Lipid nanoparticle formulations containing ionized lipids with branched structures and uses thereof

Abstract

The present invention relates to an ionized lipid containing a lipid with a branched structure, a lipid nanoparticle formulation using the same, and uses thereof. The ionized lipid of the present invention is a biodegradable lipid material having a lipid structure with a branched heteroamine structure, and lipid nanoparticles using the same can deliver nucleic acid drugs and the like with high efficiency, and therefore can be effectively used in related technical fields such as mRNA vaccines and therapeutic agents.

Description

[Technical Field]

[0001] This application claims the benefit of Application No. 10-2022-0189895, filed December 29, 2022, and Application No. 10-2023-0156562, filed November 13, 2023, which are incorporated herein by reference in their entireties. The present invention relates to lipid nanoparticle formulations containing ionizable lipids with branched structures and uses thereof. [Background technology]

[0002] In the pharmaceutical industry, drug delivery systems (DDS) aim to efficiently deliver the required amount of drug while reducing side effects and maximizing its efficacy and effectiveness. DDS can generate economic benefits comparable to new drug development and is a highly successful and valuable core technology, aiming to improve the quality of patient care through efficient drug administration.

[0003] Nucleic acids such as siRNA and mRNA are substances that can control the expression of specific proteins in the body, and are attracting attention as important tools in the treatment of cancer, genetic diseases, infectious diseases, autoimmune diseases, etc. Because nucleic acids are anionic substances with large molecular weights, they are difficult to deliver directly into cells and are easily degraded by enzymes in the blood, so research to overcome these problems is actively being conducted.

[0004] To date, the primary method for delivering nucleic acids into cells has been to mix them with positively charged lipids or polymers (called lipid-DNA conjugates (lipoplexes) and polymer-DNA conjugates (polyplexes), respectively). Lipid-DNA conjugates are widely used at the cellular level because they bind to nucleic acids and effectively deliver them into cells. However, when injected locally into the body, they often cause inflammation, and when injected intravascularly, they accumulate primarily in first-pass organs such as the lungs, liver, and spleen.

[0005] The initially developed ionizable lipid, 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), had low gene transfer efficiency and required large amounts of siRNA to achieve therapeutic effects. Subsequently, approximately 300 ionizable lipids were screened in a mouse blood coagulation factor FVII model through structural modifications of the amine head group and linker, resulting in the development of ED. 50 An optimized ionizable lipid (Dlin-MC3-DMA) with an extremely low ionization rate of 0.005 mg / kg was selected [Acc. Chem. Res. 2019]. This lipid is approximately 1,000 times more effective than existing ionizable lipids and was used as the delivery vehicle for Onpattro®, the world's first siRNA therapeutic agent for hereditary amyloidosis (hATTR), which was approved by the FDA in 2018.

[0006] To combat the COVID-19 pandemic, Moderna and Pfizer developed the first mRNA lipid nanoparticle vaccine. Currently, vaccinations have been successfully administered in several countries, including the United States and Israel. The Moderna and Pfizer coronavirus vaccines use mRNA lipid nanoparticles. The ionizable lipid in the Moderna vaccine is SM-102 (Arbutus) {{9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate}}, while the ionizable lipid in the Pfizer vaccine is ALC-0315 (Genevant) {[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)}}.

[0007] Ionizable lipids have a tertiary amine head group whose ionization degree changes depending on the body's pH and a long hydrocarbon tail connected by a linker. These lipids can then be formulated with other components to form solid lipid nanoparticle structures encapsulating nucleic acid drugs. The ionizable lipids are cationic and surround nucleic acid drugs during the low-pH formulation process. At physiological pH, they have a neutral surface charge, forming lipid nanoparticles through hydrophobic interactions. Lipid nanoparticles taken up into endosomes by endocytosis become cationic again when the endosomal pH drops, allowing them to interact with anionic lipids in the endosomal membrane and release the encapsulated nucleic acid drugs into the cell via endosomal escape.

[0008] As prior art, Korean Patent Publication No. 10-2020-0040586 discloses the preparation of ionizable lipids containing a six-membered heterocyclic amine and 1,2-epoxydodecane into lipid nanoparticles for in vivo drug delivery, and Korean Patent Publication Nos. 10-2022-0103968 and 10-2022-0103968 disclose various ionizable lipid and lipid nanoparticle compositions.

