title: An in vitro, high throughput, seven CYP cocktail inhibition assay for the evaluation of new chemical entities using LC-MS/MS.
abstract: A validated method for the simultaneous characterization of xenobiotic compound-mediated inhibition of seven major cytochrome P450 (CYP) enzymes in pooled human liver microsomes through the use of specific CYP probe substrates (cocktail assay) with low protein content, and a rapid, three minute LC-MS/MS analytical method is described. The specific CYP substrates used in this cocktail assay included phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4/5). The LC-MS method incorporated the aforementioned seven CYP substrates along with their respective major metabolites, and one internal standard, labetalol. In a cross-validation analysis, the concentrations of each CYP probe substrate in the assay had minimal effect (i.e., inhibition or activation) on the other CYP activities. Furthermore, the assay conditions for the multiple probe substrate, ie., cocktail assay, were validated against the single probe substrate assay using 18 compounds with known CYP inhibition liabilities and 10 proprietary compounds. The inhibitory constant (Ki) determined with this cocktail assay was highly correlated (R(2) ≥ 0.77 for each individual probe substrate) with that of the single probe substrate assay for all 27 CYP inhibitors. This seven CYP inhibition cocktail assay has increased the efficiency to assess compounds for inhibition of the major CYP isoforms in a high throughput, drug discovery setting.
authors.affiliation: Array BioPharma Inc., 3200 Walnut Street, Boulder, CO 80301, USA. jotten@arraybiopharma.com
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Otten
authors.forename: Gary P
authors.initials: GP
authors.lastname: Hingorani
authors.forename: Dylan P
authors.initials: DP
authors.lastname: Hartley
authors.forename: Scott D
authors.initials: SD
authors.lastname: Kragerud
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Franklin
mesh_terms: D002853:Chromatography, Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects* / Q000201:enzymology; D015335:Molecular Probes / Q000378:metabolism; D015203:Reproducibility of Results; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D015335:Molecular Probes; D003577:Cytochrome P-450 Enzyme System