Short Communication CYTOCHROME P450 2C8 (CYP2C8)-MEDIATED HYDROXYLATION OF AN ENDOTHELIN ETA RECEPTOR ANTAGONIST IN HUMAN LIVER MICROSOMES

In vitro studies were performed to identify the human cytochrome P450 enzyme(s) involved in the hydroxylation (isopropyl moiety) of a previously reported endothelin ETA receptor antagonist, compound A [( )-(5S,6R,7R)-2-isopropylamino-7-{4-methoxy-2-[(2R)3-methoxy-2-methylpropyl]}-5-(3,4-methylenedioxyphenyl)cyclopenteno(1,2-b) pyridine 6-carboxylic acid]. Several lines of evidence indicated that the reaction was mainly catalyzed by CYP2C8. Of the 10 recombinant cytochrome P450 isoforms tested, only CYP2C8 exhibited hydroxylase activity. In agreement, inhibitory antibodies selective for CYP2C8 attenuated (>95%) the hydroxylase activity in human liver microsomes, whereas antibodies and chemical inhibitors selective for other cytochrome P450 isoforms had a minor or no effect on the reaction. In addition, the formation of the hydroxy metabolite correlated well with CYP2C8selective paclitaxel 6 -hydroxylation (r 0.92; p < 0.0001) and amodiaquine N-de-ethylation (r 0.91; p < 0.0001) in a bank of human liver microsomes (n 15 organ donors). Finally, compound A hydroxylase activity conformed to Michaelis-Menten kinetics, and the Km (Michaelis constant) in human liver microsomes was similar to that of CYP2C8 ( 10 M). It is concluded that the hydroxylation of compound A is mainly catalyzed by CYP2C8, and thus the reaction can possibly serve as an alternative marker assay for CYP2C8 in human liver microsomes. The cytochromes P450 (P450s) are a superfamily of hemoproteins that catalyze the oxidative metabolism of a wide range of xenobiotics as well as endogenous compounds (Rendic, 2002). In humans, the CYP2C subfamily consists of four isoforms (CYP2C8, 2C9, 2C18, 2C19), which are present in the liver and in many extrahepatic tissues (Klose et al., 1999). Of the four CYP2C isoforms, much attention has been focused on CYP2C9, CYP2C19, and their involvement in catalysis of many clinically important compounds. CYP2C9-mediated metabolism of numerous drugs, such as warfarin (S-enantiomer), phenytoin, tolbutamide, losartan, and celecoxib, is well documented. Likewise, CYP2C19-catalyzed oxidation of S-mephenytoin, antidepressants, and proton pump inhibitors (e.g., omeprazole) has been described (Rendic, 2002; Rodrigues and Rushmore, 2002). In recent years, there has been a growing interest in the involvement of CYP2C8 in drug metabolism. CYP2C8 has been reported to play a role in the biotransformation of paclitaxel, cerivastatin, rosiglitazone, troglitazone, verapamil, amodiaquine, amiodarone, and zopiclone (Baldwin et al., 1999; Becquemont et al., 1999; Tracy et al., 1999; Yamazaki et al., 1999; Ohyama et al., 2000; Li et al., 2002; Wang et al., 2002). In the case of cerivastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor withdrawn from the market in 2001, patients were shown to have elevated cerivastatin levels in plasma when gemfibrozil was coadministered (Backman et al., 2002). The inhibition of cerivastatin metabolism by gemfibrozil was, in part, mediated by CYP2C8 (Prueksaritanont et al., 2002; Wang et al., 2002). It is therefore of interest to prospectively evaluate drug candidates as inhibitors of CYP2C8. Toward this end, paclitaxel 6 hydroxylation has been used as a marker assay for CYP2C8 (Rahman et al., 1994), and, more recently, amodiaquine N-de-ethylation has been proposed (Li et al., 2002). In the present study, the oxidative metabolism of compound A, a potent endothelin ETA receptor antagonist (Okada et al., 2000), was studied after incubation with human liver microsomes. Preliminary data indicated that the hydroxylation on the isopropyl moiety (Fig. 1) was catalyzed almost exclusively by CYP2C8. Therefore, we sought to determine the P450s involved in the reaction using inhibitory antibodies, chemical inhibitors, correlation analysis, and recombinant human P450 proteins. Materials and Methods Chemicals and Biologicals. Compound A and ( )-N-3-benzyl-phenobarbital were synthesized at Banyu Pharmaceutical Co. (Ibaraki, Japan) and in-house (Merck Research Laboratories, West Point, PA), respectively. NADPH, verapamil, amodiaquine, paclitaxel, bactin III, sulfaphenazole, quinidine, troleandomycin, and furafylline were obtained from Sigma-Aldrich (St. Louis, MO). 6 -Hydroxypaclitaxel was purchased from BD Biosciences Discovery Labware (Bedford, MA). Human liver microsomes prepared from individual or pooled (n 20) subjects were purchased from BD Biosciences Discovery Labware, Tissue Transformation Technologies (Edison, NJ), XenoTech, LLC (Lenexa, KA), and In Vitro Technologies, Inc. (Baltimore, MD). Microsomes prepared from insect cells infected with baculovirus expressing human P450 isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5) were obtained in-house. Inhibitory mouse anti-human P450 antibodies selective for CYP2A6, CYP2C, CYP2D6, and CYP3A were prepared in-house (Mei et al., 1999, 2002), and anti-human CYP1A2, CYP2B6, 1 Abbreviations used are: P450, cytochrome P450; LC-MS, liquid chromatography/mass spectrometry; Compound A, ( )-(5S,6R,7R)-2-isopropylamino-7-{4methoxy-2-[(2R)-3-methoxy-2-methylpropyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno(1,2-b) pyridine 6-carboxylic acid. Address correspondence to: Bennett Ma, Department of Drug Metabolism, WP 75A-203, Merck Research Laboratories, West Point, PA 19486. E-mail: [email protected] 0090-9556/04/3205-473–478$20.00 DRUG METABOLISM AND DISPOSITION Vol. 32, No. 5 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 1379/1149050 DMD 32:473–478, 2004 Printed in U.S.A. 473 at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from