Division of Molecular Toxicology, LACDR, Leiden/Amsterdam Center for Drug Research, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Chem Res Toxicol. 2010 Sep 20;23(9):1467-76. doi: 10.1021/tx100131f.
The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.
药物代谢物与 GSH 的结合被认为是一种重要的解毒机制，可以自发进行，也可以由谷胱甘肽 S-转移酶（GSTs）介导。如果 GSTs 在 GSH 结合中起重要作用，那么 GSTs 的遗传缺陷可能是导致药物反应（ADRs）的危险因素，这些反应是由反应性药物代谢物引起的。到目前为止，氯氮平（一种导致特发性药物反应（IDRs）的药物）的反应性代谢物的 GSTs 在解毒中的作用尚未研究。在本研究中，我们研究了四种重组人 GST（hGST A1-1、hGST M1-1、hGST P1-1 和 hGST T1-1）催化人肝微粒体和药物代谢 P450 BM3 突变体 P450 102A1M11H 体外形成的氯氮平反应性代谢物与 GSH 的结合能力。与先前的研究一致，在没有 GSTs 的情况下，从氯氮平的硝𬭩离子中鉴定出三种 GSH 结合物。在三种 GST 存在的情况下，hGST P1-1、hGST M1-1 和 hGST A1-1 均强烈增加了所有生物活化系统测试中的总 GSH 结合。用 hGST P1-1 观察到最高的活性，而 hGST M1-1 和 hGST A1-1 则显示出稍低的活性。多态性 hGST T1-1 对催化反应性氯氮平代谢物的 GSH 结合没有表现出任何活性。有趣的是，添加 GSTs 导致反应性氯氮平代谢物 GSH 结合的区域选择性发生重大变化，这可能是由于 hGSTs 的不同活性位点几何形状所致。发现的两种 GSH 结合物完全依赖于 GSTs 的存在。氯取代氯氮平的硝𬭩离子，迄今为止仅在体内研究中观察到，似乎是 hGST P1-1 催化 GSH 结合的主要途径，而 hGST A1-1 和 hGST M1-1 也显示出显著的活性。第二种 GSH 结合物以前也仅在体内研究中发现，也由 hGST P1-1 形成，并且在较小程度上由 hGST A1-1 形成。这些结果表明，人 GSTs 可能在氯氮平反应性中间物的失活中起重要作用。因此，需要进一步研究以调查 hGST P1-1 和 hGST M1-1 的遗传多态性是否导致对氯氮平诱导的药物不良反应的个体易感性差异。
