TY - JOUR T1 - A SIMPLE SEQUENTIAL INCUBATION METHOD FOR DECONVOLUTING THE COMPLICATED SEQUENTIAL METABOLISM OF CAPRAVIRINE IN HUMANS JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1438 LP - 1445 DO - 10.1124/dmd.105.005413 VL - 33 IS - 10 AU - Hai-Zhi Bu AU - Ping Kang AU - Ping Zhao AU - William F. Pool AU - Ellen Y. Wu Y1 - 2005/10/01 UR - http://dmd.aspetjournals.org/content/33/10/1438.abstract N2 - Capravirine, a non-nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1, undergoes extensive oxygenations to numerous sequential metabolites in humans. Because 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. In brief, 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 nonlabeled 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 percentage contribution of a precursor metabolite to the formation of each of its sequential metabolites (called sequential contribution) and the percentage 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., nonlabeled 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. The American Society for Pharmacology and Experimental Therapeutics ER -