[0009] Therefore, the present inventors have made extensive efforts to develop novel particles that have excellent drug encapsulation efficiency and can efficiently deliver anionic drugs, nucleic acids, etc. to target organs or cells. As a result, they prepared lipid nanoparticles containing ionized lipids with a branched structure, and confirmed that these lipid nanoparticles can encapsulate nucleic acid drugs with high efficiency and induce immunity through the encapsulated nucleic acids, thereby completing the present invention. Summary of the Invention

[0010] It is an object of the present invention to provide novel ionizable lipid compounds having branched structures. Another object of the present invention is to provide lipid nanoparticles comprising said ionizable lipid compounds. Another object of the present invention is to provide a drug delivery composition comprising the lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof.

[0011] Another object of the present invention is to provide a use of the composition for a drug delivery system comprising the lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof.

[0012] Another object of the present invention is to provide a drug delivery method comprising the step of administering to a subject the drug delivery composition comprising the lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof.

[0013] In order to achieve the above object, the present invention provides an ionizable lipid compound represented by the following formula 1, or a pharmaceutically acceptable salt thereof. [ka] [In the formula, R1 and R2 are each independently -H, -C 1-10 Alkyl, -Y, or -C 1-10 Alkyl-NR A R B is one selected from R3 and R4 are each independently -H, -C 1-10 Alkyl, -Y, or -C 1-10 Alkyl-NR A R B or are bonded to each other to form a 4- to 8-membered heterocycloalkyl containing two N atoms, However, at least one of R1 to R4 is -C 1-10 Alkyl-NR A R B and R A and R B are each independently -H, -C 1-10 alkyl, or -Y; n is an integer from 0 to 6, at least one -Y substituent is present; Y is expressed by the following [Equation 2], When more than one Y is present, they may be different; [ka] o, p, and q each independently represent any integer from 1 to 12.]

[0014] The present invention also provides use of an ionizable lipid compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of lipid nanoparticles for drug delivery. The present invention also provides use of an ionizable lipid compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug delivery system comprising lipid nanoparticles.

[0015] The present invention also provides lipid nanoparticles comprising an ionizable lipid compound or a pharmaceutically acceptable salt thereof. The present invention also provides a drug delivery composition comprising lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof.

[0016] The present invention also provides the use of a composition comprising lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof as a drug delivery system. The present invention also provides a drug delivery method, comprising the step of administering to a subject a drug delivery composition comprising the lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof. [Effects of the Invention]

