Wang Michael Zhuo, Wu Judy Qiju, Bridges Arlene S, Zeldin Darryl C, Kornbluth Sally, Tidwell Richard R, Hall James Edwin, Paine Mary F
School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Drug Metab Dispos. 2007 Nov;35(11):2067-75. doi: 10.1124/dmd.107.016428. Epub 2007 Aug 20.
CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.
包括CYP4F2和CYP4F3B在内的CYP4F酶最近被证明是催化抗寄生虫前药帕夫拉米定（DB289）在人肝微粒体中进行初始氧化O-去甲基化的主要酶。正如低口服生物利用度所表明的那样，DB289可能在肠道以及肝脏中经历首过生物转化。我们使用人肠微粒体（HIM）来表征催化DB289初始O-去甲基化生成中间代谢物M1的肠道酶。HIM中M1的形成由细胞色素P450（P450）酶催化，1-氨基苯并三唑的强效抑制以及对NADPH的需求证明了这一点。表观K（m）和V（max）值分别为0.6至2.4 microM和0.02至0.89 nmol/min/mg蛋白质（n = 9）。在所评估的P450化学抑制剂中，酮康唑最为有效，抑制M1形成达66%。两种P450介导的花生四烯酸代谢抑制剂HET0016（N-羟基-N'-（4-正丁基-2-甲基苯基）甲脒）和17-十八碳炔酸以浓度依赖性方式抑制M1形成（高达95%）。用针对CYP4F2产生的抗体进行免疫抑制显示对M1形成有浓度依赖性抑制（高达92%），而针对CYP3A4/5和CYP2J2的抗体则几乎没有或只有适度影响。在一组缺乏可检测到的CYP4A11蛋白表达的HIM中，M1形成速率与花生四烯酸ω-羟基化速率密切相关（r（2） = 0.94，P < 0.0001，n = 12）。定量蛋白质印迹分析显示这些HIM中有明显的CYP4F表达，平均（范围）为7（3 - 18）pmol/mg蛋白质。我们得出结论认为，肠道CYP4F酶可能在DB289和其他外源性物质的首过生物转化中起作用。
