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

RNA-based therapeutics and vaccines represent a novel and ex-panding 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, exten-sive 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, 14 C-containing Lipid 5 ([ 14 C]Lipid 5) and radiolabeled metabolites ([ 14 C]metabolites) were rapidly distributed, with peak concentrations reached within 1 hour in most tissues. After 10 hours, [ 14 C]Lipid 5 and [ 14 C]metabolites concentrated primarily in the urinary and digestive tracts. By 24 hours, [ 14 C]Lipid 5 and [ 14 C]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. [ 14 C]Lipid 5 and [ 14 C]metabolites were completely cleared within 168 hours (7 days). Biodistribution profiles were similar between QWBA and LC-MS/ MS techniques, pigmented and nonpigmented rats


Introduction
RNA-based therapeutics and vaccines represent a novel and expanding class of medicines that have been invaluable during the coronavirus disease 2019  pandemic. The global deployment of mRNA COVID-19 vaccines was a prominent milestone for those modalities, building confidence in mRNA research and further substantiating investigations of other mRNA-based medicines, such as enzyme replacement and enhanced cell therapies (Damase et al., 2021). The success of these mRNA therapies depends on their encapsulation in lipid nanoparticles (LNPs), assisting in the transport of exogenous nucleic acids to tissues while simultaneously preventing degradation by endo-and exonucleases (Guevara et al., 2020;Hou et al., 2021). Lipid nanoparticles are noncovalent assemblies of multiple molecular components and are typically composed of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-functionalized lipids (Lee et al., 2013;Hassett et al., 2019;Hou et al., 2021).It has been established that xenobiotic components, such as PEG-functionalized and ionizable lipids stabilize LNP formulation throughout manufacturing and storage, assist in cell surface binding, cellular uptake, and facilitate mRNA endosomal escape (Chan et al., 2012;Shi et al., 2021;Kalyanram et al., 2022). However, studies that assess the in vivo fates of LNP-mRNA formulations have focused on the mRNA distribution, and only to a much lesser extent, the biodistribution and clearance of LNP components and their metabolites (Hou et al., 2021). A comprehensive analysis to evaluate the biodistribution and clearance of both the mRNA and LNP constituents is crucial to fully assess the safety and efficacy of these components in both preclinical and clinical settings and inform the continued development of improved mRNA-LNP based medicines.
As studies into LNP formulations continue, emerging evidence suggests that LNPs have the potential for organ-and tissue-specific tropism of mRNA-based medicines (Rostami et al., 2014;Veiga et al., 2020;Tomb acz et al., 2021). The tissue-specific distribution could potentially be facilitated by multiple factors, including LNP particle size, lipid components, and incorporation of specific targeting proteins, such as monoclonal antibodies on the LNP surface (Veiga et al., 2020;Zhang et al., 2021;Di et al., 2022). Although these properties of LNPs are enticing for future drug development, reports have identified variability in delivery of these constructs with greater inflammatory responses associated with the use of xenobiotic lipids which is not apparent with naturallyoccurring LNP components (Ndeupen et al., 2021). As naturally occurring LNP components (e.g., lipids, cholesterol, cholesterol esters) undergo in vivo processing similar to that of endogenous analogs and xenobiotic lipid clearance routes are typically unknown (Scioli Montoto et al., 2020), a thorough assessment of safety and reactogenicity after LNP administration is needed. It is thus crucial that studies focus on both the primary biodistribution of the intact mRNA-LNP as well as the redistribution of its constituents and metabolites to evaluate the immunogenic potential of the intact mRNA-LNP formulation, its constituents, and their metabolites.
