Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s
Dermot F. McGinnity, Alison J. Parker, Matthew Soars,1 and Robert J. Riley
Department of Physical & Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdom
Drug Metabolism and Disposition. Vol. 28, Issue 11, 1327-1334, November 2000.
A fully automated assay to determine the enzymology of drug oxidation by the major human hepatic cytochrome P450s (CYPs; CYP1A2,-2C9, -2C19, -2D6, and -3A4) coexpressed functionally in Escherichiacoli with human NADPH-P450 reductase has been developed and validated.Ten prototypic substrates were chosen for which clearance wasprimarily CYP-dependent, and the activities of these five majorCYPs were represented. A range of intrinsic clearance (CLint)values were obtained for substrates in both pooled human livermicrosomes (HLM; 1-380 µl · min1mg1) and recombinant CYPs (0.03-7 µl · min1pmol1) and thus the percentage contribution of individual CYPs towardtheir oxidative metabolism could be estimated. All the assignmentswere consistent with the available literature data. Tolbutamidewas metabolized by CYP2C9 (70%) and CYP2C19 (30%), diazepam byCYP2C19 (100%), ibuprofen by CYP2C9 (90%) and CYP2C19 (10%), andomeprazole by CYP2C19 (68%) and CYP3A4 (32%). Metoprolol and dextromethorphanwere primarily CYP2D6 substrates and propranolol was metabolizedby CYP2D6 (59%), CYP1A2 (26%), and CYP2C19 (15%). Diltiazem, testosterone,and verapamil were metabolized predominantly by CYP3A4. In addition,the metabolite profile for the CYP-dependent clearance of severalmarkers determined by mass spectroscopy was as predicted fromthe literature. There was a good correlation between the sum ofindividual CYP CLint and HLM CLint (r2 = 0.8, P < .001) for the substrates indicating that recombinantCYPs may be used to predict HLM CLint data. This report demonstratesthat recombinant human CYPs may be useful as an approach for theprediction of the enzymology of human CYP metabolism early inthe drug discoveryprocess.