ATLA 27, 579–638, 1999 579 The Use of Long-term Hepatocyte Cultures for Detecting Induction of Drug Metabolising Enzymes: The Current Status ECVAM Hepatocytes and Metabolically Competent Systems Task Force Report 1 Sandra Coecke,1 Vera Rogiers,2 Martin Bayliss,3 José Castell,4 Johannes Doehmer,5 Gérard Fabre,6 Jeffrey Fry,7 Armin Kern8 and Carl Westmoreland3 1ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra (VA), Italy; 2Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium; 3GlaxoWellcome Research and Development, Park Road, Ware, Hertfordshire SG12 ODP, UK; 4Unidad de Hepatologia Experimental, Hospital Universitario La Fe, Avda de Campanar 21, 46009 Valencia, Spain; 5Institut für Toxikologie und Umwelthygiene, Technische Universität München, Lazarettstrasse 62, 80636 Munich, Germany; 6Preclinical Metabolism and Pharmacokinetics, Sanofi Recherche, Rue du Professeur Blayac 371, 34184 Montpellier Cédex 04, France; 7School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK; 8Drug Metabolism and Isotope Chemistry, Bayer, Aprather Weg 18a, 42096 Wuppertal, Germany Summary — In this report, metabolically competent in vitro systems have been reviewed, in the context of drug metabolising enzyme induction. Based on the experience of the scientists involved, a thorough survey of the literature on metabolically competent long-term culture models was performed. Following this, a prevalidation proposal for the use of the collagen gel sandwich hepatocyte culture system for drug metabolising enzyme induction was designed, focusing on the induction of the cytochrome P450 enzymes as the principal enzymes of inter- est. The ultimate goal of this prevalidation proposal is to provide industry and academia with a metabolically competent in vitro alternative for long-term studies. In an initial phase, the prevalidation study will be limited to the investigation of induction. However, proposals for other long-term applications of these systems should be forwarded to the European Centre for the Validation of Alternative Methods for consideration. The prevalidation proposal deals with several issues, including: a) species; b) practical prevalidation methodology; c) enzyme inducers; and d) advantages of working with independent expert laboratories. Since it is preferable to include other alternative tests for drug metabolising enzyme induction, when such tests arise, it is recommended that they meet the same level of development as for the collagen gel sand- wich long-term hepatocyte system. Those tests which do so should begin the prevalidation and validation process. Key words: long-term culture, hepatocytes, drug metabolism, enzyme regulation, induction, cytochrome P450, CYP, collagen gel culture, organotypic culture, co-culture, spheroids. This is the first report of the ECVAM Task Force on Hepatocytes and Metabolically Competent Systems and rep- resents the agreed conclusions of its members as individual scientists. Address for correspondence and reprints: Dr S. Coecke, ECVAM, TP 580, Institute for Health & Consumer Pro- tection, European Commission Joint Research Centre, 21020 Ispra (VA), Italy. 580 S. Coecke et al. Introduction tion on the enzyme induction capacities of a newly developed chemical entity. Informa- One of the first priorities for the European tion on the inducing properties of a new Centre for the Validation of Alternative chemical entity is usually obtained through Methods (ECVAM) was to become familiar animal studies, by using the mouse, rat, dog with the current status of non-animal test and/or monkey, which have been repeatedly development and validation. Furthermore, it exposed to the chemical for some days. Dur- was important to define the potential for the ing these in vivo experiments, large numbers incorporation of alternative tests into regula- of animals and large amounts of the test tory procedures. Therefore, ECVAM imple- compound are required. Thus, the availabil- mented a series of workshops detailing ity of a simple, robust and reproducible in specific topics, in which small groups of vitro model could help to expedite the drug experts reviewed and discussed the current development process. status of various in vitro tests and their The development of alternatives to the potential use. The first of these workshops, existing in vivo studies is encouraged by the The Practical Applicability of Hepatocyte trend toward an increasing acceptance of in Cultures in Routine Testing, was held in vitro studies by regulatory authorities: “. a Angera, Italy, in 1993, under the co-chair- negative result in vitro (no interaction iden- manship of Bas Blaauboer, José Castell and tified) is reassuring and can generally elimi- Vera Rogiers (1). After this workshop, a task nate the need for further clinical evaluation” force was established under the chairman- (2). Therefore, the current ECVAM proposal ship of Vera Rogiers. The following report focuses on the prevalidation of one or more summarises the results of a number of task metabolically competent in vitro systems force meetings held in Brussels. In these with the specific aim of assessing enzyme meetings, it was decided to focus on the induction. workshop report recommendations 6, 7 and 9. For information and clarity, extracts of these three recommendations are given Drug Metabolising Enzyme Regulation below. Classically, the definition of induction is the “The relatively poor maintenance of stable de novo synthesis of new enzyme (protein) biotransformation activities during hepato- molecules as a result of increased transcrip- cyte culture was identified as a major limita- tion of the respective gene following an tion of the current in vitro systems. Studies appropriate stimulus. However, an increase are required to standardise the culture con- in enzyme activity can also be observed as a ditions, in order to optimise the maintenance result of the stabilisation of an enzyme by a of various hepatocyte-specific functions.” substrate. Inhibition is defined either as an (Recommendation 6) acute decrease in activity toward a particular “The maintenance of hepatocyte-specific substrate by another simultaneously present functions during long-term culture should be compound, or a time-dependent decrease in explored further, in particular in co-cultures the amount of drug metabolising enzyme by and in three-dimensional hepatocyte culture several factors, which can include chemical systems.” (Recommendation 7) injury or a disease process (3). Knowledge of induction and inhibition of “It is essential that a minimum battery of drug metabolism could assist the prediction tests be agreed, with which to judge the of clinically significant effects such as unde- effects of different methods of isolation, sirable drug interactions and metabolism- maintenance and culture on the quality and mediated toxicity. Induction of these functionality of the hepatocytes.” (Recom- biotransformation enzymes might lead to mendation 9) enhanced toxicity because of the formation Industry has identified, as a priority, the of reactive metabolites. Furthermore, need for a metabolically competent in vitro enzyme induction can provoke a variety of system, as an alternative to the in vivo effects, including hepatic hypertrophy and model, for the examination of the induction secondary thyroid neoplasms (4). In clinical potential of new chemical entities. Regula- practice, in the context of pharmacokinetic tory agencies can require specific informa- drug–drug interactions, the metabolic clear- Drug metabolising enzyme induction 581 ance of a given drug can be enhanced and can ing to the cytosolic aryl hydrocarbon (Ah) result in reduced efficacy (5). Inhibition can receptor and translocating to the nucleus lead to enhanced toxicity, due to reduced where a ternary complex is formed with the clearance and a consequent increase in Ah receptor nuclear translocator (9, 10). plasma or tissue levels. From the viewpoint In contrast, the mechanism of gene induc- of drug therapy, to avoid potential drug–drug tion of CYP2 genes by PB and other struc- interactions and metabolism-mediated toxic- turally diverse inducing agents is regulated ity, it is desirable to develop a new drug can- by different mechanisms, without the didate that is not a potent enzyme inducer or involvement of a cytosolic receptor. A spe- inhibitor. Therefore, detailed knowledge of cific PB-responsive receptor has not been the potential of a new chemical entity to identified (9, 11). PB uses phosphorylation as either induce or inhibit drug metabolising a switch to increase the affinity of the tran- enzymes is important. scription factor(s) (10). Drug metabolising enzymes have been Ethanol regulates the expression of divided into two groups. Phase I reactions CYP2E1 by post-translational stabilisation, introduce or unmask a functional group in making it resistant to proteolytic digestion substrate molecules (for example, xenobi- (10). otics and endogenous compounds). These Glucocorticoids regulate the expression of can then be eliminated or further conjugated CYP3A genes through a receptor-mediated by Phase II reactions, the result being the mechanism involving the glucocorticoid formation of soluble compounds that are receptor. However, this receptor is appar- more readily excretable (6). ently not required for induction by The most prominent Phase I enzymes are metyrapyrone, and a complete understand-