Identification of the reactive metabolites of fenclozic acid in bile duct cannulated rats.

Fenclozic acid (Myalex) was developed by ICI pharmaceuticals in the 1960s for the treatment of rheumatoid arthritis and was a promising compound with a good preclinical safety profile and efficacy. While it did not show adverse hepatic effects in preclinical animal tests or initial studies in man [ Chalmers et al. Ann. Rheum. Dis. 1969 , 28 , 595 and Chalmers et al. Ann. Rheum. Dis. 1969 , 28 , 590 ], it was later withdrawn from clinical development. Hepatotoxicity was observed in humans at daily doses of 400 mg but was not replicated in any of the animal species tested. Rodrigues et al. [ Arch. Toxicol. 2013 , 87 , 1569 ] published a mechanistic investigation using modern in vitro assays/techniques in order to investigate the hepatotoxicity; however, only the covalent binding in rat, dog, and human microsomes was identified as a potential indicator for hepatoxicity. Metabolites associated with or responsible for covalent binding could not be detected, likely due to the low in vitro metabolic turnover of fenclozic acid in microsomes. Foulkes [ J. Pharmacol. Exp. Ther. 1970 , 172 , 115 ] investigated the in vivo metabolism of fenclozic acid which included a rat bile duct cannulated (BDC) study characterizing the biliary and urinary metabolites; however, no reactive metabolites were identified. This study aimed to reinvestigate the in vivo metabolism of fenclozic acid in rat, with a focus on identifying any reactive metabolites that could explain the in vitro covalent binding in microsomes observed across the species. Using modern analytical techniques, we were successful in identifying an epoxide reactive metabolite, which upon conjugation with glutathione (GSH), formed up to 16 GSH-related products including positional and diastereoisomers. Not including the GSH related conjugates, 7 additional metabolites were identified compared to these previous metabolism studies.