Wang Michael Zhuo, Saulter Janelle Y, Usuki Etsuko, Cheung Yen-Ling, Hall Michael, Bridges Arlene S, Loewen Greg, Parkinson Oliver T, Stephens Chad E, Allen James L, Zeldin Darryl C, Boykin David W, Tidwell Richard R, Parkinson Andrew, Paine Mary F, Hall James Edwin
Division of Molecular Pharmaceutics, School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Drug Metab Dispos. 2006 Dec;34(12):1985-94. doi: 10.1124/dmd.106.010587. Epub 2006 Sep 22.
DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 microM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.
DB289 [2,5-双(4-脒基苯基)呋喃-双-O-甲基偕胺肟] 通过连续的氧化O-去甲基化和还原N-去羟基化反应生物转化为强效抗寄生虫二脒DB75 [2,5-双(4-脒基苯基)呋喃]。先前的研究表明，N-去羟基化反应由细胞色素b5/NADH-细胞色素b5还原酶催化。负责催化DB289 O-去甲基化途径的酶尚未确定。我们报告了一项体外代谢研究，以表征人肝微粒体（HLMs）中催化DB289初始O-去甲基化（形成M1）的酶。1-氨基苯并三唑的强效抑制作用证实M1的形成由细胞色素P450酶催化。HLMs形成M1依赖于NADPH，其Km和Vmax分别为0.5微摩尔和3.8纳摩尔/分钟/毫克蛋白质。初步筛选表明，重组CYP1A1、CYP1A2和CYP1B1是M1形成的有效催化剂。然而，这三种酶均不负责HLMs形成M1。进一步筛选表明，重组CYP2J2、CYP4F2和CYP4F3B也可催化M1的形成。一种针对CYP4F2的抗体，它同时抑制CYP4F2和CYP4F3B，抑制了HLMs形成M1的91%。两种细胞色素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，有效抑制HLMs形成M1。用依巴斯汀和针对CYP2J2的抗体进行的抑制研究表明，CYP2J2不参与HLMs形成M1。此外，酮康唑优先抑制CYP4F2，但不抑制CYP4F3B，并部分抑制HLMs形成M1。我们得出结论，CYP4F酶（如CYP4F2、CYP4F3B）是负责HLMs形成M1的主要酶。这些发现表明，在人肝脏中，CYP4F亚家族成员不仅可生物转化内源性化合物，还可生物转化外源性化合物。