Studies investigating mRNA-LNP biodistribution have traditionally used methods such as intracellular enzymatic reporters to track the delivery of intact encapsulated mRNA into host cells (Hassett et al., 2019;Aldosari et al., 2021;Delehedde et al., 2021;Yang et al., 2021). Such methods provide information on the biodistribution of intact mRNA-LNPs, but because the reporting capacity of these techniques is dependent solely on the viability and successful delivery of the intact mRNA, this method has limited use in determining the in vivo fates of constituents, metabolites, or of damaged mRNA-LNPs (Blakney et al., 2019). To address the limitations associated with conventional LNP biodistribution studies, we evaluated the distribution of Heptadecane-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5), a xenobiotic amino lipid comprising a slightly polar head group and an aliphatic lipid tail. Lipid 5 is distributed within the inner core of LNPs where it assists in mRNA stabilization (Fig. 1). Analysis techniques included quantitative whole body autoradiography (QWBA) due to its capabilities allowing for assessing distribution profiles of intact and dismantled LNPs depending on the use of suitably radiolabeled constituents. This analysis method in combination with traditional metabolism and elimination studies provide an overview of the physiologic fates of Lipid 5 and Lipid 5-containing LNPs. QWBA analysis was performed in male and female rats after intravenous administration of radiolabeled Lipid 5 ([ 14 C]Lipid 5)-containing LNPs encapsulating non-translating factor IX (NTFIX) mRNA. This report includes the results of QWBA performed to assess the distribution of Lipid 5 and its 14 C-containing metabolites ([ 14 C]metabolites) after intravenous administration of an NTFIX mRNA-encapsulating, [ 14 C]Lipid 5-containing LNP. Traditional biodistribution assessments of lipid, mRNA, and expressed protein from an intravenously administered therapeutic LNP of similar lipid composition that encapsulates similarly sized mRNAs were compared to that of the 14 C-containing LNPs. The ability of QWBA to track both intact [ 14 C]Lipid 5 and its [ 14 C]metabolites provides a detailed understanding of the in vivo distribution and persistence of Lipid 5-derived compounds and makes it complementary to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques used for biodistribution studies in LNP component in vivo fate assessments. In addition, this study assessed the impact of endogenous pigmentation (i.e., melanin; (Solon, 2015) on the biodistribution profiles of Lipid 5 and its radiolabeled metabolites in pigmented (Long-Evans) and nonpigmented (Sprague Dawley) rats. Details regarding Lipid 5 metabolism and elimination following intravenous administration in male and female rats will be reported separately (Burdette et al., manuscript in press).

Materials and Methods
Reagents and Materials. All chemicals used were of reagent grade unless stated otherwise. Lipid 5 was synthesized as previously described (Sabnis et al., 2018) and labeled with 14 C at the proximal carbon of the ethanolamine aliphatic chain linked to the tertiary nitrogen (Eurofins; Columbia, MO; Fig. 1). The radiolabeled site was selected as it was identified to be the most protected from known ester hydrolysis and b-oxidation degradation pathways for Lipid 5 (Burdette  et al., manuscript in press). The purity of Lipid 5 and [ 14 C]Lipid 5 were determined as 95% chemical purity and 99% radiochemical purity, respectively. mRNA and LNP Production. Complete N1-methylpseudouridine-substituted mRNA was synthesized in vitro from a linearized DNA template containing the 5 0 untranslated region, the 3 0 untranslated region, and a poly-A tail, as described previously (Sabnis et al., 2018). After purification, mRNA (NTFIX for [ 14 C]Lipid 5-containing LNPs and similarly sized mRNA for Lipid 5containing LNPs) was diluted in citrate buffer (Teknova) and frozen for subsequent use. LNP formulations were prepared by ethanol drop nanoprecipitation as described previously (Sabnis et al., 2018). In short, Lipid 5 or [ 14 C]Lipid 5, dipalmitoylphosphatidylcholine, cholesterol, and PEGylated lipid (CordenPharma) were dissolved in ethanol (Merck KGaA) and combined with acidified mRNA at a ratio of 3:1 (aqueous:ethanol). The product was diluted in citrate-buffered saline, maintained, and combined with a Tris buffer (pH 7.75) followed by a matched buffered excipient solution. The resulting formulation was filtered through an 0.8/0.2 mm filter (Pall Corporation), buffer exchanged into sucrose containing Tris buffer (pH 7.4), and concentrated using tangential flow filtration. Both radiolabeled and non-radiolabeled LNPs had similar lipid composition, size (60-120 nm), storage, and serum stability regardless of encapsulated mRNA (dynamic light scattering on a DynaPro Plate Reader [Wyatt; Goleta, CA]). The Lipid 5 content of the radiolabeled LNPs included 7.5% [ 14 C]Lipid 5. The mRNA concentration and encapsulation were measured via a RiboGreen fluorescence assay (Thermo Fisher Scientific) and specific activity was measured via liquid scintillation. The formulation was stored at À80 C.
