Received for publication May 4, 2005.
Revised June 29, 2005.
Accepted for publication June 30, 2005.

A SIMPLE SEQUENTIAL INCUBATION METHOD FOR DECONVOLUTING THE COMPLICATED SEQUENTIAL METABOLISM OF CAPRAVIRINE IN HUMANS

Abstract

Capravirine, a non-nucleoside reverse transcriptase inhibitor for the treatment of HIV type 1, undergoes extensive oxygenations to numerous sequential metabolites in humans. Since several possible oxygenation pathways may be involved in the formation and/or sequential metabolism of a single metabolite, it is very difficult or even impossible to determine the definitive pathways and their relative contributions to the overall metabolism of capravirine using conventional approaches. For this reason, a human liver microsome-based sequential incubation method has been developed to deconvolute the complicated sequential metabolism of capravirine. Briefly, the method includes three fundamental steps: 1) 30-min primary incubation of [¹⁴C]capravirine, 2) isolation of [¹⁴C]metabolites from the primary incubate, and 3) 30-min sequential incubation of each isolated [¹⁴C]metabolite supplemented with an ongoing (30 min) microsomal incubation with non-labeled capravirine. Based on the extent of both the disappearance of the isolated precursor [¹⁴C]metabolites and the formation of sequential [¹⁴C]metabolites, definitive oxygenation pathways of capravirine were assigned. In addition, the percent contribution of a precursor metabolite to the formation of each of its sequential metabolites (called sequential contribution) and the percent contribution of a sequential metabolite formed from each of its precursor metabolites (called precursor contribution) were determined. An advantage of this system is that the sequential metabolism of each isolated [¹⁴C]metabolite can be monitored selectively by radioactivity in the presence of all relevant metabolic components (i.e., non-labeled parent and its other metabolites). This methodology should be applicable to mechanistic studies of other compounds involving complicated sequential metabolic reactions when radiolabeled materials are available.

Key words: CYP3A, cytochrome P450, HPLC, liver microsomes, mass spectrometry, metabolite identification

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