human-derived liver cells for drug development. The major problems can be summarized as follows: (a) tissue availability; (b) preservation of tissues and cells (cryopreservation); (c) logistic problems (transport, distribution, organization); (d) data variability; (e) ethical and legal aspects; (f) uniformity of the methods used; and (g) characterization and quality assessment of the preparations, tissues and model. Clearly, all these problems need to be resolved. Their nature, however, is quite different. While problems a-e are almost exclusively linked to the use of human cells, the limitations f-g are of a more general nature (Fentem, 1994; Guillouzo et ul., 199.5;Gurney and Balls, 1993; Rogiers, 1993). Before well-characterized liver cell banks can be established at national or European level, all these problems must be addressed. et al., 1988) and vitrogen (Waxman et al., 1990) and by co-culturing hepatocytes with rat liver epithelial cells of primitive biliary origin (BCguC et al., 1984). Other highly promising three-dimensional models are hepatocytes cultured in a collagen gel sandwich configuration (Dunn et al., 1989) or cultured as liver spheroids (Roberts and Soames, 1993; Tong et al., 1992). The ability of these more sophisticated hepatocyte culture systems to maintain the expression and inducibility of the major phase 1 and phase 2 drugmetabolizing enzymes needs to be investigated thoroughly so that informed conclusions can be drawn regarding their relative advantages and disadvantages and to establish how each compares with conventional culture systems and with the liver in vivo. In addition, such information would ensure that results derived from metabolic experiments with each system could be interpreted correctly. However, it should be emphasized that, although the pharmaceutical industry needs improved culture systems, the emphasis should be on convenience, and complicated systems may be of little practical use. Improvements in this direction could include the following: Improvement of existing hepatocyte culture systems The availability of well defined extracellular matrices, such as matrigel produced from an established cell line rather than by extraction from Englebreth-Holm-Swarm mouse sarcoma, a procedure which is not acceptable for ethical and scientific reasons. A rigorous comparison of the effectiveness of various commercially available, extracellular matrices. The development of a co-culture system of hepatocytes with immortalized helper cells. Although the basic model, as developed by the Guillouzo group (BCguCet al., 1984) has been identified as a promising model in the ECVAM report on hepatocytes (Blaauboer et al., 1994b), it could be substantially simplified by the development of standardized immortalized biliary epithelial cells expressing LRP (liver regulatory protein) (Corlu et al., 1991) or of appropriately transfected cell lines. Further development and improvement of threedimensional systems cultured in hydrated collagen I gels. A promising possibility is the use of immobilization gels, a simpler technique than the collagen gel sandwich configuration (Koebe et al., 1994) and one that may be more feasible for use in the pharmaceutical industry. Recent studies in several laboratories have demonstrated that, to maintain their differentiated state in vitro, hepatocytes require a complex environment, the expression of liver-specific functions being regulated not only by exogenous factors but also by cell-matrix and cell-cell interactions (Guillouzo et al., 1990; Rogiers, 1993). Encouraging results have been obtained by culturing hepatocytes on biologically-derived support matrices such as matrigel (Schuetz It must be emphasized, however, that only those culture models that express for 2-3 wk the key phase I and phase 2 drug-metabolizing enzymes at levels that reflect those in vivo, or at least in relative amounts comparable with those in vivo, will be of significant importance. In addition, an adequate response to inducing and inhibiting agents must be considered as being essential for the improvement of existing hepatocyte culture systems. In vitra-in civo correlation Results obtained using short- and long-term hepatocyte cultures are not directly applicable to the in vice situation. A serious drawback is the absence of in vivo data on tissue drug concentrations in man. In addition, the absence of elimination pathways in cell culture models may well generate exposure conditions poorly predictive of the in vivo situation. This can lead to a misinterpretation of in vitro data. For example, the concentrations used in hepatocyte cultures may be irrelevant to the situation in civo. One possibility is that cells may be exposed to much lower concentrations in viva because the drug cannot easily reach the cells. This would result in an overestimation of