Accelerated Communication Metabolic Activation of Nevirapine in Human Liver Microsomes: Dehydrogenation and Inactivation of Cytochrome P450 3A4

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 KI 31 M and kinact 0.029 min , respectively. It is proposed that formation of the quinone methide intermediate may represent a rate-limiting step in the initiation of nevirapine-mediated hepatotoxicity. Nevirapine (NVP) (Fig. 1) is the first non-nucleoside reverse transcriptase inhibitor approved by the U.S. Food and Drug Administration (FDA) for use in combination therapy of HIV-1 infection. Currently, nevirapine remains one of the most prescribed antiretroviral drugs in the developing countries, both to prevent vertical transmission and in combination therapy (Lockman et al., 2007). Despite its therapeutic benefits, treatment with nevirapine has been associated with a significant incidence of hepatotoxicity and skin rash (Pollard et al., 1998). An FDA black box warning label was placed on nevirapine in 2000 due to concerns regarding its hepatotoxicity. Although the exact mechanism of nevirapine hepatotoxicity is not clearly understood, a probable causal link between nevirapine metabolites and skin rash has been established (Popovic et al., 2006). In humans, nevirapine undergoes significant hepatic metabolism mainly by 2-, 3-, 8-, and 12-hydroxylation followed by glucuronidation of these hydroxyl metabolites (Riska et al., 1999). The 2-, 3-, and 8-hydroxylation can presumably lead to formation of para-quinone imine intermediates after further oxidation (Fig. 1). The metabolite 12-hydroxynevirapine has the potential to be sulfated followed by loss of sulfate to form a reactive quinone methide (Fig. 1). In addition, 4-carboxynevirapine, formed from further oxidation of 12-hydroxynevirapine, can generate acyl glucuronide, which has the potential to bind to cellular proteins. In human liver microsomal incubations, similar hydroxylation metabolites were observed (Erickson et al., 1999). CYP3A4 was identified as the major enzyme involved in formation of 2and 12-hydroxynevirapine, whereas CYP2B6 was the predominant enzyme forming 3and 8-hydroxynevirapine (Erickson et al., 1999). However, no glutathione (GSH) conjugates of nevirapine or its hydroxyl metabolites have been reported, and the identities of putative reactive metabolites of nevirapine remain unknown. More recently, 12-hydroxynevirapine has been proposed as a factor in nevirapine hepatocarcinogenicity (Antunes et al., 2008) as well as skin rash (Chen et al., 2008). The reactivity of 12-sulfoxynevirapine was demonstrated by formation of multiple DNA adducts in the reactions with 12-mesyloxynevirapine (Antunes et Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.024851. □S The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. ABBREVIATIONS: NVP, nevirapine; GSH, glutathione; DNVP, 12-trideutero-nevirapine; DMSO-d6, (dimethyl sulfoxide)-D6; HLM, human liver microsome; P450, cytochrome P450; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PI, precursor ion; EPI, enhanced production; HPLC, high-performance liquid chromatography. 0090-9556/09/3707-1557–1562$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 7 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 24851/3484155 DMD 37:1557–1562, 2009 Printed in U.S.A. 1557 http://dmd.aspetjournals.org/content/suppl/2009/04/13/dmd.108.024851.DC1 Supplemental material to this article can be found at: at A PE T Jornals on M ay 2, 2017 dm d.aspurnals.org D ow nladed from