Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis.
Increasing reports of time-dependent inhibition of cytochrome P450 (P450) suggest further emphasis on interpreting the consequences, either from a pharmacokinetic or toxicological perspective. Two automated, time-dependent inhibition assays with a liquid chromatography-tandem mass spectrometric endpoint are presented. The initial assay utilizes human liver microsomes, a single concentration of inhibitor, and a single preincubation time of 30 min. Phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam are used as substrates for CYP1A2, 2C9, 2C19, 2D6, and 3A4, and the assay differentiates between reversible and irreversible inhibition. The second assay uses individual recombinant human P450s, six inhibitor concentrations, and three time points to accurately define kinact and KI. A good correlation is demonstrated between kinact/KI and partition ratio, indicating that both terms are related in describing the efficiency of enzyme inactivation. Despite the single preincubation time point of 30 min used in the initial assay, a good relationship has been found to exist between the unbound IC50 estimated from this initial screen and the kinact/KI ratio derived from the more extensive subsequent single P450 assay. The higher throughput human liver microsomal assay can therefore generate IC50 values that can be used to predict the pharmacokinetic impact on cotherapies from the estimated kinact/KI ratio, predicted human dose, and pharmacokinetics.
Atkinson
Anthony
A
Department of Physical and Metabolic Science, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK.
Kenny
Jane R
JR
Grime
Ken
K
D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001331:Automation; D002853:Chromatography, Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004008:Diclofenac / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008617:Mephenytoin / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010615:Phenacetin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C045793:CYP2C19 protein, human; D019388:Cytochrome P-450 CYP1A2; D065731:Cytochrome P-450 CYP2C19; D010615:Phenacetin; D008617:Mephenytoin