Short Communication Identification of Enzymes Responsible for Primary and Sequential Oxygenation Reactions of Capravirine in Human Liver Microsomes

Capravirine, a new non-nucleoside reverse transcriptase inhibitor, undergoes extensive oxygenation reactions, including N-oxidation, sulfoxidation, sulfonation, and hydroxylation in humans. Numerous primary (mono-oxygenated) and sequential (di-, tri-, and tetraoxygenated) metabolites of capravirine are formed via the individual or combined oxygenation pathways. In this study, cytochrome P450 enzymes responsible for the primary and sequential oxygenation reactions of capravirine in human liver microsomes were identified at the specific pathway level. The total oxygenation of capravirine is mediated predominantly (>90%) by CYP3A4 and marginally (<10%) by CYP2C8, 2C9, and 2C19 in humans. Specifically, each of the two major mono-oxygenated metabolites C23 (sulfoxide) and C26 (N-oxide), is mediated predominantly (>90%) by CYP3A4 and slightly (<10%) by CYP2C8, the minor tertiary hydroxylated metabolite C19 by CYP3A4, 2C8, and 2C19, and the minor primary hydroxylated metabolite C20 by CYP3A4, 2C8, and 2C9. However, all sequential oxygenation reactions are mediated exclusively by CYP3A4. Due to their relatively insignificant contributions of C19 and C20 to total capravirine metabolism, no attempt was made to determine relative contributions of cytochrome P450 enzymes to the formation of the two minor metabolites. Capravirine (AG1549 or S-1153), a new non-nucleoside reverse transcriptase inhibitor that was under development for the oral treatment of human immunodeficiency virus type 1 (De Clercq, 2001, 2002), undergoes extensive oxygenation reactions in humans after oral administration of capravirine alone or in combination with ritonavir (Bu et al., 2004), as well as in human liver microsomes (Bu et al., 2005). The oxygenation pathways of capravirine in humans are restricted to N-oxidation at the pyridinyl nitrogen atom, sulfoxidation, sulfonation, and hydroxylation at the isopropyl group (Fig. 1). Four primary (mono-oxygenated) and numerous sequential (di-, tri-, and tetra-oxygenated) metabolites of capravirine are formed via the aforementioned individual or combined oxygenation pathways. Because several possible oxygenation pathways may be involved in the formation and/or sequential oxygenation of a single metabolite, it is impossible to determine the definitive pathways and their relative contributions to the overall oxygenation of capravirine using conventional approaches. For this reason, a simple, efficient sequential incubation method has been developed to deconvolute the complicated sequential oxygenation of capravirine (Bu et al., 2005). In the previous study, the definitive oxygenation pathways of capravirine were identified and the percentage contribution of a precursor metabolite to the formation of each of its sequential metabolites (called sequential contribution), as well as the percentage contribution of a sequential metabolite formed from each of its precursor metabolites (called precursor contribution), were estimated (Fig. 1). The purpose of the present study was to identify the P450 enzyme(s) responsible for each of the primary and sequential oxygenation reactions of capravirine in human liver microsomes. Materials and Methods Materials. [C]Capravirine ( 99% radiochemical purity) was synthesized at Pfizer (St. Louis, MO). Human liver microsomes (pooled from 14 donors) were prepared at Pfizer (Groton, CT). Human P450 Supersomes 1A2, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1, flavin-containing monooxygenase (FMO) Supersomes FMO1, FMO3, and FMO5 (expressed in baculovirus-insect cells), and monoclonal antibody inhibitory to human CYP3A4 were purchased from BD Gentest (Woburn, MA). Ritonavir was synthesized at Pfizer (Groton, CT). Ketoconazole, quercetin, sulfaphenazole, and ticlopidine were received from Sigma-Aldrich. Seven authentic metabolites of capravirine (C15, C19, C20, C22, C23, C25B, and C26, Fig. 1) were synthesized at Pfizer (San Diego, CA). All other commercially available reagents and solvents were of either analytical or HPLC grade. Microsomal Metabolism. [C]Capravirine (2 M) was incubated for varying times (0–1 h) at 37°C in an incubation system consisting of 100 mM potassium phosphate buffer (pH 7.4), 0.2 mg of human liver microsomes, and 1 mM NADPH in a final volume of 1 ml. After a 5-min preincubation, reactions were initiated by the addition of NADPH. Reactions were terminated by the addition of 2 ml of ice-cold acetonitrile. Samples were vortexed and centrifuged for 10 min. The supernatants were transferred into polypropylene tubes for evaporation to dryness under N2 at 40°C. The residues were reconstituted in 110 l of 20:80 (v/v) methanol/20 mM ammonium acetate (pH 4), and aliquots (100 l) were injected into an HPLC-MS-RAM system, as described in the following Metabolite Profiling section, for analysis. Single Inhibition. [C]Capravirine (2 M) was coincubated with ritonavir or ketoconazole at concentrations ranging from 0 to 2 M in human liver microsomes for 30 min at 37°C. All other incubation conditions and sample preparation procedures were the same as described above (Microsomal Metabolism). The inhibitor concentrations were chosen based on their ability to inhibit CYP3A4 with relative specificity (von Moltke et al., 1998). Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.011189. ABBREVIATIONS: P450, cytochrome P450; FMO, flavin-containing monooxygenase; HPLC, high-performance liquid chromatography; MS, mass spectrometry; RAM, radioactivity monitoring. 0090-9556/06/3411-1798–1802$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 11 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 11189/3148788 DMD 34:1798–1802, 2006 Printed in U.S.A. 1798 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from