Systemic Exposure, Metabolism, and Elimination of [¹⁴C]-Labeled Amino Lipid, Lipid 5, after a Single Administration of mRNA Encapsulating Lipid Nanoparticles to Sprague-Dawley Rats

The emerging therapeutic modality of lipid nanoparticle (LNP)-encapsulated mRNAs has demonstrated promising clinical results when used as vaccines and is currently being tested in formulations for a wide range of targeted chronic disease treatments. These therapeutics are multicomponent assemblages of well-characterized naturally occurring molecules in addition to xenobiotic molecules, whose in vivo distributions are poorly understood. Here, the metabolic outcome and in vivo elimination of Lipid 5, a key xenobiotic amino lipid in LNP formulations, were assessed after intravenous administration of 14C-labeled Lipid 5 to Sprague Dawley rats. Intact Lipid 5 was predominantly cleared from plasma within 10 h after dosing, with only small quantities (<1% of 14C dose) of a single diacid metabolite detected after 10 h. Lipid 5 was rapidly metabolized via ester hydrolysis into aliphatic alcohols and diacidic amino head group moieties, which were further metabolized via β-oxidation. Overall, >90% of the administered Lipid 5-derived 14C was recovered in urine (65%) and feces (35%), predominantly as oxidative metabolites, within 72 h after dosing, indicating rapid renal and hepatic elimination. In vitro metabolite identification after incubation with human, nonhuman primate, and rat hepatocytes showed similar metabolites to those found in vivo. No meaningful differences were observed in Lipid 5 metabolism or elimination by sex. In conclusion, Lipid 5, a critical amino lipid component of LNPs for mRNA therapeutic delivery, showed minimal exposure, rapid metabolism, and near-complete elimination of 14C metabolites in rats. Significance Statement Lipid 5 is a key component of lipid nanoparticles used for the delivery of mRNA-based medicines; understanding the rates and routes of its clearance is crucial to assessing its long-term safety in LNP technology. This study conclusively established the rapid metabolism, and near- complete elimination of intravenously administered [14C]Lipid 5 in rats via both liver and kidney as oxidative metabolites derived from ester hydrolysis and subsequent β-oxidation.