[0017] The present invention relates to an ionized lipid containing a lipid with a branched structure, a lipid nanoparticle formulation using the same, and uses thereof. The ionized lipid of the present invention is a biodegradable lipid material having a lipid structure with a branched heteroamine structure, and lipid nanoparticles using this ionized lipid can deliver nucleic acid drugs and the like with high efficiency, and therefore can be effectively used in related technical fields such as mRNA vaccines and therapeutic agents. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the synthesis process of novel ionizable lipids with branched structures. [Figure 2] FIG. 2 shows the MS spectrum results for confirming the synthesis of ionizable lipids. [Figure 3] FIG. 3 is a graph showing the luminescence gene expression levels and cytotoxicity of ionizable lipid nanoparticles in Hela cells. [Figure 4] FIG. 4 is a graph showing the luminescence gene expression levels and cytotoxicity of ionizable lipid nanoparticles in HEK 293 cells. [Figure 5] FIG. 5 is a graph showing AST levels in mouse blood after administration of EW244-E-7 lipid nanoparticles. [Figure 6] FIG. 6 is a graph showing ALT levels in mouse blood after administration of EW244-E-7 lipid nanoparticles. [Figure 7] FIG. 7 shows the bioluminescence of mFluc-loaded EW221-E-7 lipid nanoparticles intravenously injected into mice. [Figure 8] FIG. 8 shows the bioluminescence of mFluc-loaded EW244-E-7 lipid nanoparticles intravenously injected into mice. [Figure 9] FIG. 9 shows the bioluminescence of mFluc-loaded EW221-E-7 lipid nanoparticles injected intramuscularly into mice. [Figure 10] FIG. 10 shows the bioluminescence of mFluc-loaded EW244-E-7 lipid nanoparticles injected intramuscularly into mice. [Figure 11] FIG. 11 is a graph showing the expression levels of EPO and MCP-1 after injection of EW244-E-7 lipid nanoparticles loaded with hEPO mRNA into mice. [Figure 12] FIG. 12 is a graph showing the expression levels of MCP-1 after mice were injected with EW244-E-7 lipid nanoparticles loaded with hEPO mRNA. [Figure 13]Figure 13 is a graph showing neutralizing antibody titers after injection of mice with EW244-E-7 lipid nanoparticles loaded with COVID-19 spike mRNA. [Figure 14] Figure 14 is a graph showing IFN-gamma induction after injection of mice with EW244-E-7 lipid nanoparticles loaded with COVID-19 spike mRNA. [Figure 15] Figure 15 is a graph showing neutralizing antibody titers against the Wuhan strain (S) after injecting mice with lipid nanoparticles loaded with in-silico spike mRNA. [Figure 16] FIG. 16 is a graph showing neutralizing antibody titers against the Indian strain (Delta) after injection of mice with lipid nanoparticles loaded with in-silico spike mRNA. [Figure 17] FIG. 17 is a graph showing neutralizing antibody titers against Omicron (BA.5) after injection of mice with lipid nanoparticles loaded with in-silico spiked mRNA. [Figure 18] FIG. 18 is a graph showing IFN-gamma induction after injection of mice with lipid nanoparticles loaded with in-silico spiked mRNA. [Figure 19] FIG. 19 is a graph showing IgG antibody titers after injection of mice with EW244-E-7 lipid nanoparticles loaded with mRNA encoding RSV. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below. The present invention provides an ionizable lipid compound represented by the following formula 1, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R1 and R2 are each independently -H, -C 1-10 Alkyl, -Y, or -C 1-10 Alkyl-NR A R Bis one selected from R3 and R4 are each independently -H, -C 1-10 Alkyl, -Y, or -C 1-10 Alkyl-NR A R B or are bonded to each other to form a 4- to 8-membered heterocycloalkyl containing two N atoms, However, at least one of R1 to R4 is -C 1-10 Alkyl-NR A R B and R A and R B are each independently -H, -C 1-10 alkyl, or -Y; n is an integer from 0 to 6, at least one -Y substituent is present; Y is expressed by the following [Equation 2], When more than one Y is present, they may be different; [ka] o, p, and q each independently represent any integer from 1 to 12.]

[0020] In another embodiment of the present invention, In the above formula 1, R1 and R2 are each independently -H, -C 1-6 Alkyl, -Y, or -C 1-6 Alkyl-NR A R B is one selected from R3 and R4 are each independently -H, -C 1-6 Alkyl, -Y, or -C 1-6 Alkyl-NR A R B or are bonded to each other to form a 4- to 8-membered heterocycloalkyl containing two N atoms, However, at least one of R1 to R4 is -C 1-6 Alkyl-NR A RB and R A and R B are each independently -H, -C 1-6 alkyl, or -Y; n is an integer from 0 to 4, there are 2 to 6 -Y substituents; Y is expressed by the following [Equation 2]: Each Y can be different; [ka] o and p each independently represent an integer of 1 to 9; q is an integer of 1 to 5.

[0021] In another embodiment of the present invention, In the above formula 1, R1 and R2 are each independently -H, -C 1-4 Alkyl, -Y, or -C 1-4 Alkyl-NR A R B is one selected from R3 and R4 are each independently -H, -C 1-4 Alkyl, -Y, or -C 1-4 Alkyl-NR A R B or are bonded to each other to form a 5- to 6-membered heterocycloalkyl containing two N atoms, However, at least one of R1 to R4 is -C 1-4 Alkyl-NR A R B and R A and R B are each independently -H, -C 1-4 alkyl, or -Y; n is an integer of 1 to 2, there are 3 to 6 -Y substituents; Y is expressed by the following [Equation 2]: Each Y can be different; [ka] o and p each independently represent an integer of 1 to 9; q is an integer of 1 to 5.

[0022] In another embodiment of the present invention, In the above formula 1, R1 and R2 each independently represent -H, -C 1-4 Alkyl, -Y, or -C 1-4 Alkyl-NR A R B is one selected from R3 and R4 are each independently -H, -C 1-4 Alkyl, -Y, or -C 1-4 Alkyl-NR A R B or are bonded to each other to form a 5- to 6-membered heterocycloalkyl containing two N atoms, However, at least one of R1 to R4 is -C 1-4 Alkyl-NR A R B and R A and R B are each independently -H, -C 1-4 alkyl, or -Y; n is an integer of 1 to 2, there are 3 to 6 -Y substituents; Y is expressed by the following [Equation 2]: Each Y can be different; [ka] o and p each independently represent an integer of 3 to 9; q is an integer of 1 to 3.

