title: Automated definition of the enzymology of drug oxidation by the major human drug metabolizing cytochrome P450s.
abstract: 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 Escherichia coli with human NADPH-P450 reductase has been developed and validated. Ten prototypic substrates were chosen for which clearance was primarily CYP-dependent, and the activities of these five major CYPs were represented. A range of intrinsic clearance (CL(int)) values were obtained for substrates in both pooled human liver microsomes (HLM; 1-380 microl. min(-1)mg(-1)) and recombinant CYPs (0.03-7 microl. min(-1)pmol(-1)) and thus the percentage contribution of individual CYPs toward their oxidative metabolism could be estimated. All the assignments were consistent with the available literature data. Tolbutamide was metabolized by CYP2C9 (70%) and CYP2C19 (30%), diazepam by CYP2C19 (100%), ibuprofen by CYP2C9 (90%) and CYP2C19 (10%), and omeprazole by CYP2C19 (68%) and CYP3A4 (32%). Metoprolol and dextromethorphan were primarily CYP2D6 substrates and propranolol was metabolized by 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 several markers determined by mass spectroscopy was as predicted from the literature. There was a good correlation between the sum of individual CYP CL(int) and HLM CL(int) (r(2) = 0.8, P <.001) for the substrates indicating that recombinant CYPs may be used to predict HLM CL(int) data. This report demonstrates that recombinant human CYPs may be useful as an approach for the prediction of the enzymology of human CYP metabolism early in the drug discovery process.
authors.affiliation: Department of Physical & Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdom.
authors.forename: D F
authors.initials: DF
authors.lastname: McGinnity
authors.forename: A J
authors.initials: AJ
authors.lastname: Parker
authors.forename: M
authors.initials: M
authors.lastname: Soars
authors.forename: R J
authors.initials: RJ
authors.lastname: Riley
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D004364:Pharmaceutical Preparations / Q000378:metabolism*
chemical_list: D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System