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 heptadecan-9-yl 8-((2-hydroxyethyl) (8-(nonyloxy)-8-oxooctyl)amino)octanoate (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 hour after dosing, with only small quantities (<1% of 14C dose) of a single diacid metabolite detected after 10 hour. Lipid 5 was rapidly metabolized via ester hydrolysis into aliphatic alcohols and diacidic amino head group moieties, which were further metabolized via ß-oxidation. Overall, >90% of the administered Lipid 5-derived 14C was recovered in urine (65%) and feces (35%), predominantly as oxidative metabolites, within 72 hour 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: Heptadecan-9-yl 8-((2-hydroxyethyl) (8-(nonyloxy)-8-oxooctyl)amino)octanoate (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 lipid nanoparticle 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 ß-oxidation.

Biodistribution of Lipid 5, mRNA, and Its Translated Protein Following Intravenous Administration of mRNA-Encapsulated Lipid Nanoparticles in Rats.

RNA-based therapeutics and vaccines represent a novel and expanding class of medicines, the success of which depends on the encapsulation and protection of mRNA molecules in lipid nanoparticle (LNP)-based carriers. With the development of mRNA-LNP modalities, which can incorporate xenobiotic constituents, extensive biodistribution analyses are necessary to better understand the factors that influence their in vivo exposure profiles. This study investigated the biodistribution of heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5)-a xenobiotic amino lipid-and its metabolites in male and female pigmented (Long-Evans) and nonpigmented (Sprague Dawley) rats by using quantitative whole-body autoradiography (QWBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. After intravenous injection of Lipid 5-containing LNPs, 14C-containing Lipid 5 ([14C]Lipid 5) and radiolabeled metabolites ([14C]metabolites) were rapidly distributed, with peak concentrations reached within 1 hour in most tissues. After 10 hours, [14C]Lipid 5 and [14C]metabolites concentrated primarily in the urinary and digestive tracts. By 24 hours, [14C]Lipid 5 and [14C]metabolites were localized almost exclusively in the liver and intestines, with few or no concentrations detected in non-excretory systems, which is suggestive of hepatobiliary and renal clearance. [14C]Lipid 5 and [14C]metabolites were completely cleared within 168 hours (7 days). Biodistribution profiles were similar between QWBA and LC-MS/MS techniques, pigmented and nonpigmented rats, and male and female rats, excluding the reproductive organs. In conclusion, the rapid clearance through known excretory systems, with no evidence of redistribution for Lipid 5 or accumulation of [14C]metabolites, provides confidence for the safe and effective use of Lipid 5-containing LNPs. SIGNIFICANCE STATEMENT: This study demonstrates the rapid, systemic distribution of intact and radiolabeled metabolites of Lipid 5, a xenobiotic amino lipid component of novel mRNA-LNP medicines, and its effective clearance without substantial redistribution after intravenous administration; additionally, findings were consistent between different mRNAs encapsulated within LNPs of similar composition. This study confirms the applicability of current analytical methods for lipid biodistribution analyses, and taken together with appropriate safety studies, supports the continued use of Lipid 5 in mRNA-medicines.