Metabolic activation and cytotoxicity of ibudilast mediated by CYP3A4

Ibudilast (IBD) is a relatively nonselective inhibitor of phosphodiesterase, commonly used for treating asthma, progressive multiple sclerosis and other neuropathological pain conditions. Although IBD was considered safe and harmless to human health, its clinical use might be associated with reported increases of serum AST and ALT as well as liver weight. However, the mechanisms behind such liver injury are still unknown. The purpose of this work was to investigate metabolic activation of IBD and to define correlation between bioactivation and hepatotoxicity of IBD. Two oxidative metabolites, IBD-derived glutathione (GSH) conjugates (M1, M2), N-acetyl-L-cysteine (NAC) conjugates (M3, M4), and cysteine (Cys) conjugates (M5, M6) were detected in mouse liver microsomes fortified with IBD (100 μM) and trapping agents GSH, NAC, or Cys, respectively, and two GSH conjugates (M1 and M2), one NAC conjugate (M4) and one Cys conjugate (M5) were detected. Similar observation was obtained in human liver microsomal incubations. The formation of M1-M6 was NADPH-dependent. Moreover, biliary GSH conjugates and urinary NAC conjugates derived from IBD were detected in mice given IBD intragastrically at 100 mg/kg. The metabolism study suggested the formation of an epoxide intermediate. In addition, the epoxide intermediate was found to react with cysteine residues of hepatic protein in a dose-dependent manner. Further studies indicate that CYP3A4 dominated the metabolic activation of IBD. Exposure of primary hepatocytes to IBD resulted in decreased cell survival. Pretreatment of mice hepatocytes with ketoconazole attenuated the susceptibility to the cytotoxicity of IBD (25-400 μM). The reactive epoxide intermediate might correlate the hepatotoxicity induced by IBD. This work revealed the reactive epoxide intermediate might correlate the hepatotoxicity induced by IBD, and would provide new insights into the mechanisms behind the adverse reactions taking place in clinical use of IBD, especially for the reported liver injury.