In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200 μM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10 mg/kg or 25 mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400 μM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.