Intravenous Administration of [ 14 C]Lipid 5-Containing mRNA-LNP to Rats. Sprague Dawley (SD; 5 males and 5 females) or Long-Evans (LE; 5 males and 5 females) rats were intravenously administered 2 mg/kg [ 14 C]Lipid 5-containing LNP encapsulating a single, nontranslating mRNA (NTFIX; Eurofins) over a 10-minute period (35 uCi dosed per animal). One male and 1 female SD and LE rat were euthanized at 1, 10, 24, 48, and 168 hours postdose to assess [ 14 C]Lipid 5 biodistribution from peak systemic exposure to near complete systemic clearance based on previous experiments. Animals were euthanized via carbon dioxide anesthetic (7-L chamber flushed with CO 2 [99.9% purity] at a 3-L/min flow rate with 42.8% volume displacement) and immersed in a hexane/ dry ice bath for approximately 6 to 11 minutes. Animal carcasses were drained, blotted dry, embedded in 5% carboxymethylcellulose (Sigma-Aldrich), and stored at -10 to -30 C until further use. All research involving animals was conducted at Charles River Laboratories in accordance with CR-Mattawan and Moderna, Inc. Animal Care and Use Committee guidelines.
QWBA Analysis. Prior to sectioning, frozen standards comprising carboxymethylcellulose and food coloring (N $ 4) were used to ensure section thickness quality control. Frozen embedded rat carcasses were sectioned in approximately 30 mm slices through the sagittal plane and captured on adhesive tape. Sections that displayed optimal visibility of tissues and biologic fluids of interest were scanned subsequent to exposure to phosphor imaging screens.  dmd.aspetjournals.org quantified by using image densitometry (MCID Image Analysis software version 7.1) and standardized to calibration controls; a standard curve was constructed from the integrated response (MDC/mm 2 ) and 14 C calibration standard concentrations. A lower limit of quantification of 40-ng equivalents/g was established based on radioactivity measured from the lowest calibration standard used divided by the specific activity of the dose formulation (mCi/mg). Artifacts were excluded as necessary from the analysis during image processing.
Quantification of Lipid 5 by LC-MS/MS. Quantification of Lipid 5containing rat blood samples was determined via high-performance LC coupled with LC-MS/MS and validated as per FDA Bioanalytical Method Validation guidelines for industry (US Food and Drug Administration, 2018). For the analysis of rat tissue samples, a qualification of analytical method was performed via accuracy and precision, selectivity, and recovery of Lipid 5 in brain, heart, liver, spleen, lung, kidney, stomach, small intestine, bone marrow, testes, skeletal muscle, ovaries, uterus, full thickness tail skin, eyes, thymus, jugular vein, and lymph node tissue homogenates. Tissue homogenate samples were prepared by suspending 200 mg tissue in 4% bovine serum albumin followed by bead homogenization. Resulting homogenates underwent acetonitrile:methanol (1:1) protein precipitation and centrifugation at 4,000 rpm for 10 minutes at 5 C, and the resulting supernatant was used for further analysis. Samples were analyzed via reverse-phase (C8) LC on an Agilent 1200 HP liquid chromatograph coupled with a Sciex Triple Quad API 5000 LC-MS/MS system. The MS/MS was operated at positive-ion polarity and multiple-reaction-monitoring scanning mode; acquisitions of Lipid 5-specific signals were identified via 710.7 m/z-to >472.5 m/zspecific transition. Concentrations of Lipid 5 were determined via linear regression of a Lipid 5 calibration curve (0.5 to 500 ng/ml); the lower limit of quantitation of Lipid 5 in plasma and tissues was 0.500 ng/ml. A 95% mean recovery of Lipid 5 from tissues was observed as evaluated with low and high quality control standards.
