A Simple Sequential Incubation Method for Deconvoluting the Complicated Sequential Metabolism of Capravirine in Humans

Capravirine undergoes extensive oxygenation in humans. Since several pathways may be involved in the formation and/or sequential metabolism of a single metabolite, it is 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. 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 in the presence of all relevant metabolic components. This methodology can be used to investigate sequential metabolism of other compounds when radiolabeled materials are available.