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

Department of Pharmacokinetics, Dynamics & Metabolism (H.-Z.B., P.K., P.Z., W.F.P., E.Y.W.), Pfizer Global Research and Development, San Diego, California

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.

This article has been cited by other articles:

				H.-Z. Bu, P. Zhao, P. Kang, W. F. Pool, E. Y. Wu, and B. V. Shetty Evaluation of Capravirine as a CYP3A Probe Substrate: In Vitro and in Vivo Metabolism of Capravirine in Rats and Dogs Drug Metab. Dispos., September 1, 2007; 35(9): 1593 - 1602. [Abstract] [Full Text] [PDF]

				H.-Z. Bu, P. Zhao, P. Kang, W. F. Pool, and E. Y. Wu Identification of Enzymes Responsible for Primary and Sequential Oxygenation Reactions of Capravirine in Human Liver Microsomes Drug Metab. Dispos., November 1, 2006; 34(11): 1798 - 1802. [Abstract] [Full Text] [PDF]