Bioactivation of Sitaxentan in Liver Microsomes, Hepatocytes, and Expressed Human P450s with Characterization of the Glutathione Conjugate by Liquid Chromatography Tandem Mass Spectrometry

Abstract

Sitaxentan is a selective endothelin-A receptor antagonist that was marketed as Thelin in several European countries and Canada for pulmonary arterial hypertension. Sitaxentan was undergoing further clinical trials in the United States but due to four deaths and one case of liver transplantation from severe liver toxicity that appeared to be idiosyncratic in nature, it was withdrawn worldwide in December, 2010. Sitaxentan contains a 1,3-benzodioxole ring that undergoes enzymatic demethyleneation to an ortho-catechol metabolite that can further oxidize to a reactive ortho-quinone metabolite. Here, we report the detection and mass spectral characterization of a glutathione conjugate of this sitaxentan quinone reactive metabolite that was trapped in vitro using mouse, rat, dog, and human liver microsomes supplemented with NADPH and glutathione and that was also observed in rat and human hepatocytes. Using human liver microsomes, we also demonstrated that P450 3A4 undergoes time-dependent inhibition. Density functional calculations on the catechol metabolite of sitaxentan indicated that the reaction leading to the quinone was thermodynamically favorable with an enthalpy change of -6.3 kcal/mol. Using density functional methodology, we modeled the attack of glutathione on the quinone with an S-methyl thiolate anion which allowed us to predict, based on the difference in transition state energies, that the 2-position on the phenyl ring was more likely than the 5-position as the site of glutathione conjugation. Overall, our results demonstrated that sitaxentan is capable of facile formation of a reactive ortho-quinone metabolite capable of reacting with glutathione and may rationalize the idiosyncratic nature of the hepatotoxicity that led to its withdrawal.

Multiple Compound-Related Adverse Properties Contribute to Liver Injury Caused by Endothelin Receptor Antagonists

“…Potentiated toxicity to THLE-3A4 cells has been observed with numerous other drugs that are bioactivated to reactive intermediates via CYP3A4 (Thompson et al, 2012;Foster et al, 2013;Gustafsson et al, 2014). Sitaxentan is metabolized by CYP3A4 (Erve et al, 2013) and exhibited a high in vitro f CVB to human hepatocytes; therefore, a likely explanation for the potentiated toxicity to THLE-3A4 cells is the formation of a cytotoxic reactive intermediate in these cells. Investigations of sitaxentan biotransformation in human liver microsomes revealed timedependent inhibition of CYP3A4 and formation of an unstable metabolite that was trapped by addition of reduced GSH (Erve et al, 2013).…”

“…Sitaxentan is metabolized by CYP3A4 (Erve et al, 2013) and exhibited a high in vitro f CVB to human hepatocytes; therefore, a likely explanation for the potentiated toxicity to THLE-3A4 cells is the formation of a cytotoxic reactive intermediate in these cells. Investigations of sitaxentan biotransformation in human liver microsomes revealed timedependent inhibition of CYP3A4 and formation of an unstable metabolite that was trapped by addition of reduced GSH (Erve et al, 2013). The GSH adduct was proposed to be formed via an ortho-quinone intermediate, and two potential sites for the formation of such an intermediate were proposed, one of which was more thermodynamically favored (Supplemental Scheme 1).…”

In Vitro and In Vivo Characterization of Reactive Intermediates of Corynoline

“…Although corynoline was reported to cause cytotoxicity (Choi et al, 2007), metabolism of corynoline has not been carefully investigated. Corynoline contains two 1,3-benzodioxole groups, and many 1,3-benzodioxole-containing compounds were found to induce toxicities (Erve et al, 2013). Metabolic activation is suggested to initiate the process of the toxicities induced by these toxic compounds.…”

“…Time-dependent inhibition is often a criterion for irreversible enzyme inhibition. Covalent modification of the P450 apoprotein, alkylation of the heme moiety, or destruction of the heme group via the metabolic intermediate complex (Erve et al, 2013) is considered to trigger the enzyme inactivation. Our observation of ortho-quinone intermediate provided the evidence for a possible mechanism by which the reactive species alkylated the apoprotein and/ or heme of the host enzyme.…”

“…Sitaxentan, a selective endothelin-A receptor antagonist, was withdrawn from the global market by Pfizer in December 2010 due to its idiosyncratic hepatotoxicity. A similar ortho-quinone metabolite of sitaxentan was recently characterized and was suggested to be associated with the reported hepatic toxicity (Erve et al, 2013).…”