Integration of in vitro approaches to predict drug metabolism and interactions in man in development of pharmaceuticals
Investigation of in vitro human liver-derived drug-metabolising test systems as to their capability to predict in vivo metabolism and pharmacokinetics of pharmaceuticals
The systems were characterised systematically for their substrate specificity and kinetics, level of expression of drug metabolising enzymes, at both the RNA and protein level, and their responses to inducing compounds as appropriate. Where necessary, specific reagents, such as enzyme-specific substrates and inhibitors, were developed for these purposes. A unique aspect of the study was the rigorous "blinded" evaluation of the routes, rates and specificity of metabolism and drug interactions of four unknown compounds supplied by industrial partners. Comparison of the in vitro data with observations obtained in-vivo revealed that the candidate systems performed generally satisfactorily in prediction of various aspects of in vivo behaviour of the four compounds. The applications for which the different systems are most suited were established and guidance on their use are being prepared. The project has provided a pre-validation step for further development and testing (validation) of an optimal (integrated) approach, which will allow the effective prediction of the metabolism of drugs in vivo during drug discovery, during the safety evaluation of NCE's and in the design and interpretation of early clinical trials. The aim of the EUROCYP project was to integrate current and new information on human liver-derived test systems and models for predicting the metabolism and metabolic interactions of drugs and other chemicals and to compare the results with those in vivo. During the course of the project, the following objectives were achieved: -Selection and characterisation of enzyme-specific substrates and reagents (chemicals, antibodies, cDNAs). -Characterisation of systems of different levels of biological complexity: enzyme active site models, subcellular fractions, intact cells, tissue slices. -Genetic manipulation of these systems to improve their utility. -The systematic comparison of these systems with each other and with human in vivo data. The choice of systems was based on a number of criteria, including: specificity, kinetic fidelity, stability and technical feasibility. In the attached table some of the important semi quantitative characteristics of the candidate systems have been compared. CYP enzymes in each four-candetate systems (Microsomes, Slices, Hepatocytes, Recombinants) were characterized with respect to presence/absence, variability, stability, and inducibility. All of the systems are suitable for short-term studies of drug metabolism. However, during longer term culture, in both hepatocytes and liver slices there is differential loss of P450 enzyme content and activity. This can be reduced in hepatocytes by co-culture or alginate bead entrapment. With liver slices, additional research will needed to establish whether such preservation is possible. Both cultured hepatocytes and liver slices can provide a useful system for assessing the induction potential of drugs and other chemicals. Each of the candidate systems was assessed blind for its ability to predict the routes and rates of metabolism of four unknowns ("NCE's" - new chemical entities; drugs in late stage devleopment or early clinical use). The performance of the various candidate systems in predicting in vivo metabolite patterns, metabolising enzymes and kinetics of the four NCE's were found to be generally from good to excellent, with very few exceptions. In general, it was possible to predict most of the salient features of the metabolism and kinetics of the four NCE's by the majority of the candidate systems. The "in vitro systems", microsomal fraction, recombinant expressed P450 enzymes and homology models, were better suited to the determination of P450 specificity, whilst the intact cell systems (hepatocytes and liver slices) were more suitable for predicting the totality of the metabolic pathways and in vivo clearance. The applications of several systems are necessary in order to provide an integrated picture of the fate and metabolic interactions of a new drug.