Quantification of mRNA by bDNA Assay. mRNA quantification in rats administered mRNA-LNPs was performed using a QuantiGene 2.0 kit (Thermo Fisher Scientific). Rat plasma and homogenized tissue samples were collected and lysed prior to analysis. Calibration standards and quality control samples were prepared in both pooled sera and pooled tissue homogenates and processed similarly to tissue samples. All resulting samples underwent a minimum 100-fold dilution in lysis buffer (Thermo Fisher Scientific) prior to conducting of branched DNA assay. Coated plates were exposed to the diluted rat samples overnight at 55 C, washed, exposed to a pre-AMP working solution for 1 hour at 55 C, and to a label probe working solution for 1 hour at 50 C. The resulting plate was washed, and the quality control 2.0 substrate added to each well, which was incubated for 5 minutes at room temperature. Data acquisition was performed using a Spectramax luminometer (Molecular Devices), captured on Softmax PRO Gxp software (Molecular Devices; version 5.4.6) and further analyzed using Watson LIMS (Thermo Fisher Scientific; version 7.6.1 HF1). The lower  Quantification of Protein by LC-MS/MS. Quantification of translated protein product in rats administered Lipid 5-containing mRNA-LNPs were performed via LC-MS/MS. Rat tissue samples were homogenized in T-PER (Thermo Fisher Scientific), reduced with 2 ml of 100 mM TCEP, and denatured by incubating at 100 C for 10 minutes. The samples were then cooled to room temperature and alkylated by adding 4 ml of 50 mM iodoacetamide. Subsequent protein digestion was performed by exposing the alkylated sample to a trypsin solution (0.2 mg/ml) at a final ratio of 1:20 (trypsin:protein) at 45 C for 1 hour. Resulting digested samples were centrifuged at 14,000 rpm (18,407 xg) for 10 minutes and the supernatant combined with an isotopically labeled internal standard. Samples were analyzed using a Triple Quad API 7500 MS system (Sciex). The lower limit of quantitation of protein in tissues was 20.0 pg/ml. PK and PD Analysis. Pharmacokinetic parameters (mRNA and Lipid 5) and pharmacodynamic parameters (protein) were calculated using noncompartmental analysis (Phoenix WinNonlin version 8.3).

Results
Biodistribution of [ 14 C]Lipid 5 and its 14 C-Containing Metabolites in SD and LE Rats. Radioactivity appeared rapidly, with maximal concentrations measured in several tissues within the first hour after administration of [ 14 C]Lipid 5-containing LNPs ( Fig. 2A; Supplemental  Fig. 1). In SD rats, the liver (20,000 ng equivalents test article/g), adrenal gland (12,000 ng equivalents test article/g), spleen (8320 ng equivalents test article/g), and small-intestinal contents (6800 ng equivalents test article/g) had the highest distributions of radioactivity compared with other tissues at 1 hour (Fig. 3). By 10 hours ( Fig. 2B and Supplemental Fig. 2), a general decrease in radioactivity was observed throughout most tissues compared with the 1-hour time point, with the maximum distribution at 10 hours observed within the large-intestinal contents (14,500 ng equivalents test article/g), cecum contents (7,530 ng equivalents test article/g), urine (7,990 ng equivalents test article/g), and liver (3,880 ng equivalents test article/g) for both male and female