the toxicity of the drug for this cell type. Alternatively, some drugs may accumulate in certain organs, tissues or cell types and an in vitro test system such as hepatocyte cultures would then lead to an underestimation of their toxic properties. Analysis of potential solutions Hepatocyte-based zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in vitro models in pharmacotoxicology 689 zyxwvuts Development of new in vitro models: immortalized hepatocy tes As mentioned previously, the development of fully characterized primary hepatocyte culture systems that better reflect the ability of the liver in uivo to metabolize foreign compounds would be of tremendous value to the pharmaceutical industry. However, as only limited numbers of cells, with a relatively short lifespan, can be produced from each experimental animal, the extensive use of hepatocyte cultures as in vitro models requires repeated isolations of cells, a technically demanding operation, and would thus be time consuming, expensive and wasteful of animals. In addition, slight variations between the quality of different preparations of cells may affect the results obtained. Such problems would be overcome by the availability of a continuously dividing hepatocyte cell line. Unfortunately, existing hepatoma cell lines express low levels of many drug-metabolizing enzymes, and even the most promising of these (e.g. Faza 967 and Hep G2) fall a long way short of the ideal. Attempts have been made recently to immortalize hepatocytes (MacDonald et al., 1994). An in vitro approach, applicable to human hepatocytes, is to transform primary cell cultures with a gene coding for a protein, such as the SV40 large T antigen, that will cause the hepatocytes to divide. An alternative in vivo approach, applicable to rodent hepatocytes, is to use transgenic animals that already carry the immortalizing gene as a transgene. The groups of Phillips and Shephard (Kramer et al., 1994) are currently attempting to establish conditionally immortalized, differentiated hepatocyte cell lines derived from transgenic mice harbouring a temperature-sensitive mutant of the SV40 large T antigen gene under the control of an inducible major histocompatibility complex H-2Kb class I promoter (Jat et al., 1991). In theory, cells isolated from these mice should divide and be in a dedifferentiated state at the permissive temperature and differentiated at the non-permissive temperature. If successful, the system would provide a continuously dividing, minimally transformed cell line, the functions of which should correspond closely to those of adult mouse hepatocytes in uivo. From these mice a population of dividing cells that retain the morphological characteristics of differentiated adult hepatocytes and secrete albumin into the culture medium has already been obtained. Results from quantitative RNase protection assays demonstrate that the cells express CYPlA2-a highly liver-specific cytochrome P-450 that is readily lost during hepatocyte culture-and retain the ability to induce the expression of this gene in response to the appropriate foreign compounds. The cells have been maintained in culture for 30 passages and subsequently several cloned cell lines have been obtained, many of which exhibit the characteristic morphology of adult mouse hepatocytes. The state of differentiation of each cell line must, however, be investigated by determining the expression of a variety of functions associated with highly differentiated hepatocytes. Although it is expected that at least some of the immortalized hepatocyte cell lines produced by the transgenic approach described above will prove more satisfactory for pharmacotoxicological studies than primary cultures of hepatocytes, they may not reflect fully the capacity to metabolize foreign compounds exhibited by hepatocytes in vivo. However, by stably transforming the cells with DNA sequences encoding liver-enriched transcription factors or selected drugmetabolizing proteins, it should be possible to alter their phenotype so that it resembles more closely that of an adult hepatocyte. Immortalized cells produced by the approaches described above appear promising, but much work remains to be done before such cells can be made generally available. However, it is clear that such models should be developed for the future. In particular, such models of human and rodent origin would provide an excellent tool for the study of drug metabolism. It would markedly increase the quality of pharmacotoxicological data that could be obtained from in vitro systems and result in substantial savings in time and money. Consequently, the development of immortalized human and rodent hepatocytes, possibly supplemented by means of stable transformation, would be of real benefit to the pharmaceutical industry. Development of molecular probes and assay