Sun Hao, Moore Chad, Dansette Patrick M, Kumar Santosh, Halpert James R, Yost Garold S
Lead Generation Group, Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Global Research and Development, Pfizer, Inc., Groton, Connecticut, USA.
Drug Metab Dispos. 2009 Mar;37(3):672-84. doi: 10.1124/dmd.108.022707. Epub 2008 Dec 12.
4-Chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (indapamide), an indoline-containing diuretic drug, has recently been evaluated in a large Phase III clinical trial (ADVANCE) with a fixed-dose combination of an angiotensin-converting enzyme inhibitor, perindopril, and shown to significantly reduce the risks of major vascular toxicities in people with type 2 diabetes. The original metabolic studies of indapamide reported that the indoline functional group was aromatized to indole through a dehydrogenation pathway by cytochromes P450. However, the enzymatic efficiency of indapamide dehydrogenation was not elucidated. A consequence of indoline aromatization is that the product indoles might have dramatically different therapeutic potencies. Thus, studies that characterize dehydrogenation of the functional indoline of indapamide were needed. Here we identified several indapamide metabolic pathways in vitro with human liver microsomes and recombinant CYP3A4 that include the dehydrogenation of indapamide to its corresponding indole form, and also hydroxylation and epoxidation metabolites, as characterized by liquid chromatography/mass spectrometry. Indapamide dehydrogenation efficiency (V(max)/K(m)=204 min/mM) by CYP3A4 was approximately 10-fold greater than that of indoline dehydrogenation. In silico molecular docking of indapamide into two CYP3A4 crystal structures, to evaluate the active site parameters that control dehydrogenation, produced conflicting results about the interactions of Arg212 with indapamide in the active site. These conflicting theories were addressed by functional studies with a CYP3A4R212A mutant enzyme, which showed that Arg212 does not seem to facilitate positioning of indapamide for dehydrogenation. However, the metabolites of indapamide were precisely consistent with in silico predictions of binding orientations using three diverse computer methods to predict drug metabolism pathways.
4-氯-N-(2-甲基-1-吲哚啉基)-3-氨磺酰基苯甲酰胺（吲达帕胺）是一种含吲哚啉的利尿药物，最近在一项大型III期临床试验（ADVANCE）中进行了评估，该试验采用血管紧张素转换酶抑制剂培哚普利的固定剂量联合用药，结果显示可显著降低2型糖尿病患者发生主要血管毒性的风险。吲达帕胺最初的代谢研究报告称，吲哚啉官能团通过细胞色素P450的脱氢途径芳构化为吲哚。然而，吲达帕胺脱氢的酶促效率尚未阐明。吲哚啉芳构化的一个结果是产物吲哚可能具有截然不同的治疗效力。因此，需要开展研究来表征吲达帕胺功能性吲哚啉的脱氢过程。在此，我们利用人肝微粒体和重组CYP3A4在体外鉴定了几种吲达帕胺代谢途径，包括吲达帕胺脱氢生成其相应的吲哚形式，以及通过液相色谱/质谱表征的羟基化和环氧化代谢物。CYP3A4对吲达帕胺的脱氢效率（V(max)/K(m)=204 min/mM）比吲哚啉脱氢效率高约10倍。为评估控制脱氢的活性位点参数，将吲达帕胺与两种CYP3A4晶体结构进行了计算机模拟分子对接，结果显示活性位点中Arg212与吲达帕胺的相互作用存在相互矛盾的结果。通过对CYP3A4R212A突变酶的功能研究解决了这些相互矛盾的理论，该研究表明Arg212似乎无助于吲达帕胺脱氢的定位。然而，吲达帕胺的代谢物与使用三种不同计算机方法预测药物代谢途径的计算机模拟结合取向预测结果精确一致。