rats (Fig. 3). By the 24-hour time point (Supplemental Fig. 3), the majority of the radioactivity had been eliminated from the animals of both sexes, with the large-intestinal contents (4550 ng equivalents test article/g), cecum contents (1230 ng equivalents test article/g), small-intestinal contents (930 ng equivalents test article/g), and liver (892 ng equivalents test article/g) displaying modest to medium levels of radioactivity in both male and female rats compared with both the 1-hour and 10-hour time points (Fig. 3; Supplemental Tables 1 and 2). Through 48 hours (Supplemental Fig. 4) and 168 hours ( Fig. 2C; Supplemental Fig. 5), radioactivity measurements were low or indistinguishable from background in most tissues; minimal levels of radioactivity were measured in the small-intestinal contents (284 ng equivalents test article/g), liver (227 ng equivalents test article/g), and large-intestinal contents (192 ng equivalents test article/g) at 48 hours, with negligible amounts detected in the adrenal gland (139 ng equivalents test article/g), spleen (71 ng equivalents test article/g), and liver (59 ng equivalents test article/g) at 168 hours for both male and female rats. Detectable amounts of radioactivity were measured in the ovaries of female rats at 48 and 168 hours (225 and 169 ng equivalents test article/g, respectively), while levels were undetectable in testes at the same timepoints. The biodistribution of Lipid 5 and [ 14 C]metabolites) were similar between SD and LE rats at all time points assessed (Supplemental Figs. 6-10; Supplemental Tables 3 and 4), including having comparable distribution between pigmented and nonpigmented tissue for most time points.
Biodistribution of Lipid 5, mRNA, and Protein Products in SD Rats Using a Therapeutic mRNA-LNP. Rapid biodistribution of intact Lipid 5 was seen in most tissues, achieving maximum observed concentration (T max ) at the earliest time tested (0.16 hours)  Fig. 4) with only the liver (Fig. 4B) and spleen (Fig. 4G) reaching T max levels at 1 hour post dose. The highest Lipid 5 tissue concentrations (C max ) were recorded in the plasma (54,700 ± 12,800 ng/ml; Fig. 4A), followed by the lungs (14,600 ± 2450 ng/g; Fig. 4H), kidney (9410 ± 2640 ng/g; Fig. 4C), and liver (8050 ± 846 ng/g; Fig. 3B) as compared with other tissues. The highest areas under the curve (AUC last ) values observed for Lipid 5 were in the plasma and spleen, at 85,700 ± 14,800 hour*ng/mL and 26,900 ± 2120 hour*ng/g, respectively, followed by the liver, lungs, and kidney at 25,400 ± 2060 hour*ng/g, 21,000 ± 3660 hour*ng/g, and 10,100 ± 1460 hour*ng/g, respectively. Total tissue distribution as it relates to plasma (tissue-toplasma AUC ratio) was measured maximally, at 31.4%, 29.6%, 24.5%, and 11.8%, for the spleen, liver, lungs, and kidney, respectively, and to a lesser extent, within the jejunum, stomach, testes, heart, thymus, uterus, brain, and muscle in descending order of distribution levels (Table 1). Insufficient data were available for accurate determination of pooled lymph nodes, eye, ovary, and femur bone marrow exposure profiles. By 168 hours, most tissues were completely cleared of Lipid 5, with the longest half-life (T 1/2 ) of Lipid 5 reported in the lungs at 23.1 hours; the T 1/2 in liver was 9.30 hours.
