Sinal C J, Bend J R
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada.
Chem Res Toxicol. 1995 Jan-Feb;8(1):82-91. doi: 10.1021/tx00043a011.
The capacity for metabolic intermediate (MI) complex formation as a mechanism of action for the isozyme-selective cytochrome P450 (P450) inhibitors N-benzyl-1-aminobenzotriazole (BBT), N-(alpha-methylbenzyl)-1-aminobenzotriazole (alpha MB), and N-(alpha-ethylbenzyl)-1-aminobenzotriazole (alpha EB) was investigated in hepatic microsomes from untreated, phenobarbital-induced, and beta-naphthoflavone-induced guinea pigs. Similar to other complex forming amines, MI complex formation was observed as an absorbance maximum at approximately 455 nm by optical-difference spectroscopy, was dependent upon incubation with NADP(H), and was dissociable by the addition of 50 microM potassium ferricyanide. MI complexes formed by BBT, alpha MB, and alpha EB were also dissociable by sedimentation and resuspension, as well as in the presence of limiting concentrations of NADP(H). Maximal complexation with the three compounds was observed in microsomes from phenobarbital-induced guinea pigs where the initial rate of complex formation was dependent upon inhibitor concentration and apparent Km values of 108 +/- 44, 338 +/- 96, and 84 +/- 15 microM for BBT, alpha MB, and alpha EB, respectively, were found. Inclusion of 1 mM glutathione in the incubation mixtures had a significant attenuating effect upon complex formation, suggesting the involvement of an electrophilic, reactive intermediate. Complex formation was not observed with the three inhibitors in pulmonary microsomes from either guinea pigs or rabbits. MI complexation is not likely to contribute to the mechanism-based inactivation of guinea pig hepatic P450 2Bx, the homologue of rabbit P450 2B4, due to the irreversible inactivation of this isoform at very low inhibitor concentrations, the lack of glutathione attenuation of this destruction, the instability of formed MI complexes, and the absence of MI complex formation with guinea pig or rabbit pulmonary P450.
研究了同工酶选择性细胞色素P450（P450）抑制剂N-苄基-1-氨基苯并三唑（BBT）、N-（α-甲基苄基）-1-氨基苯并三唑（αMB）和N-（α-乙基苄基）-1-氨基苯并三唑（αEB）形成代谢中间体（MI）复合物作为其作用机制的能力，实验对象为未经处理、经苯巴比妥诱导和经β-萘黄酮诱导的豚鼠肝脏微粒体。与其他形成复合物的胺类相似，通过光差光谱法观察到MI复合物在约455nm处有最大吸光度，其形成依赖于与NADP（H）一起孵育，并且可通过加入50μM铁氰化钾而解离。由BBT、αMB和αEB形成的MI复合物也可通过沉淀和重悬以及在有限浓度的NADP（H）存在下解离。在苯巴比妥诱导的豚鼠微粒体中观察到与这三种化合物的最大络合，其中复合物形成的初始速率依赖于抑制剂浓度，并且分别发现BBT、αMB和αEB的表观Km值为108±44、338±96和84±15μM。在孵育混合物中加入1mM谷胱甘肽对复合物形成有显著的减弱作用，表明有亲电反应性中间体参与。在豚鼠或兔子的肺微粒体中，这三种抑制剂未观察到复合物形成。MI复合物形成不太可能是豚鼠肝脏P450 2Bx（兔P450 2B4的同源物）基于机制失活的原因，因为该同工型在非常低的抑制剂浓度下即发生不可逆失活、谷胱甘肽对这种破坏没有减弱作用、形成的MI复合物不稳定以及在豚鼠或兔子肺P450中未形成MI复合物。
