Grimm S W, Bend J R, Halpert J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, USA.
Drug Metab Dispos. 1995 May;23(5):577-83.
The kinetics of mechanism-based inactivation of phenobarbital-inducible rabbit hepatic cytochromes P450 2B4 and 2B5 by N-benzyl-(BBT) and N-alpha-methylbenzyl (alpha MB) 1-aminobenzotriazole were investigated using reconstituted P450 2B4, a stable heterologous expression system, and hepatic microsomes. Low micromolar concentrations of the 1-aminobenzotriazole derivatives caused reversible inhibition as well as rapid inactivation of reconstituted P450 2B4 and recombinant P450 2B4 and 2B5. In contrast, even at a 1000-fold higher concentration, aminobenzotriazole inactivated the expressed P450 2B enzymes less rapidly. Preincubation of phenobarbital-induced hepatic microsomes with BBT and alpha MB resulted in concentration-dependent decreases in marker activities of P450 2B4 and 2B5, benzyloxyresorufin O-debenzylase and androstenedione 15 alpha-hydroxylase, respectively. BBT caused the inactivation of P450 2B4 and 2B5 in hepatic microsomes with apparent Kl values of 1.9 and 2.4 microM and maximal rate constants of 0.29 and 0.18 min-1, respectively. alpha MB inactivated both P450 2B enzymes with similar Kl values (approximately 7 microM) and maximal rate constants only slightly higher for 2B4 compared with 2B5 (0.68 vs. 0.55 min-1). Similar P450 2B selectivity of BBT and alpha MB in both hepatic microsomes and the stable expression system further validates this new expression system and the use of the selective markers identified for 2B4 and 2B5 in hepatic microsomes. The results also provide a mechanistic basis for the high potency of the N-aralkylated 1-aminobenzotriazole derivatives in vivo and suggest that treatments that inactivate 2B4 will also lead to 2B5 inactivation.
利用重组P450 2B4（一种稳定的异源表达系统）和肝微粒体，研究了N-苄基-（BBT）和N-α-甲基苄基（αMB）1-氨基苯并三唑对苯巴比妥诱导的兔肝细胞色素P450 2B4和2B5基于机制的失活动力学。低微摩尔浓度的1-氨基苯并三唑衍生物可引起重组P450 2B4以及重组P450 2B4和2B5的可逆抑制和快速失活。相比之下，即使浓度高1000倍，氨基苯并三唑对表达的P450 2B酶的失活速度也较慢。用BBT和αMB对苯巴比妥诱导的肝微粒体进行预孵育，分别导致P450 2B4和2B5的标记活性、苄氧基试卤灵O-脱苄酶和雄烯二酮15α-羟化酶的浓度依赖性降低。BBT导致肝微粒体中P450 2B4和2B5失活，表观Kl值分别为1.9和2.4μM，最大速率常数分别为0.29和0.18 min-1。αMB使两种P450 2B酶失活，Kl值相似（约7μM），2B4的最大速率常数仅比2B5略高（0.68对0.55 min-1）。BBT和αMB在肝微粒体和稳定表达系统中对P450 2B具有相似的选择性，进一步验证了这种新的表达系统以及在肝微粒体中为2B4和2B5鉴定的选择性标记物的用途。结果还为N-芳烷基化1-氨基苯并三唑衍生物在体内的高效性提供了机制基础，并表明使2B4失活的处理也将导致2B5失活。