In the evaluation of the distribution of the LNP mRNA component (Table 2; Fig. 5), T max was reported within 1 hour for the majority of tissues, with the jejunum (Fig. 5B) and eyes (Fig. 5E) reaching T max at 4 hours, and the axillary and inguinal lymph nodes (Fig. 5G) reaching T max at 48 hours. The highest mRNA distribution (C max ) occurred in plasma (18,200 ± 3450 ng/ml; Fig. 5A), liver (8530 ± 1340 ng/g; Fig. 5B), lungs (4500 ± 1070 ng/g; Fig. 5H), and spleen (3030 ± 1130 ng/g; Fig. 5G) compared with other tissues examined. The highest AUC last values were also reported in the spleen (202,000 ± 21,400 hour*ng/g), plasma (146,000 ± 14,600 hour*ng/mL), liver (34,000 ± 3340 hour*ng/g), and lungs (18,900 ± 2040 hour*ng/g). Compared with all tissues, the spleen showed the greatest tissue-to-plasma ratio, at 138%, followed by the liver at 23.3% and the lungs at 12.9%, with the remaining tissues at a less than 6.05% tissue-to-plasma ratio (including the kidney, heart, muscle, jejunum, testes, stomach, thymus, uterus, brain, femur, and brain marrow in descending order of ratios; Table 2). Insufficient data were available to accurately determine axillary and popliteal lymph node, eye, and ovary AUC values. Half-life values ranged from 4.76 to 55.1 hours in all tissues where estimable. The spleen (Fig. 5G) had the longest calculated T 1/2 values, of 55.1 hours, among all tissues examined; the T 1/2 of mRNA in liver was 8.15 hours.
Protein expressed from LNP-encapsulated mRNA (Table 3; Fig. 6) was rapidly detected in the thymus (TE max of 0.83 hour; Fig. 6G), while the uterus (Fig. 6F) and kidneys (Fig. 6C) only reached TE max at 72 hours. The highest E max was observed in the liver (44.2 ± 7.06 mg/g; Fig. 6B) and ovaries (14.7 mg/g; Fig. 6F) compared with the other tissues examined. Protein AUEC values were highest in the liver (1,520 ± 157 hour*mg/g; Fig. 6B), and kidney (72.1 ± 23.0 hour*mg/g; Fig. 6C) followed by the lung, jejunum, heart, thymus, spleen, uterus, stomach, testes, and brain in descending order of protein levels (Table 3). Insufficient data were available to accurately determine ovary, muscle, femur bone marrow, eye, and pooled lymph node AUEC exposure profiles. ) digestive system, c) urinary system, d) muscoskeletal system, e) nervous system, f) reproductive system, g) immune system, h) respiratory system. Protein was detected for at least 72 hours in the majority of tissues, and the longest half-life for protein was observed in the lungs, at 121 hours. The protein T 1/2 in liver was 35.3 hours.

Discussion
The technology behind mRNA-based medicines has developed rapidly over the last decade and, in the face of the COVID-19 pandemic, mRNA medicines have demonstrated their safety and effectiveness as vaccines (Polack et al., 2020;Baden et al., 2021). While there now exists a greater understanding regarding the utility of mRNA-based medicines, the biodistribution profiles of LNPs and their constituents as well as the drivers influencing their biodistribution are less well understood. With the development of novel therapeutic and prophylactic mRNA-LNP modalities, many of which incorporate xenobiotic constituents, an in-depth biodistribution profile analysis is necessary to understand the in vivo fates and factors that influence the in vivo exposure of these medicines and their constituents. These data will inform the evolving clinical evaluation strategy as well as providing insight into future mRNA-LNP product development. In this study, we showed that after intravenous administration of Lipid 5, a xenobiotic amino lipid used in LNP formulations rapidly distributes throughout rat tissues in vivo, with exposure directed primarily toward the liver, spleen, lung, and kidneys. In addition, we showed that Lipid 5 and its [ 14 C]metabolites are undetectable in most tissues within 7 days (168 hours). Our findings showed similar distribution profiles in both male and female, pigmented and nonpigmented rats, suggesting minimal influence of sex or pigmentation in exposure of Lipid 5 and its [ 14 C]metabolites. The findings support the use of non-radioactive analyses in rat biodistribution models to assess the efficacy and safety of Lipid 5-containing LNPs in a preclinical setting.
