title: Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC-MS.
abstract: The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC-MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC-MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM-LC-MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC(50)s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC(50)s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC(50)s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC(50)s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC-MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug-drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.
authors.affiliation: Wyeth Research, P.O. Box CN 8000, Princeton, NJ 08543-8000, United States. Electronic address: DIL@WYETH.COM.
authors.forename: Li
authors.initials: L
authors.lastname: Di
authors.affiliation: Wyeth Research, P.O. Box CN 8000, Princeton, NJ 08543-8000, United States.
authors.forename: Edward H
authors.initials: EH
authors.lastname: Kerns
authors.affiliation: Wyeth Research, P.O. Box CN 8000, Princeton, NJ 08543-8000, United States.
authors.forename: Susan Q
authors.initials: SQ
authors.lastname: Li
authors.affiliation: Wyeth Research, P.O. Box CN 8000, Princeton, NJ 08543-8000, United States.
authors.forename: Guy T
authors.initials: GT
authors.lastname: Carter
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004008:Diclofenac / Q000378:metabolism; D015195:Drug Design; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004983:Ethanolamines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D008874:Midazolam / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D003374:Coumarins; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D005456:Fluorescent Dyes; D007527:Isoenzymes; D011994:Recombinant Proteins; D004008:Diclofenac; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008874:Midazolam