Donepezil is a reversible acetylcholinesterase inhibitor that is currently the most commonly prescribed drug for the treatment of Alzheimer’s disease. In general, donepezil is known as a safe and well-tolerated drug, and it was not associated with liver abnormalities in several clinical trials. However, rare cases of drug-related liver toxicity have been reported since it has become commercially available. Few studies have investigated the metabolic profile of donepezil, and the mechanism of liver damage caused by donepezil has not been elucidated. In this study, the in vitro metabolism of donepezil was investigated using liquid chromatography–tandem mass spectrometry based on a non-targeted metabolomics approach. To identify metabolites, the data were subjected to multivariate data analysis and molecular networking. A total of 21 donepezil metabolites (17 in human liver microsomes, 21 in mice liver microsomes, and 17 in rat liver microsomes) were detected including 14 newly identified metabolites. One potential reactive metabolite was identified in rat liver microsomal incubation samples. Metabolites were formed through four major metabolic pathways: (1) O-demethylation, (2) hydroxylation, (3) N-oxidation, and (4) N-debenzylation. This study indicates that a non-targeted metabolomics approach combined with molecular networking is a reliable tool to identify and detect unknown drug metabolites.

A liver injury was recently reported for saxagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor. However, the underlying mechanisms of saxagliptin-induced liver injury remain unknown. This study aimed to evaluate whether saxagliptin, a potent and selective DPP-4 inhibitor that is globally used for treating type 2 diabetes mellitus, binds to the nucleophiles in vitro. Four DPP-4 inhibitors, including vildagliptin, were evaluated for comparison. Only saxagliptin and vildagliptin, which both contain a cyanopyrrolidine group, quickly reacted with L-cysteine to enzyme-independently produce thiazolinic acid metabolites. This saxagliptin–cysteine adduct was also found in saxagliptin-administered male Sprague–Dawley rats. In addition, this study newly identified cysteinyl glycine conjugates of saxagliptin and 5-hydroxysaxagliptin. The observed metabolic pathways were hydroxylation and conjugation with cysteine, glutathione, sulfate, and glucuronide. In summary, we determined four new thiazoline-containing thiol metabolites (cysteine and cysteinylglycine conjugates of saxagliptin and 5-hydroxysaxagliptin) in saxagliptin-administered male rats. Our results reveal that saxagliptin can covalently bind to the thiol groups of cysteine residues of endogenous proteins in vivo, indicating the potential for saxagliptin to cause drug-induced liver injury.