The tissue distribution analysis using [ 14 C]Lipid 5-containing LNPs showed rapid distribution of Lipid 5 and its [ 14 C]metabolites throughout all tissues tested within the first hour, localizing primarily in the digestive and urinary systems (Figs. 2 and 3). Ten hours after intravenous LNP administration, the majority of the remaining radioactivity was eliminated from the peripheral tissues and was concentrated primarily in the digestive system (intestines, intestinal lumen, and other organs that facilitate metabolism and excretion of xenobiotics, such as the liver (Kok-Yong and Lawrence, 2015). Notably, the levels observed at the 10-hour timepoint were much lower than that of the same tissues 1 hour after the [ 14 C]Lipid 5-containing LNP was administered. By 24 hours, the majority of radioactivity was exclusively distributed within the digestive system, with minimal 14 C detected in other organ systems. The localization and progression over time of 14 C in the liver, small intestine, and large intestine is suggestive of hepatobiliary clearance with [ 14 C]Lipid 5 and its [ 14 C]metabolites excreted into bile and eliminated via feces (Kok-Yong and Lawrence, 2015); this predicted clearance pathway is similar to previous studies examining LNP clearance using fluorescent-dye-labeled LNPs (Press et al., 2014). The localization of 14 C in the kidneys and intra-bladder urine is also consistent with observation from other studies where the majority of radioactivity (62%) from labeled Lipid 5-containing LNPs administered to SD rats were excreted through urine, primarily as oxidative metabolites produced via ester dmd.aspetjournals.org hydrolysis and b-oxidation of Lipid 5 (Burdette et al., manuscript in press). Excluding the digestive and urinary system, other organs with high observed levels of radioactivity were the adrenal glands, lungs, spleen, and lymph nodes, which were consistent with biodistribution profiles previously reported for RNA encapsulating LNPs (Chen et al., 2019; https://www.ema.europa.eu/en/documents/assessment-report/comirnatyepar-public-assessment-report_en.pdf; Di et al., 2022). In female SD and LE rats, high concentrations of Lipid 5 were observed in the ovaries, but with insufficient duration of exposure to calculate an AUC. This observed exposure could potentially be attributed to accumulation within the cumulus cell layers surrounding the oocytes, as has been reported for nanoparticle delivery systems previously (Hou and Zhu, 2017); however, detailed analysis is needed to confirm that hypothesis. Excluding reproductive organs, similar trends in 14 C biodistribution were observed between male and female rats, with differences primarily occurring within the first 10 hours after administration of the LNPs. The study did not find high or increasing concentrations of 14 C in non-excretory systems post initial administration in male and female SD and LE rats, which suggests that both [ 14 C]Lipid 5 and its [ 14 C]metabolites are rapidly eliminated rather than extensively redistributed throughout the body, consistent with observations from other studies (Caldwell et al., 1995). The lack of an observed difference in 14 C biodistribution between pigmented (LE) and nonpigmented (SD) rats suggests a minimal influence of endogenous pigmentation such as melanin on intravenously administered Lipid 5 exposure. These findings are expected to be translatable to Lipid 5-containing LNP in vivo exposure studies in other animal species. Biodistribution analysis of Lipid 5 by using LC-MS/MS was consistent with biodistributions from the QWBA studies. Lipid 5 and mRNA exposure after administration of mRNA-LNPs concentrated primarily within the plasma, liver, spleen, lungs, and kidney, with the most rapid clearance occurring in the plasma, The longest duration of exposure was in the spleen, consistent with previous reports for mRNA-encapsulating LNPs (Lee et al., 2010;Bahl et al., 2017). As described previously, the differential tissue uptake of intact LNPs into cells in combination with dissimilar tissue perfusion rates may provide an explanation for the observed inter-tissue exposure and timing differences (Currie, 2018). Also, the delay between peak protein expression (Table 3; Fig. 6) and peak Lipid 5 (Table 1; Fig. 4) and mRNA exposure (Table 2; Fig. 5) is consistent with the necessity for cellular uptake and endosomal release prior to target protein translation (Alfagih et al., 2020). Although these may be plausible explanations for the observations made, additional research is needed to test these mechanistic hypotheses.
