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Sequential Metabolism Is Responsible for Diltiazem-Induced Time-Dependent Loss of CYP3A

Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, La Jolla Laboratories, La Jolla, California (P.Z., C.A.L.); and Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington (K.L.K.)

Kinetic parameters (k_inact and K_I) obtained in microsomes are often used to predict time-dependent inactivation. We previously reported that microsomal inactivation kinetic parameters of diltiazem underpredicted CYP3A inactivation in hepatocytes. In this study, we evaluated the contributions of inactivation and reversible inhibition of CYP3A by diltiazem and its N-desmethyl (MA) and N,N-didesmethyl (MD) metabolites. In human liver microsomes, MA was a more potent time-dependent inactivator of CYP3A than its parent drug, with apparent k_inact approximately 4-fold higher than that of diltiazem at a microsomal protein concentration of 0.2 mg/ml. MD did not inactivate CYP3A. Inactivation of CYP3A by diltiazem was dependent on microsomal protein concentration (25, 36, and 41% decrease in CYP3A activity at 0.2, 0.4, and 0.8 mg/ml microsomal protein, respectively, incubated with 10 µM diltiazem over 20 min), whereas inactivation by MA did not seem to be protein concentration-dependent. MA and MD were reversible inhibitors of CYP3A with competitive K_i values of 2.7 and 0.2 µM, respectively. In cryopreserved hepatocytes incubated with diltiazem, time-dependent loss of CYP3A was accompanied by increased formation of MA and MD, with the MA level similar to its K_I at higher diltiazem concentrations. In addition, the metabolites appeared to be accumulated inside the cells. In summary, time-dependent CYP3A inactivation by MA seems to be the major contributor responsible for the loss of CYP3A in human liver microsomes and human hepatocytes incubated with diltiazem. These findings suggest that prediction of CYP3A loss based solely on microsomal inactivation parameters of parent drug may be inadequate.