Abstract

Nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, has been associated with incidences of skin rash and hepatotoxicity in patients. Although the mechanism of idiosyncratic hepatotoxicity remains unknown, it is proposed that metabolic activation of nevirapine and subsequent covalently binding of reactive metabolites to cellular proteins play a causative role. Studies were initiated to determine whether nevirapine undergoes cytochrome P450 (P450)-mediated bioactivation in human liver microsomes to electrophilic intermediates. Liquid chromatography-tandem mass spectrometry analysis of incubations containing nevirapine and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed the formation of a GSH conjugate derived from the addition of the sulfydryl nucleophile to nevirapine. No other GSH conjugates were detected, including conjugates of oxidized metabolites of nevirapine. These findings are consistent with a bioactivation sequence involving initial P450-catalyzed dehydrogenation of the aromatic nucleus with a 4-methyl group in nevirapine to an electrophilic quinone methide intermediate, which is subsequently attacked by glutathione yielding the sulfydryl conjugate. Formation of the nevirapine GSH conjugate was primarily catalyzed by heterologously expressed recombinant CYP3A4 and, to a lesser extent, CYP2D6, CYP2C19, and CYP2A6. In addition, the quinone methide reactive metabolite was a mechanism-based inactivator of CYP3A4, with inactivation parameters K_I = 31 μM and k_inact = 0.029 min^–1, respectively. It is proposed that formation of the quinone methide intermediate may represent a rate-limiting step in the initiation of nevirapine-mediated hepatotoxicity.