Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories Ltd., 16-1 Minami, Akasaka, Kainan, Japan.
Drug Metab Dispos. 2010 Sep;38(9):1486-92. doi: 10.1124/dmd.110.033274. Epub 2010 May 25.
The cynomolgus monkey is used in drug metabolism studies, because of its evolutionary closeness to human, including cytochrome P450. Cynomolgus monkey CYP2D17, highly homologous to human CYP2D6, has been identified and characterized. Here, we report characterization of another CYP2D, CYP2D44, identified in cynomolgus monkey liver. The CYP2D44 cDNA contained an open reading frame of 497 amino acids sharing high sequence identity (87-93%) with other primate CYP2Ds. CYP2D44 mRNA was predominantly expressed in liver, similar to CYP2D17 mRNA. CYP2D17 and CYP2D44 form a gene cluster in the genome, similar to human CYP2Ds. Metabolic assays of the CYP2D17 and CYP2D44 proteins heterologously expressed in Escherichia coli indicated that CYP2D44 metabolized human CYP2D6 substrates, bufuralol and dextromethorphan (bufuralol 1'-hydroxylation and dextromethorphan O-demethylation) but to a lesser extent than CYP2D17. Kinetic analysis of dextromethorphan metabolism indicated that the apparent K(m) and V(max) of CYP2D17 and CYP2D44 catalyzed O-demethylation were similar, and, the V(max) values of CYP2D17 and CYP2D44 catalyzed N-demethylation (which human CYP2D6 catalyzes much less effectively) were similar, but the apparent K(m) of the CYP2D44 reaction was higher. Western blot analysis showed that CYP2D proteins were expressed in cynomolgus and rhesus monkey liver as well as in human and marmoset liver. Similar to CYP2D6, CYP2D44 copy number varied among the eight cynomolgus monkeys and four rhesus monkeys used in this study. These results indicated that CYP2D44, together with CYP2D17, had functional characteristics similar to those of human CYP2D6 but measurably differed in dextromethorphan N-demethylation, suggesting its importance for CYP2D-dependent drug metabolism in macaque.
食蟹猴常用于药物代谢研究，因为它在进化上与人类接近，包括细胞色素 P450。食蟹猴 CYP2D17 与人 CYP2D6 高度同源，已被鉴定和表征。在这里，我们报告了在食蟹猴肝脏中鉴定的另一种 CYP2D，CYP2D44 的特征。CYP2D44 cDNA 包含一个 497 个氨基酸的开放阅读框，与其他灵长类 CYP2D 具有高度序列同一性（87-93%）。CYP2D44 mRNA 主要在肝脏中表达，与 CYP2D17 mRNA 相似。CYP2D17 和 CYP2D44 在基因组中形成基因簇，与人类 CYP2D 相似。在大肠杆菌中异源表达的 CYP2D17 和 CYP2D44 蛋白的代谢测定表明，CYP2D44 代谢人 CYP2D6 底物布氟洛尔和右美沙芬（布氟洛尔 1'-羟化和右美沙芬 O-去甲基化），但程度低于 CYP2D17。右美沙芬代谢的动力学分析表明，CYP2D17 和 CYP2D44 催化的 O-去甲基化的表观 K(m)和 V(max)相似，而 CYP2D17 和 CYP2D44 催化的 N-去甲基化（人 CYP2D6 催化效率较低）的 V(max)值相似，但 CYP2D44 反应的表观 K(m)较高。Western blot 分析表明，CYP2D 蛋白在食蟹猴和恒河猴肝脏以及人和狨猴肝脏中表达。与 CYP2D6 相似，CYP2D44 的拷贝数在本研究中使用的 8 只食蟹猴和 4 只恒河猴中存在差异。这些结果表明，CYP2D44 与 CYP2D17 一起具有与人 CYP2D6 相似的功能特征，但在右美沙芬 N-去甲基化方面存在可测量的差异，表明其对食蟹猴中 CYP2D 依赖性药物代谢的重要性。
