In this study, we delineated the poorly characterized metabolism of anamorelin, a growth hormone secretagogue receptor agonist, in vitro using human liver microsomes (HLM), based on classical molecular networking (MN) and feature-based molecular networking (FBMN) from the Global Natural Products Social Molecular Networking platform. Following the in vitro HLM reaction, the MN analysis showed 11 neighboring nodes whose information propagated from the node corresponding to anamorelin. The FBMN analysis described the separation of six nodes that the MN analysis could not achieve. In addition, the similarity among neighboring nodes could be discerned via their respective metabolic pathways. Collectively, 18 metabolites (M1-M12) were successfully identified, suggesting that the metabolic pathways involved were demethylation, hydroxylation, dealkylation, desaturation, and N-oxidation, whereas 6 metabolites (M13a*-b*, M14a*-b*, and M15a*-b*) remained unidentified. Furthermore, the major metabolites detected in HLM, M1 and M7, were dissimilar from those observed in the CYP3A4 isozyme assay, which is recognized to be markedly inhibited by anamorelin. Specifically, M7, M8, and M9 were identified as the major metabolites in the CYP3A4 isozyme assay. Therefore, a thorough investigation of metabolism is imperative for future in vivo studies. These findings may offer prospective therapeutic opportunities for anamorelin.
Keywords: GHSR agonist; LC-MS/MS; anamorelin; metabolism; molecular networking.