Both QWBA and LC-MS/MS analyses performed on similar LNPs encapsulating different mRNAs, showed comparable biodistribution profiles for Lipid 5, even with QWBA analyses also including the presence of [ 14 C]metabolites. This demonstrates the applicability of standard non-radioactive methods for assessing LNP and LNP component tissue distribution for Lipid 5-containing LNPs. Since QWBA allows for the  dmd.aspetjournals.org quantification of labeled parent molecules and metabolites (Solon and Kraus, 2001), the similarities observed between QWBA (Lipid 5 and its [ 14 C]metabolites) and LC-MS/MS analysis (intact Lipid 5 only) suggest that [ 14 C]metabolite distribution and persistence in tissues do not differ greatly from that of the parent Lipid 5. Additionally, increasing concentrations of [ 14 C]Lipid 5 and its [ 14 C]metabolites were not observed following the initial increase post administration, suggesting that redistribution of the xenobiotic lipid, and its [ 14 C]metabolites, is not expected. This study also reported similar biodistribution profiles for two different mRNA constructs of similar sizes encapsulated into LNPs with the same lipid constituents, suggesting that the LNP composition rather than the mRNA component is primarily responsible for the biodistribution profile of these mRNA-LNPs. The different mRNA-LNPs delivered support the extrapolation of findings to other mRNA-LNPs of similar morphology and composition. However, this may differ for mRNA-LNPs of other sizes, surface charge, lipid composition, or surface modifications, such as antibodies, receptors, or ligands.
The modest sample size used for QWBA analyses, with only two animals (1 male and 1 female) assessed for each time point, is in alignment with the industry standard while also minimizing the use of study animals (Solon and Kraus, 2001). Regardless, consistency in the data obtained between individuals supports the robustness of the study design to investigate the in vivo analysis of Lipid 5-containing LNPs. This study contributes to the interpretation and design of preclinical pharmacology and toxicology studies with the potential of extrapolating findings for clinical study considerations (European Medicines Agency, 2021). The extrapolations are supported by established pharmacology and toxicology mechanisms that are shared between rodents and humans (European Medicines Agency, 2021). The similar Lipid 5 biodistribution profiles observed, regardless of pigmentation or sex, and lack of evidence of redistribution of LNP-derived Lipid 5 or its [ 14 C]metabolites demonstrate that the biodistribution strategy used within this study provides a clear understanding of systemic exposure and thus informs Lipid 5-based LNP clinical safety and drug-drug interactions. The rapid elimination of all Lipid 5-derived radioactivity from the body in combination with the mechanistic understanding of metabolism and clearance via multiple, ubiquitous, high-capacity systems as detailed in previous studies (Sabnis et al., 2018;Burdette et al., manuscript in press). These findings in combination with traditional cytochrome P450s enzyme superfamilies and drug transporter studies (Ci et al., manuscript in preparation) support the overall, low probability of drugdrug interactions with co-administered medications and thus informs the continued use of Lipid 5 therapeutic LNPs.
The results from this study demonstrate that Lipid 5-containing LNPs are distributed throughout tissues after initial intravenous administration and Lipid 5 is rapidly eliminated thereafter. The time course and radioactivity distribution patterns of [ 14 C]Lipid 5-containing LNPs, suggest that Lipid 5 and its [ 14 C]metabolites undergo elimination by both biliary and renal routes with no indication of redistribution or prolonged metabolite exposure. Furthermore, biodistribution profiles were consistent between pigmented and nonpigmented male and female rats, indicating that neither sex nor pigmentation influences Lipid 5 exposure. The findings of this in vivo preclinical study support the translatability of Lipid 5 exposure profiles in standard rat biodistribution studies to humans as a complement to appropriately designed safety studies due to shared absorption, distribution, metabolism, and excretion pathways.  ) digestive system, c) urinary system, d) muscoskeletal system, e) nervous system, f) reproductive system, g) immune system, h) respiratory system.