[0023] In another embodiment of the present invention, the compound may be selected from the following compounds: [ka]

[0024] The ionizable lipid refers to an amine-containing lipid that can be easily protonated, for example, a lipid that changes charge state depending on the ambient pH. The ionizable lipids may be protonated (positively charged) at a pH below the pKa of the cationic lipid and substantially neutral at a pH above the pKa.

[0025] In the present invention, ionizable lipids are ionizable compounds with lipid-like properties that serve to efficiently encapsulate drugs (e.g., anionic drugs and / or nucleic acids) into lipid nanoparticles via electrostatic interactions with the drugs.

[0026] The ionizable lipid of the present invention can be used in the form of a pharmaceutically acceptable salt, and preferably, the salt is an acid addition salt with a pharmaceutically acceptable free acid.Inorganic and organic acids can be used as free acids.Usable inorganic acids include hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid, and usable organic acids include citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, and aspartic acid.

[0027] The ionizable lipids according to the present invention include not only pharmaceutically acceptable salts, but also all salts, isomers, hydrates, and solvates that can be prepared by conventional methods.

[0028] The ionizable lipid compound having a branched structure of the present invention has excellent drug delivery efficiency because it migrates to the cell membrane and disrupts the structure of the cell membrane, thereby promoting endosomal escape, or because the branched structure can reduce the rate of ester hydrolysis or enzymatic degradation.

[0029] The present invention also provides lipid nanoparticles comprising an ionizable lipid compound or a pharmaceutically acceptable salt thereof. The lipid nanoparticles may further comprise phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates.

[0030] The phospholipids act to encapsulate and protect the core formed by the interaction of the ionizable lipid and the drug within the lipid nanoparticles, and bind to the phospholipid bilayer of the target cells, facilitating crossing of the cell membrane and escape from the endosome for intracellular delivery of the drug.

[0031] Phospholipids may be used without limitation as long as they can promote fusion of lipid nanoparticles according to the embodiments. For example, phospholipids include dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg yolk phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), ... The phospholipid may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], etc. Specifically, the phospholipid is DOPE, and lipid nanoparticles containing DOPE may be effective for mRNA delivery (excellent drug delivery efficiency for mRNA).

[0032] Cholesterol provides morphological rigidity to the lipid charges within the lipid nanoparticles and is distributed in the core and on the surface of the nanoparticles, enhancing the stability of the nanoparticles. Lipid-PEG (polyethylene glycol) conjugates, lipid-PEG, PEG-lipid, or lipid-PEG refer to a conjugated lipid and PEG, meaning a lipid with a hydrophilic polymer, polyethylene glycol (PEG), attached to one end. Lipid-PEG conjugates contribute to the stability of nanoparticles within lipid nanoparticles in serum and prevent aggregation between nanoparticles. Lipid-PEG conjugates also protect nucleic acids from degradative enzymes during in vivo delivery, thereby enhancing the stability of nucleic acids in the body and potentially extending the half-life of drugs encapsulated in nanoparticles.

[0033] In lipid-PEG conjugates, PEG can be directly conjugated to the lipid or can be linked to the lipid via a linker moiety. Any linker moiety suitable for linking PEG to a lipid can be used, including, for example, ester-free and ester-containing linker moieties. Ester-free linker groups include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, etc., or a combination thereof (e.g., a linker containing both a carbamate linker moiety and an amide linker moiety). Ester-containing linker moieties include, but are not limited to, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0034] The lipid in the lipid-PEG conjugate can be any lipid that can be bound to polyethylene glycol, and can also be a phospholipid and / or cholesterol, which are other components of lipid nanoparticles. Specifically, the lipid in the lipid-PEG conjugate can be ceramide, dimyristoylglycerol (DMG), succinoyldiacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), cholesterol, or the like, and more specifically, C16-PEG2000 ceramide.

[0035] PEG in lipid-PEG conjugates is a hydrophilic polymer that inhibits plasma protein adsorption, prolongs the circulation time of lipid nanoparticles, prevents aggregation, and acts as a stealth mechanism in vivo, preventing nanoparticle degradation.

