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AUTOMATED ASSESSMENT OF TIME-DEPENDENT INHIBITION OF HUMAN CYTOCHROME P450 ENZYMES USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY ANALYSIS

Department of Physical and Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, United Kingdom

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 k_inact and K_I. A good correlation is demonstrated between k_inact/K_I 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 k_inact/K_I 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 k_inact/K_I ratio, predicted human dose, and pharmacokinetics.

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