title: Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development.
abstract: We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug-probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k(obs)) at a single concentration of test compounds because the k(obs) of eleven CYP3A inactivators at 10 microM in the assay system nearly corresponded to k(inact)/K(I) values, an indicator of a compound's propensity to alter the activity of a CYP in vivo (R(2) = 0.97). Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.
authors.affiliation: Drug Metabolism and Pharmacokinetic Research Laboratory, Mitsubishi Tanabe Pharma Corporation, Toda, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Mori
authors.forename: H
authors.initials: H
authors.lastname: Hashimoto
authors.forename: H
authors.initials: H
authors.lastname: Takatsu
authors.forename: M
authors.initials: M
authors.lastname: Tsuda-Tsukimoto
authors.forename: T
authors.initials: T
authors.lastname: Kume
mesh_terms: D000638:Amiodarone / Q000031:analogs & derivatives / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors*; D001681:Biological Assay / Q000379:methods*; D065729:Cytochrome P-450 CYP2C9; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065692:Cytochrome P-450 CYP3A Inhibitors; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D012015:Reference Standards; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065692:Cytochrome P-450 CYP3A Inhibitors; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C036116:desethylamiodarone; D000638:Amiodarone; D007538:Isoniazid