Hutzler J Matthew, Steenwyk Rick C, Smith Evan B, Walker Gregory S, Wienkers Larry C
Pfizer, Pharmacokinetics, Dynamics, and Metabolism (PDM), St. Louis, Missouri, USA.
Chem Res Toxicol. 2004 Feb;17(2):174-84. doi: 10.1021/tx034199f.
The kinetics for inactivation of cytochrome P450 2D6 by (1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) were characterized, and the mechanism was determined in an effort to understand the observed time-based inactivation. Loss of dextromethorphan O-demethylase activity following coincubation with EMTPP followed pseudo-first-order kinetics and was both NADPH- and EMTPP-dependent. Inactivation was characterized by an apparent Ki of 5.5 microM with a maximal rate constant for inactivation (kinact) of 0.09 min(-1), a t1/2 of 7.7 min, and a partition ratio of approximately 99. P450 2D6 inactivation was unaffected by coincubation with exogenous nucleophiles or reactive oxygen scavengers and was protected by the competing inhibitors N-4-(trifluoromethyl)benzyl quinidinium bromide and quinidine. After a 30 min incubation with 100 microM EMTPP, dextromethorphan O-demethylase activity was decreased approximately 76%, with a disproportionate loss ( approximately 35%) in carbon monoxide binding. Additional mechanistic studies showed no evidence of either metabolite inhibitory complex formation or heme adduction. However, a P450 2D6 apoprotein adduct was characterized that had a mass shift relative to unadducted P450 2D6 apoprotein consistent with the molecular mass of EMTPP (353 Da). In vitro metabolism studies revealed that EMTPP is susceptible to P450 2D6-mediated hydroxylation and dehydrogenation, postulated to both form via initial hydrogen atom abstraction from the alpha-carbon of the imidazole ethyl substituent. Additional studies demonstrated that while a dehydrogenated EMTPP metabolite was apparently stable and observable, we propose that a thermodynamic partitioning may exist, which results in formation of a second dehydrogenated imidazo-methide-like metabolite that may serve as the reactive species causing mechanism-based inactivation of P450 2D6. Last, trapping studies with EMTPP yielded an N-acetyl cysteine conjugate, which upon tandem MS and NMR analysis revealed adduction to the alpha-carbon of the imidazole ethyl substituent. Overall, evidence suggests that nucleophilic attack of an imidazo-methide-like intermediate by a P450 2D6 active site residue leads to apoprotein adduction and consequent inactivation.
对（1-[(2-乙基-4-甲基-1H-咪唑-5-基)甲基]-4-[4-(三氟甲基)-2-吡啶基]哌嗪（EMTPP）使细胞色素P450 2D6失活的动力学进行了表征，并确定了其机制，以了解观察到的基于时间的失活现象。与EMTPP共同孵育后右美沙芬O-脱甲基酶活性的丧失遵循假一级动力学，且依赖于NADPH和EMTPP。失活的表观抑制常数（Ki）为5.5微摩尔，最大失活速率常数（kinact）为0.09分钟⁻¹，半衰期为7.7分钟，分配比约为99。与外源性亲核试剂或活性氧清除剂共同孵育对P450 2D6失活没有影响，且其可被竞争性抑制剂N-4-(三氟甲基)苄基溴化奎尼丁和奎尼丁保护。与100微摩尔EMTPP孵育30分钟后，右美沙芬O-脱甲基酶活性降低约76%，一氧化碳结合有不成比例的损失（约35%）。额外的机制研究未发现代谢物抑制复合物形成或血红素加合的证据。然而，鉴定出一种P450 2D6脱辅基蛋白加合物，其相对于未加合的P450 2D6脱辅基蛋白有质量位移，与EMTPP的分子量（353道尔顿）一致。体外代谢研究表明，EMTPP易受P450 2D6介导的羟基化和脱氢作用影响，推测这两种反应均通过从咪唑乙基取代基的α-碳上最初夺取氢原子而形成。额外的研究表明，虽然一种脱氢的EMTPP代谢物显然稳定且可观察到，但我们提出可能存在一种热力学分配，导致形成第二种脱氢的咪唑甲川样代谢物，其可能作为导致P450 2D6基于机制失活的活性物种。最后，用EMTPP进行的捕获研究产生了一种N-乙酰半胱氨酸缀合物，经串联质谱和核磁共振分析显示其加合到咪唑乙基取代基的α-碳上。总体而言，证据表明P450 2D6活性位点残基对咪唑甲川样中间体的亲核攻击导致脱辅基蛋白加合并随之失活。
