QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: dmd.108.022707v1 37/3/672    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sun, H. Articles by Yost, G. S. Search for Related Content PubMed PubMed Citation Articles by Sun, H. Articles by Yost, G. S.

Dehydrogenation of the Indoline-Containing Drug 4-Chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (Indapamide) by CYP3A4: Correlation with in Silico Predictions

Lead Generation Group, Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Global Research and Development, Pfizer, Inc., Groton, Connecticut (H.S.); Université Paris Descartes, Unité de Formation et de Recherche Biomédicale, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8601, Paris, France (P.M.D.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California (J.R.H.); Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri (S.K.); and Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah (C.M., G.S.Y.)

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.