Metabolism of the ionizable lipid SM-102 in liver microsomes, 2D hepatocytes, and 3D liver spheroids.

Recent advances in lipid nanoparticle (LNP) delivery systems have enabled the development of mRNA vaccines and oligonucleotide therapeutics. Reliable hepatic in vitro systems are essential for the evaluation of the biodegradation of LNP components. SM-102 is an important ionizable lipid used in Moderna's LNP-mRNA vaccine. In this study, we compared the metabolism of SM-102 using 3 in vitro hepatic models, including liver microsomes, 2D hepatocyte cultures, and 3D liver spheroids prepared from primary rodent hepatocytes and HepG2 cells. An ultra-high-performance liquid chromatography high-resolution mass spectrometry method was developed to detect and characterize SM-102 metabolites. A 2,4-dinitrophenylhydrazine derivatization method was developed to further capture and characterize the potential carbonyl products. Following incubation of SM-102 with liver microsomes from human, monkey, dog, rat, and mouse, 7 metabolites were identified, with oxidation to carbonyl-containing metabolites representing the predominant pathway. In mouse 2D hepatocytes and 3D liver spheroids, 6 metabolites were detected, and the major metabolic pathways included N-dealkylation (M6), acetylation (M7), and hydrolysis (M8). In rat 2D hepatocyte cultures, 6 metabolites were detected, with M6 and M8 as the major metabolites. In contrast, β-oxidation-related metabolites (M10 and M11) were the major metabolites detected in rat 3D hepatocyte spheroids. Comparable metabolic profiles of SM-102 were observed in HepG2 2D cultures and 3D spheroids, with hydrolysis and N-dealkylation as the predominant metabolic pathways. Taken together, this study characterized the metabolic profiles of SM-102 across 3 in vitro hepatic models, and comparable metabolic profiles were observed between 2D hepatocytes and 3D spheroids prepared from primary mouse hepatocytes and HepG2 cells. These findings provide useful information for selecting appropriate in vitro models to ensure the reliable evaluation of ionizable lipid biodegradation. SIGNIFICANCE STATEMENT: This study compared the metabolic profiles of SM-102 across 3 hepatic models. 2D hepatocyte and 3D liver spheroid identified hydrolysis, N-dealkylation, and β-oxidation as major metabolic pathways for SM-102. In contrast, liver microsomes predominantly drove the oxidative metabolism. These findings highlight the necessity of cell-based models for the reliable evaluation of ionizable lipid biodegradation.