Ma Bennett, Polsky-Fisher Stacey L, Vickers Stanley, Cui Donghui, Rodrigues A David
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
Drug Metab Dispos. 2007 Aug;35(8):1301-7. doi: 10.1124/dmd.107.014753. Epub 2007 Apr 25.
In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human gamma-aminobutyric acid(A) receptor alpha2 and alpha3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. Eadie-Hofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K(m) (3.4 and 4.5 microM, respectively) and a high K(m) (12.7 and 40.0 microM, respectively) component. For both metabolites, the high K(m) component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (>75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 microM. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.
进行了体外代谢研究，以确定参与7-(1,1-二甲基乙基)-6-(2-乙基-2H-1,2,4-三唑-3-基甲氧基)-3-(2-氟苯基)-1,2,4-三唑并[4,3-b]哒嗪（TPA023，一种人γ-氨基丁酸 A 受体α2和α3亚基的选择性激动剂）生物转化的人细胞色素 P450 酶。TPA023 与 NADPH 强化的人肝微粒体孵育导致形成叔丁基羟基 TPA023、N-去乙基 TPA023 和三种次要代谢物。在 CYP3A 选择性抑制抗体和化学抑制剂存在下，叔丁基羟基化和 N-去乙基化反应均受到极大抑制（>85%），表明 CYP3A 亚家族成员在 TPA023 代谢中起重要作用。富含 CYP3A5 的人肝微粒体中叔丁基羟基化和 N-去乙基化的 Eadie-Hofstee 图显示出低 Km（分别为 3.4 和 4.5 microM）和高 Km（分别为 12.7 和 40.0 microM）成分。对于这两种代谢物，在含有最低水平 CYP3A5 的微粒体制剂池中未观察到高 Km 成分。肝微粒体与米非司酮（对 CYP3A4 的选择性大于 CYP3A5）预孵育极大地抑制了叔丁基羟基化和 N-去乙基化（>75%）；然而，富含 CYP3A5 的肝微粒体池中的残余活性明显更高（p < 0.0005）。此外，当底物浓度从 4 microM 增加到 40 microM 时，在富含 CYP3A5 和缺乏 CYP3A5 的制剂存在下均观察到叔丁基羟化酶和 N-去乙基酶活性的残余水平升高。一致的是，重组 CYP3A5 催化的代谢物形成由双相模型描述。结论是 CYP3A4 在 TPA023 代谢中起主要作用，并且 CYP3A5 在该化合物浓度较高时也可能起作用。
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