Metabolic Activation and Cytotoxicity of Donepezil Induced by CYP3A4

-desmethyl DNP (M1), the oxidative metabolite of DNP, was characterized by chemical synthesis, LC-MS/MS, and nuclear magnetic resonance. A reactive quinone methide resulting from the metabolism of DNP was captured by glutathione (GSH) fortified in liver microsomal incubations after exposure to DNP, and the resulting GSH conjugate (M2) was detected in the bile of rats receiving DNP. Recombinant human P450 enzyme incubation studies demonstrated that CYP3A4 was the principal enzyme responsible for the production of M1 and M2. The generation of M2 declined in rat primary hepatocytes pretreated with ketoconazole, an inhibitor of CYP3A4, which also decreased the vulnerability of rat primary hepatocytes to DNP-caused cytotoxicity. These findings suggest that the quinone methide metabolite may contribute to the cytotoxicity and hepatotoxicity caused by the DNP.