[0036] The lipid nanoparticles may contain ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate in a molar ratio of 15-35:15-35:40-60:0.1-5, preferably 25-40:10-25:40-60:0.5-3, and more preferably 25-30:17-22:50-55:1-2.

[0037] The present invention also provides a drug delivery composition comprising lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof. The anionic drug may be at least one selected from the group consisting of a peptide, a protein drug, a protein-nucleic acid construct, and an anionic biopolymer-drug conjugate.

[0038] The nucleic acid may be at least one selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), guide ribonucleic acid (gRNA), single-stranded guide ribonucleic acid (sgRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

[0039] The drug delivery composition may contain a physiologically active substance, such as an anionic drug and / or a therapeutic nucleic acid, encapsulated in lipid nanoparticles, and the physiologically active substance, such as an anionic drug and / or a therapeutic nucleic acid, is stably and efficiently encapsulated in the lipid nanoparticles, thereby enabling the delivery composition to exhibit excellent therapeutic effects. Another advantage is that the type of drug encapsulated in the lipid nanoparticles can be changed depending on the therapeutic purpose.

[0040] The lipid nanoparticles may have anionic drugs and / or nucleic acids encapsulated therein. The lipid nanoparticles encapsulated with anionic drugs and / or nucleic acids are the same as those described for the lipid nanoparticles above.

[0041] The weight ratio of the ionizable lipid to the drug (anionic drug, nucleic acid, or a combination thereof) contained in the lipid nanoparticles can be 1 to 20:1, preferably 1 to 15:1, 1 to 10:1, and more preferably 7.5 to 10:1.

[0042] In the present invention, the drug delivery composition can be used as a pharmaceutical composition for the prevention and treatment of diseases. The pharmaceutical composition may be administered systemically or locally, specifically by an administration route selected from the group consisting of intradermal, subcutaneous, intramuscular, intraocular, intraarticular, intracerebroventricular, intraspinal, oral, intravenous, intratracheal, intraperitoneal, intranasal, intrauterine administration, or any combination thereof.

[0043] The pharmaceutical composition is administered in a pharmacologically effective amount. In the present invention, the term "pharmacologically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical care. The effective dose level can be determined based on factors including the type and severity of the disease, drug activity, drug sensitivity, administration timing, administration route and excretion rate, treatment duration, concomitant medications, and other factors well known in the medical field. The pharmaceutical composition according to one embodiment can be administered as a sole therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and in a single dose or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves maximum efficacy with the minimum amount without side effects, which can be easily determined by those skilled in the art. Specifically, the pharmacologically effective amount according to the present invention varies depending on the patient's age, sex, and weight, and can be administered daily, every other day, or in divided doses 1 to 3 times daily. However, the dosage can be increased or decreased depending on the administration route, degree of obesity, sex, weight, age, etc., and the dosage does not limit the scope of the present invention.

[0044] The present invention also provides the use of a composition comprising lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof as a drug delivery system. Furthermore, the present invention provides a drug delivery method comprising the step of administering to a subject a drug delivery composition comprising lipid nanoparticles and an anionic drug, a nucleic acid, or a combination thereof.

[0045] Subjects include, but are not limited to, all mammals, including humans, non-human primates, dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, rats, and mice. Administration can be systemic or local, and may be selected from the group consisting of intradermal, subcutaneous, intramuscular, intraocular, intraarticular, intracerebroventricular, intraspinal, oral, intravenous, intratracheal, intraperitoneal, intranasal, intrauterine, or any combination thereof.

[0046] In specific examples and experimental examples, the inventors synthesized ionizable lipids with biodegradable functional groups introduced into the heteroamine structure (see Figure 1) and confirmed that the ionizable lipids could be successfully synthesized (see Figure 2). Furthermore, lipid nanoparticles encapsulating nucleic acids were prepared by dissolving cholesterol, phospholipids, and lipid-PEG conjugates in the synthesized ionizable lipids and mixing the lipids with mRNA at a volume ratio of 10:1. The prepared nucleic acid-encapsulated lipid nanoparticles exhibited favorable particle size and uniformity for drug release, as well as excellent drug encapsulation efficiency.

