title: Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes.
abstract: In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.
authors.affiliation: Department of Drug Metabolism, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
authors.forename: I
authors.initials: I
authors.lastname: Chu
authors.forename: L
authors.initials: L
authors.lastname: Favreau
authors.forename: T
authors.initials: T
authors.lastname: Soares
authors.forename: C c
authors.initials: Cc
authors.lastname: Lin
authors.forename: A A
authors.initials: AA
authors.lastname: Nomeir
mesh_terms: D001274:Atmospheric Pressure; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis*; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D003917:Dextrorphan / Q000737:chemistry / Q000494:pharmacology; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005069:Evaluation Studies as Topic; D006801:Humans; D006915:Hydroxytestosterones / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006915:Hydroxytestosterones; D003917:Dextrorphan; C025591:6 beta-hydroxytestosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human