title: An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point.
abstract: The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.
authors.affiliation: Pfizer Global Research and Development, Pharmacokinetics Dynamic and Metabolism, Sandwich, Kent, UK CT13 9NJ, United Kingdom. kuresh.youdim@pfizer.com
authors.forename: Kuresh A
authors.initials: KA
authors.lastname: Youdim
authors.forename: Richard
authors.initials: R
authors.lastname: Lyons
authors.forename: Leon
authors.initials: L
authors.lastname: Payne
authors.forename: Barry C
authors.initials: BC
authors.lastname: Jones
authors.forename: Kenneth
authors.initials: K
authors.lastname: Saunders
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D001681:Biological Assay; D002853:Chromatography, Liquid / Q000379:methods*; D019388:Cytochrome P-450 CYP1A2 / Q000032:analysis / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000032:analysis / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism / Q000494:pharmacology; D004008:Diclofenac / Q000378:metabolism / Q000494:pharmacology; D004347:Drug Interactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism / Q000494:pharmacology; D008904:Miniaturization; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism / Q000494:pharmacology; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity / Q000187:drug effects; D013619:Tacrine / Q000378:metabolism / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D004008:Diclofenac; D013619:Tacrine; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; C045793:CYP2C19 protein, human; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D065731:Cytochrome P-450 CYP2C19; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D008874:Midazolam