s jbr the quantification of drug- metabolizing enzy mes Whatever the system being used for in vitro investigation of foreign compound metabolism, proper interpretation of the results obtained will be dependent on the accurate determination of the metabolic potential of the system. Because of the pronounced interindividual variation in the expression of cytochromes P-450 in humans (Palmer et al., 1990) this is particularly true for systems, such as hepatocytes or microsomal membranes, of human origin. The metabolic potential of a system can be determined easily by the quantification of the major proteins responsible for the metabolism of foreign compounds. At present, this can be achieved with greater specificity at the RNA level, and with very few exceptions the relative abundance of the mRNA reflects closely that of the corresponding protein. In the case of microsomal preparations. the concentrations of mRNAs in the source tissue can be determined. Probes for any mRNA of known sequence can be readily obtained by reverse-transcription/PCR. The technique of RNase protection can distinguish between mRNAs as similar as those encoding CYP2Bl and 2B2 (Akrawi et al., 1993) which have more than 97% sequence identity, and has a sensitivity of less than a single molecule per cell An alternative approach would be to use the technique of quantitative PCR (Gilliland et al., 1990). This V. Rogiers PI ul. 690 technique, although less accurate than RNase protection, is quicker and less technically demanding, and could easily be adapted for routine use in the pharmaceutical industry. The versatility of the method is such that it can be adapted either to quantify all members of a subfamily of phase I or phase 2 drug-metabolizing enzymes simultaneously or to measure selectively the abundance of a single member of the subfamily. Although for most quantitative purposes probes for mRNAs are adequate, the production of isoformspecific antibodies against human cytochromes P-450 and other phase 1 and 2 drug-metabolizing enzymes would be of tremendous benefit to pharmacotoxicological research. One method of attempting to raise form-specific antibodies against, for example. cytochromes P-450, is to use as antigens short synthetic peptides based on sequences that differ between closely related polypeptides. An alternative approach is that of phage-display (McCafferty et al.. 1990). which provides a powerful biological system for antibody selection. Basically, if one considers cytochromes P-450, the form of interest would be expressed in a heterologous system, such as bacteria or baculovirus, purified and then injected into a mouse. Subsequently, DNA sequences coding for antibodies are amplified by reverse-transcription/PCR from total RNA isolated from the mouse’s spleen. The antibody-encoding sequences are then linked to a sequence encoding a coat protein of a filamentous bacteriophage, thus enabling their expression as fusion proteins on the surface of the bacteriophage. A large library of recombinant phage (a phage display library) can be screened for antibodies against the original P-450 antigen. Each of the selected phages can be rescreened for cross-reactivity against a battery of other, closely-related P-450s (produced by means of heterologous expression of cloned cDNAs). This approach should enable the identification of phage that display antibodies that are specific for a particular member of a P-450 subfamily and others that will recognize all members of the subfamily. Similarly, it should be possible to identify isoform-specific inhibitory antibodies. An additional advantage to the isolation of monoclonal antibodies by this recombinant DNA approach is that. once identified and characterized, they can be produced in sufficient quantities to meet the needs of academia and the pharmaceutical industry. Consequently. the production of specific antibodies and cDNA probes for key phase 1 and phase 2 drug-metabolizing enzymes for the major species of importance in drug development (human, dog, monkey, rat and mouse) must receive priority in the near future. Establishment of’ humun liver bunks There is no doubt that human hepatocytes are the most appropriate choice to develop an in vitro model for drug development. They can give a complete picture of drug metabolism and may be of consider- able help in solving several essential questions, including the identity of isoenzymes involved in the metabolism of new and already marketed drugs. Examples in this respect are cyclosporin A (Pichard et ul., 1990 and 1992) ethynylestradiol (Guengerich, 1988), gestodene (Guengerich, 1990) lansoprazole and omeprazole (Pichard et ul., 1995). Furthermore, the inducing and inhibitory capacities of various compounds can be examined in human hepatocyte cultures. Good examples of induction and inhibition can be found in the