Mechanism-based inactivation of cytochrome P450 3A4 by mibefradil through heme destruction

Mibefradil (Posicor) was developed as a calcium channel blocker for the treatment of chronic hypertension. The compound was withdrawn from the market in 1998 because of the potential for rhabdomyolysis, renal failure, or bradycardia when it was coadministered with other drugs. Mibefradil has previously been shown to be a potent reversible (IC(50) = 0.3-2 μM) and mechanism-based (K(i) = 2.3 μM; k(inact) = 0.4 min(-1)) inhibitor of CYP3A4-catalyzed statin metabolism. At present, the mechanism of CYP3A4 inactivation by mibefradil is not known. Mechanism-based inactivation experiments and spectral studies were used to examine the mechanism of CYP3A4 inactivation by mibefradil and its major metabolite, des-methoxyacetyl mibefradil (Ro 40-5966), in vitro. Both mibefradil and Ro 40-5966 were shown to exhibit type I binding characteristics (K(s) = 0.69 ± 0.06 and 1.39 ± 0.04 μM, respectively) toward CYP3A4. Complete K(i)/k(inact) experiments were performed, revealing a rapid and irreversible decrease in CYP3A4-catalyzed 1'-hydroxymidazolam formation. Approximately 70% of CYP3A4 activity was lost in the first minute of incubation with mibefradil, and inactivation was nonlinear after 2 min. Ro 40-5966 also resulted in time-dependent inhibition of CYP3A4, albeit to a lesser extent than mibefradil. The decrease in CYP3A4 activity in the presence of mibefradil and NADPH was subsequently shown to have a good correlation with the time-dependent loss of CO binding, which, coupled with the lack of stable heme and/or apoprotein adducts, suggests heme destruction as the mechanism of inactivation of CYP3A4 by mibefradil.