ReviewHuman hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation
Introduction
As our knowledge and understanding of the species differences in the regulation and substrate specificity of the mammalian CYP450 enzymes has broadened significantly, the need for a human-relevant in vitro hepatic model system has become more apparent than ever before. Pharmaceutical scientists have attempted to utilize a number of liver-derived model systems to study drug disposition in vitro, including liver slices, immortalized cell lines, and primary hepatocytes. With the lack of phenotypic gene expression in nearly all immortalized cell lines and the limitations of liver slices, such as short-term viability and diffusional barriers, primary cultures of human hepatocytes have become the ‘gold standard’ for the in vitro testing of drugs. For nearly two decades primary cultures of human hepatocytes have been utilized for toxicological and pharmacological studies (Ferrini et al., 1997, Guillouzo, 1998, Guillouzo et al., 1997, Kern et al., 1997). Since that time an overabundance of literature has emerged that describes the various applications and methods for which they have been utilized, especially for studying drug metabolism and the induction of liver cytochrome P450 enzymes.
Within the literature, one can find a number of different in vitro approaches that have been applied successfully for assessing the metabolism and induction potential of drugs (Donato et al., 1995, Maurel, 1996a, Kern et al., 1997, Guillouzo, 1998, Li et al., 1997b, Silva et al., 1998). However, for the novice who is attempting to identify those methods and conditions that are most appropriate for a particular type of study, this may appear initially to be an overwhelming task. Likewise, there are few resources available for obtaining essential information needed to determine which is the best model system and methods for a particular purpose or type of study. This commentary attempts to address some of the more important issues and caveats which must be considered when utilizing primary cultures of human hepatocytes for drug evaluation, especially for long-term studies of gene expression (e.g., CYP450 induction or suppression). The effects of different culture conditions on the restoration and maintenance of normal hepatic structure and function in vitro will be presented, especially as they relate to testing the potential of new drugs to affect liver enzyme expression. The relationship between induction of CYP3A4 in human hepatocytes and the activation of orphan nuclear receptors, such as pregnane X receptor (PXR) and constitutively activated receptor (CAR), by xenobiotics is discussed in light of both species differences in CYP450 regulation and predicting drug interactions in humans. Specific issues and important considerations pertinent to testing NCEs in vitro utilizing primary hepatocyte cultures are discussed. Finally, the development of future guidelines for the standardization of the practices and protocols utilizing this in vitro model system is proposed.
Section snippets
Effects of culture conditions on drug-induced gene expression in primary human hepatocytes
Past progress in elucidating the optimal conditions for the long-term cultivation of rodent hepatocytes has helped define some of the key components in the matrix and medium environment that are most critical for the expression of a differentiated phenotype in vitro (LeCluyse et al., 1996a, LeCluyse et al., 1996b). As part of these developments, several diverse approaches have emerged in an effort to preserve hepatic function by manipulating the extracellular matrix composition, configuration,
Temporal changes in cytochrome CYP450 expression in vitro
The mRNA levels and activities of various CYP450 isoforms are differentially expressed over time in culture and should be taken into account when considering human hepatocytes for long-term studies of metabolism and enzyme induction. Fig. 8 shows the relationship between CYP3A4 mRNA expression and the corresponding testosterone 6β-hydroxylase catalytic activity over several days in culture. In general, there is a time-dependent decrease in the expression of mRNA for all major cytochrome P450
Inter-preparation variability in cytochrome P450 induction
Preparation-to-preparation variability in the extent of induction of various cytochrome P450 enzymes is fairly extensive in primary cultures of human hepatocytes. Fig. 9 illustrates some of the variability in CYP1A2, CYP2C9 and CYP3A4 inducibility, as represented by 7-ethoxyresorufin O-dealkylase, tolbutamide methylhydroxylase and testosterone 6β-hydroxylase activities, respectively, in microsomes prepared from multiple cultures of human hepatocytes treated with β-naphthoflavone or rifampin.
PXR and species differences in CYP3A regulation
Induction of CYP3A gene expression is caused by a variety of xenobiotics, including many drugs (Maurel, 1996b, Guzelian, 1988), and represents the basis for a number of common drug–drug interactions. Many xenobiotics have been examined for their effects on CYP3A expression and marked species differences have been revealed (Kocarek et al., 1993, Kocarek et al., 1995, Barwick et al., 1996). For example, pregnenolone 16α-carbonitrile and dexamethasone are known efficacious CYP3A inducers in
Current recommendations and future considerations
There are a multitude of techniques described in the literature for testing the potential of NCEs to induce or suppress the expression of phase I and II drug-metabolizing enzymes in primary cultures of hepatocytes. Moreover, many of these methods present results using a variety of experimental parameters and endpoints. Few comprehensive comparisons of experimental conditions using pharmacologically and toxicologically relevant endpoints have thus far been reported. As such, there is a general
In vitro screening for enzyme induction: an overview
The techniques currently available for measuring enzyme induction (in a manner that permits the identification of which particular enzymes are induced) fall into two categories: ex vivo and in vitro (Parkinson, 1996a, Parkinson, 1996b, Maurel, 1996a). The ex vivo technique is particularly applicable to studies in laboratory animals, where a drug is administered in vivo and, after a certain time (typically several days to several weeks), the liver is removed and analyzed for increased expression
Conclusions
One of the biggest challenges that continues to face pharmaceutical scientists today is the accurate prediction of in vivo disposition from in vitro data. The prospect of accurately predicting a drug’s in vivo induction potential is not unlike that which currently confronts scientists faced with predicting enzyme inhibition from data derived in vitro. Just as we have made progress in the latter area, we are becoming more capable of accurately assessing the likelihood that a particular drug will
Acknowledgements
The author would like to gratefully acknowledge the valuable contributions of Andrew Parkinson, Ajay Madan, Steve Kliewer, Bingfang Yan, Shiew-Mei Huang, Summer Jolley, D. James Coon, Darryl Gilbert and Geraldine Hamilton to the discussions and data provided within this manuscript. This work was supported in part by the Food and Drug Administration (Center for Drug Evaluation and Research) (#223-97-3004), GlaxoWellcome, DuPont Pharmaceuticals, and Parke-Davis.
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