literature: these include omeprazole and lansoprazole (Curi-Pedrosa et al., 1994; Diaz rt al., 1990) and N-substituted imidazoles (Maurice et al., 1992). respectively. Potential drug interactions may be determined efficiently, as is the case for cyclosporin A (Pichard et al., 1990). Furthermore, activation to cytotoxic and/or genotoxic metabolites can be achieved using human hepatocyte cultures. A recent example is that of cyproterone acetate (Werner et ul.. 1995). Obviously, results obtained in oitro, even when the model is based on human hepatocytes, cannot be transferred to the in cico situation without caution. A major problem, however, in using humanderived in vitro models lies in the fact that human material with which to prepare hepatocytes is not readily available, and certainly not in sufficient quantities to fulfil the real needs of the European pharmaceutical industry. A solution to this is not evident since on a European scale the availability of human liver tissue has decreased as demand for transplantation increases. However, the following may be proposed: I. More attention should be paid to public education, so that, coupled with a reassurance that any removal of liver tissue would be properly conducted and controlled, any apprehension would be replaced by willingness to collaborate. In this respect, not only should emotional difficulties be overcome but also cultural, religious and social aspects should be dealt with, since they all contribute to the decreasing availability of human liver tissue (Gurney and Balls, 1993). 2. A proper transport system should be set up at the national and European level to overcome the existing logistic problems of tissue procurement (Gurney and Balls, 1993). 3. Studies on cryopreservation and long-term cold storage of human hepatocytes should be given priority (Gurney and Balls, 1993). 4. Existing national and European legislation should be modified. Since the use of human liver tissue for experimental purposes has not been included in existing legislation (with the exception of those of France and Turkey), it is not clear whether the use by industry of human hepatocytes has a legal basis (Vercruysse, 1993). 5. The establishment and enlargement with cytosolic fractions and S-9 fractions of already existing Hepatocyte-based in uifro models in pharmacotoxicology 691 Conclusions banks of human microsomes seems feasible, as in this case storage is not a problem. The fractions The goal of the EC meeting on ‘Hepatocyte-based stored should be fully characterized for as many in vitro models and their application in pharmacotoxforms of CYP and related monooxygenase activiicology’ was to reach a general consensus with ties as possible, and for other phase 1 and phase representatives of the leading European pharmaceuti2 drug-metabolizing enzymes such as flavin-concal companies with respect to research priorities taining monooxygenases, epoxide hydrolases, of immediate importance to the industry. The UDP-glucuronyltransferases and glutathione Sagreed points of interest can be summarized as transferases, for which little information exists follows: (Wrighton zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et al., 1994). Search ,for an alternative Because of the limited availability of human material it is necessary to search for ‘the best alternative’. Comparisons can be made between the xenobiotic biotransformation capacity of different species, including humans (Mennes, 1992). This approach may identify a better model species for humans than rat. Indeed, wide differences exist between human and rat hepatocytes concerning the biotransformation patterns of many compounds (Gonzalez, 1992; Guengerich, 1994). In this respect, monkey, and possibly pig may represent better models for the human situation, although these species have not been fully investigated. The use of reliable in vitro models of these species is very desirable since unnecessary in vivo studies with higher vertebrates could be avoided. Consequently, these other models, including cell lines, should be further developed to provide the pharmaceutical industry with the best possible alternative for human hepatocytes. Development of physiologically based pharmacokinetic models for the evaluation of in vitro results with respect to the in vivo situation Physiologically-based pharmacokinetic (PBPK) models allow a quantitative description of the fate of a xenobiotic and its metabolite(s) in mammalian organisms. The PBPK modelling technique could be a useful tool to predict and determine in vivo metabolic constants (Gargas et al., 1986). Recently, incorporation of in vitro kinetic parameters, obtained for some compounds in fresh hepatocytes, into PBPK models accurately simulated in uivo pharmacokinetics (Kedderis et al., 1993). Therefore, in vitro data obtained from cultures of