It is well recognized that the nitrile group in most nitrile-containing drugs is passed through the body unchanged.Nitrile biotransformation is rare and, when observed, is a very minor metabolic pathway.However, for some cyanopyrrolidine DPP-4 inhibitors, the conversion of nitrile into carboxylic acid is a major metabolic pathway, and the predominant enzyme responsible for the formation of carboxylic acid metabolite is still unclear.In the present study, besigliptin, a potent cyanopyrrolidine DPP-4 inhibitor that is currently under clinical development for the treatment of type-2 diabetes was selected as the model drug to investigate the nitrile metabolism.The pharmacokinetics, metabolism, and excretion of besigliptin were evaluated in healthy male subjects after a single po 100 mg dose of besigliptin.A total of 16 metabolites were identified by uhra performance liquid chromatography/ quadrupole time-of-flight mass spectrometry.The major biotransformation pathways included N-demethylation (formation of M1 and its derivatives), conversion of nitrile group to carboxylic acid (formation of M2 and its derivatives), and the combination of these two pathways (M3).Carboxylic acid metabolite M2 was the major circulating component in plasma, followed by unchanged drug and M1.