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Progress Curve Analysis of CYP1A2 Inhibition: A More Informative Approach to the Assessment of Mechanism-Based Inactivation?

Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Sandwich, Kent, United Kingdom (D.A.F., C.C.); and School of Biological Sciences, University of Plymouth, Plymouth, United Kingdom (S.C.)

Mechanism-based cytochrome P450 inactivation is defined as a time- and NADPH-dependent inactivation that is not reversible upon extensive dialysis. Current methodologies use dilution approaches to estimate the rate of inactivation and offer limited mechanistic insight and are significantly influenced by experimental conditions. We investigated the potential of progress curve analysis because this experimental design allows investigation of both the reversible (K_iapp) and irreversible (K_i, K_inact) components of the reaction mechanism. The human liver microsomal CYP1A2 inactivation kinetics of resveratrol, oltipraz, furafylline, and dihydralazine (Fig. 2) were evaluated. The inactivation results for furafylline (K_i, 0.8 µM; K_inact, 0.16 min^–1) are within 2-fold to pub-lished data (K_i, 1.6 µM; K_inact, 0.19 min^–1). Resveratrol and dihydralazine results are within a 4.3-fold range of published data, which compares well with ranges of estimates of these parameters across publications (e.g., furafylline has estimates ranging of K_i from 1.6 to 22.3 µM and K_inact from 0.19 to 0.87 min^–1). This range of estimates highlights the potential caveats surrounding the existing methodologies that have been previously discussed in depth. In addition to these inactivation parameters, we have been able to demonstrate a variation in balance of reversible versus irreversible inhibition within these inactivators. Oltipraz and resveratrol have K_iapp values similar to their K_i, indicating a significant early onset reversible inhibition, whereas furafylline and dihydralazine are dominated by irreversible inactivation. This approach allows a more mechanistic investigation of an inactivator and in the future may improve the prediction of clinical drug-drug interactions.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Chemical structures of furafylline, dihydralazine, oltipraz, resveratrol, and paroxetine.

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