Human Hepatocytes As a Tool for Studying Toxicity and Drug Metabolism

292 Current Drug Metabolism, 2003, 4, 292-312 Human Hepatocytes as a Tool for Studying Toxicity and Drug Metabolism M.J. Gómez-Lechón*, M.T. Donato, J.V. Castell and R. Jover Centro de Investigación, Hospital La Fe, Avda Campanar 21, 46009-Valencia, Spain Abstract: Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment. Key Words: Cryopreservation, cytochrome P450, CYP-engineered cells, drug-drug interaction, drug metabolism, hepatocytes, hepatotoxicity, drug induction. INTRODUCTION Nowadays, the process of selecting drug candidates is becoming much more rational as studies on metabolism and After intake, drugs are usually biotransformed into new kinetics of drug candidates are implemented earlier by using chemical species that may have either toxic or therapeutic human in vitro hepatic models for these studies at the early effects. This process occurs at several sites in the organism, pre-clinical stages [7]. but the liver is the most active organ in the fate of foreign Different human-derived models are currently being used compounds [1, 2]. Although biotransformation generally is a to investigate human metabolism of drugs in vitro. On the detoxification process, drugs can also be transformed into one hand are human liver preparations like microsomes and new chemical species displaying greater pharmacological or on the other, more complex cellular systems like human toxic potential. CYP-engineered cells, hepatoma cell lines and isolated and Drug metabolism is a major determinant of drug cultured hepatocytes, which could give a more complete clearance, interindividual pharmacokinetic differences and, picture of the metabolism of a drug in man. Human CYP- indirectly, of the clinical efficacy and toxicity of drugs [3-5]. engineered cells are at present the most efficient tool for Altered pharmacokinetics can result in inadequate determining whether a given CYP can or cannot give rise to concentration of the drug at the site of action. This, in turn, a particular drug metabolite, however, these cells do not results in inappropriate pharmacodynamic action and/or allow anticipating the metabolic profile of a drug in man [8]. great variations in clinical response. Indeed, variability Several metabolic-competent cell lines have recently been reduces the safety margin [6]. Analysis of the metabolic developed by using expression vectors encoding full-length profile and hepatic clearance is important for drugs human CYP genes. Several groups succeeded in expressing eliminated by hepatic metabolism and, hence, development fully active CYP isozymes in cells that constitutively express of a new drug demands exhaustive characterisation of its such activities under the control of a strong promoter [9, 10]. metabolic profile. Therefore, the development of a new drug Nevertheless, human hepatocytes are recognized to be the requires an exhaustive characterisation not only of its closest model to human liver [11, 12]. Hepatocytes in pharmacological activity, but also knowledge of major chemically defined culture conditions express most typical enzymes involved in metabolite formation, and the potential hepatic biochemical functions, among which is the ability to enzyme inhibiting or enzyme inducing properties of the drug. metabolise drugs [1, 12, 13]. Primary hepatocytes are differentiated cells able to reproduce in vitro the response of *Address correspondence to this author at the Centro de Investigación, human liver to pathophysiological factors and are currently Hospital La Fe, Avda de Campanar 21, 46009-Valencia (Spain); Tel: +34 96 considered a valuable in vitro tool for determining drug 1973040; Fax: +34 96 1973018; E-mail: [email protected] metabolism and for assessing the risk of drug hepatotoxicity 1389-2002/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd. Human Hepatocytes as a Tool for Studying Toxicity Current Drug Metabolism, 2003, Vol. 4, No. 4 293 in man [11, 12]. The restricted accessibility to suitable liver obtaining high yields of viable human hepatocytes have been samples has greatly hindered the widespread use of primary extensively studied and improved. Livers for transplantation cultures of human hepatocytes. Moreover, cell cultures need are perfused in situ with cold University of Wisconsin (UW) to be prepared each time from liver tissue, what makes it solution to avoid warm ischemia and organ is usually even more difficult to use human hepatocytes for routine maintained under these conditions for several hours until testing. The irregular supply of human liver for cell hepatocyte isolation. There are some concerns about liver harvesting purposes has led to a need for optimised protocols samples stored in the UW solution, as cold ischemia could be for the long-term storage of hepatocytes so that available considered as a possible factor involved in the efficiency of tissue can be used more efficiently. the isolation procedure and the metabolic competence of As a consequence of the restricted access to human liver cultured cells. It was reported that hepatocytes isolated from samples, alternatives to replace or reduce the need of human fresh liver maintain liver-specific functions better than hepatocytes have been explored. The use of hepatic cell hepatocytes obtained from organ donor liver after hypo- lines, either from naturally occurring hepatomas, or from thermic storage for several hours in UW solution [29-31]. laboratory-immortalized hepatocytes have been proposed as However other studies reported that human liver, isolated an alternative model to primary cultured hepatocytes but hepatocytes or liver slices can be efficiently stored in cold hepatoma cells do not constitute a real alternative, as these solutions for several hours with minor losses of functionality cells poorly express biotransformation activities [14, 15]. [25, 33, 37]. However, promising advances are being made with these A large number of viable adult human hepatocytes can be cells by expressing transcription factors that control CYP isolated from whole livers and from a single liver lobe [34, expression and are lacking in these cells [16-19]. 35], but perfusion requires large amounts of collagenase and Consequently, researchers are exploring several strategies in during the digestion process the perfusate is often irregularly order to achieve hepatic cell lines re-expressing the whole distributed through all the tissue giving low yield. Other spectrum of human xenobiotic-metabolising enzymes, as an proteolytic enzymes, such as liberase, have been successfully alternative to primary cultures [19-21]. used to isolate hepatocytes [23, 36]. Small tissue samples obtained in the course of liver surgery or from tissue ISOLATION OF HEPATOCYTES FROM HUMAN resections for pathological examination constitute a reliable LIVER SAMPLES source of viable hepatocytes. The two-step collagenase perfusion has been also successfully employed to isolate Human liver tissue has become increasingly available for hepatocytes from these samples [12, 29, 37-39]. Whenever research purposes due, in part, to the expansion of the liver possible, a wedge-shaped biopsy of liver with a single cut transplantation programmes. Livers from organ donors, surface and readily accessible blood vessels should be which are not implanted for some reason (i.e. steatosis or obtained. An adequate cannulation of the small vessels to non-identification of an adequate recipient), can be used for ensure a complete perfusion of the piece and an efficient hepatocyte isolation. A major limitation of using human liver oxygenation of the perfusion buffer during cell isolation are for xenobiotic metabolism studies is ensuring a regular key factors to obtain high yields of viable, minimally supply of adequate

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