Investigations towards understanding the idiosyncratic nature of zileuton toxicity

In January 1997, an anti-asthma drug, zileuton, was approved by the FDA due to its effective inhibition of 5-lipoxygenase, an initial enzyme in the leukotriene pathway. Since its release, zileuton has been associated with cases of elevated hepatic enzymes prompting patient removal from therapy.; We provide evidence here that the zileuton associated liver toxicity likely involves a sequence of biotransformations leading to 2-acetylbenzothiophene (2-ABT), which is subsequently metabolized to give a reactive intermediate(s). In vitro experiments with the human lymphoblast MCL5 cell line demonstrated that 2-ABT is cytotoxic in a P450-dependent manner. Human liver microsome (HLM) incubations with 2-ABT revealed the formation of two short-lived oxidized species, "M + 16" and "M + 32". Both of these metabolites formed adducts in the presence of glutathione (GSH) or N-acetylcysteine (NAC). The adduct obtained from the singly oxidized "M + 16" species, either from incubation with GSH or NAC, was found to be unstable in acidic medium and eliminated water readily to form a new compound. Authentic synthetic standards demonstrated that 2-ABT-S-oxide 41 corresponded to the "M + 16" metabolite and that the S-oxide underwent nucleophilic addition of GSH or NAC to produce the singly oxidized adducts observed in HLM. The S-oxide adducts readily eliminated water to form a rearomatized 2-ABT-GSH adduct or 2-ABT-NAC adduct. Coelution experiments with the synthetic standard confirmed the structure of the eliminated 2-ABT-NAC adduct 43. LC/MS analyses of urine samples collected from rats dosed with zileuton indicates that 43 is a metabolite of zileuton formed in vivo.; Subsequent in vitro studies have demonstrated a role for glutathione-s-transferases in the detoxification of 2-ABT-S-Oxide. GSTM1-1 and GSTP1-1 was shown to catalyze the conjugative reaction between glutathione and 2-ABT-S-Oxide, with an apparent kcat/Km of 0.46 +/- 0.16 and 0.35 +/- 0.12 for GSTM1-1 and GSTP1-1 respectively. GSTM1-1 and GSTP1-1 were found to be irreversibly inhibited by 2-ABT-S-Oxide, while GSTA1-1 was reversibly inhibited. The irreversible inhibition of GSTM1-1 and GSTP1-1 highlights the potential for alkylation.; Taken all together, these studies provide a plausible mechanistic rationale for the hepatotoxicity associated with zileuton therapy. In particular, the data support the hypothesis of 2-ABT-S-Oxide as a reactive metabolite in zileuton toxicity.