Cytochrome P450 isoform-specific <em>in vitro</em> methods to predict drug metabolism and interactions

Abstract

Cytochromes P450 (P450, CYP) are a superfamily of enzymes that participate especially in the oxidative metabolism of various xenobiotics and endogenous compounds.

The major goal of this study was to characterise suitable methods for routine preclinical in vitro testing of new chemical entities (NCE) and to test the methods for the affinity screening of selected drugs.

In vitro methods used involve the utilisation of human liver microsomes for studies with P450-selective reference inhibitors, inhibitory antibodies and cDNA-expressed enzymes in cytochrome P450-catalysed activities and for studying the reactions of selegiline and entacapone.

In this project, the CYP-catalysed oxidative in vitro biotransformation of selegiline into its primary metabolites desmethylselegiline and l-methamphetamine and the transformation of entacapone into its in vitro metabolite N-desethylentacapone were studied. The affinities of selegiline, desmethylselegiline, l-methamphetamine, entacapone, candesartan, eprosartan, irbesartan, losartan and valsartan to P450 enzymes were also elucidated, and the selectivity of tranylcypromine as a CYP2A6-selective reference inhibitor was characterised.

The most important findings were that the methodology developed during this work is suitable for preclinical in vitro testing of NCEs and that the results obtained for the studied compounds are in line with the available in vivo data.

By the in vitro testing methodology, it is possible to target the in vivo interaction studies to the relevant groups of compounds. The in vitro methods presented in this thesis could also make the early phases of drug development more cost-effective. Further, the number of animals used for in vivo testing in preclinical metabolism and interaction studies can be markedly reduced by effectively using this methodology.