Metabolism of vesatolimod in rat, dog, and human liver microsomes: Metabolic stability assessment, metabolite identification, and interspecies comparison

Vesatolimod (GS-9620) is an agonist of Toll-like Receptor (TLR7) 7, which has been developed as an anti-hepatitis B virus (HBV) agent. The focus of the present study is on the metabolic stability evaluation and metabolite identification of GS-9620 in rat, dog, and human liver microsomes, as well as interspecies comparison. The average observed in vitro T_1/2 values were 3.06, 13.06, and 15.56 minutes in rat, dog, and human liver microsomes, respectively. The findings suggested that GS-9620 was rapidly metabolized in the presence of reductive nicotinamide adenine dinucleotide phosphate (NADPH) in rat liver microsomes (RLM), and moderately metabolized in dog liver microsomes (DLM) and human liver microsomes (HLM). Subsequently, the metabolites were characterized using an ultra-high performance liquid chromatography coupled with linear ion trap orbitrap tandem mass spectrometer (UHPLC-LTQ-Orbitrap-MS) with dd-MS² on-line data acquisition mode. Under the current conditions, a total of 18 metabolites were detected and their identities were proposed by comparing their accurate masses, fragmental ions, and retention times with those of GS-9620. Three metabolites (M2, M4, and M18) were authentically identified by using Reference Standards. In RLM, 16 metabolites were identified with M2 being the most abundant metabolite. M4, M5, and M9 were rat-specific. In DLM, 12 minor metabolites were identified with a dog-specific metabolite (M6). In HLM, GS-9620 showed similar metabolic profiles to that in DLM, and 11 minor metabolites were detected with M12 being human-specific. Based on the identified metabolites, the metabolic pathways of GS-9620 were proposed, including hydroxylation, bis-hydroxylation, dehydrogenation, and oxidative deamination.