hepatocytes of different species, including humans, can be used as biochemical parameters in PBPK models. Biochemical parameters could be the kinetic parameters (K,,,, I’,,,) of key phase 1 and phase 2 drug-metabolizing enzymes. Other data needed are anatomical and physiological variables, and physicochemical parameters of compounds. PBPK models can then be used to correlate in vitro results with the in vivo situation, and predict the behaviour of new drugs under various experimental conditions and in different species (Blaauboer et al., 1994a; Houston, 1994). This approach can be developed into a practical validated model for immediate use by the pharmaceutical industry. The European pharmaceutical industry is particulary interested in using hepatocyte-based in vitro models at an early stage of drug development. The use of such systems for risk assessment is not a high priority since it is more a long-term goal. In vitro models of practical use for the pharmaceutical industry must be either well differentiated, and reflect as closely as possible the situation in uivo with respect to their content of the major phase 1 and phase 2 drug-metabolizing enzymes, or tailor-made towards the problem under investigation. In both cases, the limitations of the models should be clearly defined. ‘. Both short-term (2-3 days) and long-term (2-3 wk) models are important. The former already exists and is of particular interest for the identification of the biotransformation pattern of new drugs. The latter does not exist and is of tremendous importance for the study of the inducing potential of a new drug, some drug interactions and the regulation of the expression of a number of drug-metabolizing enzymes. A long-term model that is well characterized as far as key phase 1 and phase 2 drug-metabolizing enzymes are concerned (enzymatic activity, protein and mRNA concentrations) requires development. Research priorities should therefore be as follows: (a) Improvement of existing hepatocyte culture models (cell-cell models, three-dimensional models, biomatrix models, etc.) with special emphasis on convenience and characterization. (b) Development of new culture models with a high priority for immortalized human hepatocytes, possibly stably transformed with DNA sequences encoding selected drug-metabolizing proteins or liver-enriched transcription factors. Species in order of importance are human, dog, monkey, rat, mouse and rabbit. The pig model is currently under investigation and could become an important alternative system. (4 As already much information exists on rodents, and in particular on rats, it is essential to compare the relative drug-metabolic performance of hepatocytes derived from man and rat cultured under identical conditions. V. Rogiers et al. 692 5. A constant and adequate supply of human hepatocytes from liver banks is of tremendous importance. However, it is generally accepted that currently, in Europe, their availability is a serious problem. Thus, high priority should be given to the development of realistic alternatives such as the official establishment of banks of microsomes from human liver and, eventually, from livers of higher mammalian species in general. 6. Associated needs required for the establishment of a bank of human liver microsomes (also hepatocytes at a later date) are: (4 Public education (b) (4 (4 (e) (0 to overcome fear and repugnance to the concept of organ donation for experimental purposes. Establishment of a good transport and distribution network. Priority for cryopreservation studies (especially if hepatocytes are involved). Standardization of methods, protocols and models. Modification of the national and European legislation on organ donation to include the use of organs for experimental purposes. with special attention being paid to the ethical aspect that this would involve. Attention for competition from the USA. Networks are already in place which can cover the demands of the European market. It would be preferable if Europe was responsible for meeting its own needs. changes in the biotransformation pattern of new drugs and the toxicity involved. I I. Interest was also expressed in the following: (a) The use of liver slices, their characterization and the need for comparative studies with cultured hepatocytes. of existing genotoxicity tests (b) Improvement with hepatocytes, looking at a mutagenic effect rather than DNA repair or adducts. with choosing the relevant (c) The problems endpoints for in zdro toxicity studies-nonspecific versus liver-specific ones. in citro models (d) The use of hepatocyte-based as bioreactors for the production of the various metabolites of new drugs. (e) The use of in vitro systems to solve toxicological problems of general interest, such as cholestasis and steatosis. 12. 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