Abstract
Efavirenz is a non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitor used in combination therapy to treat HIV-1. Efavirenz metabolism is catalyzed primarily by the polymorphic enzyme P450 2B6. Metabolism of efavirenz by P450 2B6 and the naturally occurring P450 2B6.4 mutant led to the formation of 8-hydroxyefavirenz. Efavirenz inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin activity of the wild-type P450 2B6 enzyme in a time-, concentration-, and NADPH-dependent manner. However, the P450 2B6.4 variant was not inactivated by efavirenz. The ability of efavirenz to inactivate both enzymes was investigated using cyclophosphamide and bupropion, two structurally unrelated substrates of P450 2B6, as probes. Preincubations with efavirenz decreased the ability of the wild-type enzyme to hydroxylate both substrates to similar extents but had no effect on the activities of the mutant enzyme. Interestingly, the inactivation of the wild-type enzyme was completely reversible after 24 h of dialysis as determined by heme, reduced CO spectra, and activity loss. In contrast, 8-hydroxyefavirenz, a metabolite of efavirenz, was able to inactivate both enzymes irreversibly. These data suggest that incubations of P450 2B6 and P450 2B6.4 with either the parent compound efavirenz or the metabolite 8-hydroxyefavirenz in the reconstituted system result in the formation of two different reactive intermediates that lead to losses in enzymatic activity by two different mechanisms, one reversible and one irreversible.
Footnotes
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This study was supported in part by National Institutes of Health Grants CA 16954 and T32 GM007767.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.102525.
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ABBREVIATIONS: P450, cytochrome P450; P450 2B6, N-terminally truncated P450 2B6; HIV-1, human immunodeficiency virus-1; P450 2B6.4, N-terminally truncated P450 2B6 lysine 262 arginine mutant; BSA, bovine serum albumin; EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; CPA, cyclosphosphamide; HPLC, high-performance liquid chromatography; MI, metabolic intermediate.
- Received February 6, 2006.
- Accepted April 10, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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