[0047] Next, we measured the luminescence of the lipid nanoparticles loaded with mFluc and confirmed that they had excellent intracellular nucleic acid delivery efficiency and no cytotoxicity (Figures 3 and 4). Furthermore, we administered the lipid nanoparticles to mice and confirmed that they did not cause liver toxicity in the animals (Figures 5 and 6). Furthermore, we confirmed that the lipid nanoparticles loaded with mFluc were delivered to the liver by intravenous injection in mice (Figures 7 and 8) and to the injection site by intramuscular injection (Figures 9 and 10), demonstrating that the lipid nanoparticles are suitable for in vivo drug delivery. Furthermore, we administered hEPO mRNA-loaded lipid nanoparticles to mice and confirmed that they were suitable for in vivo protein expression and had excellent early immune induction ability (Figures 11 and 12). Furthermore, when lipid nanoparticles carrying COVID-19 mRNA or respiratory syncytial virus-encoding mRNA were delivered to mice, increases in neutralizing antibody titers, IFN-gamma secretion, and IgG titers were observed (Figures 13-19), confirming that the lipid nanoparticles induce humoral or cellular immune responses.

[0048] The above results indicate that the lipid nanoparticles containing the ionized lipids having a branched structure of the present invention can deliver nucleic acid gene therapy drugs and vaccines with high efficiency and can be effectively used in lipid nanoparticle-mediated mRNA vaccines, gene therapy, and other related technical fields. [Example]

[0049] Example 1: Synthesis of ionizable lipids <1-1> Synthesis of ionizable lipids Ionizable lipids with branched structures were synthesized by introducing biodegradable functional groups into the heteroamine structure.

[0050] Specifically, 9-bromononanoic acid, DIC (1.5 equiv.), and DMAP (0.2 equiv.) were added to a DCM solvent containing 3-octanol and reacted overnight at 25 °C (Figure 1). The reaction mixture was then purified using a CombiFlash column in hexane / ethyl acetate (5:1 v / v). The solvent was evaporated, and the product was dissolved in ethanol. Next, DIPEA (1 equiv.) and an amine (0.3 equiv.) with the structural formula shown in Table 1 below were added, and the reaction mixture was reacted at 25 °C for 3 days. The reaction mixture was purified using a CombiFlash column in DCM / MeOH (9:1 v / v). This afforded novel ionizable lipids containing various amine head groups and ester linkages. The resulting ionizable lipids were named EW221-E-7, EW244-E-7, and EW246-E-7, depending on the amine type. [Table 1]

[0051] <1-2> Confirmation of ionizable lipid synthesis To confirm the ionizable lipid synthesized in Example <1-1> above, nuclear magnetic resonance analysis (NMR spectroscopy) was carried out. Specifically, 5 μg of the ionizable lipid (EW244-E-7) synthesized in Example <1-1> was diluted with 0.5 mL of CDCl (Sigma, USA) to a concentration of 100 mM. Next, 0.5 mL of the lipid solution was added to a 400 MHz NMR tube, capped and sealed with parafilm, and the NMR spectrum was measured using an Agilent 400 MHz FT-NMR (Agilent, USA).

[0052] As a result, as shown in Figure 2, it was found that the signals representing each functional group of EW244-E-7 were saturated. Furthermore, mass spectrometry (MS) was performed to identify the synthesized ionizable lipid (EW244-E-7).

[0053] Specifically, ionizable lipids were diluted with ethanol to a concentration of 0.5 ppm or less and analyzed on a 6230 LC / MS (Agilent Technologies, Palo Alto, CA, USA) using a Zorbax SB-C18 separation column (Agilent Technologies, 100 mm × 2.1 mm id, 3.5 μm). As a result, as shown in Table 2, it was confirmed that the measured mass-to-charge ratio (m / z) of the ionizable lipid (EW244-E-7) was almost identical to the calculated mass-to-charge ratio. [Table 2] From the above results, it was confirmed that ionizable lipids were successfully synthesized.

[0054] [Example 2] Preparation of lipid nanoparticles <2-1> Preparation of lipid nanoparticles carrying nucleic acids The ionizable lipid (EW244-E-7) synthesized in Example 1-1, phospholipid (DOPE) (Avanti, USA), cholesterol (cholesterol powder, BioReagent, cell culture-compatible, ≥99%, Sigma, Korea), and lipid-PEG conjugate (C16-PEG2000 ceramide (Avanti, USA)) were dissolved in ethanol at a molar ratio of 26.5:20:52:1.5 (Table 3). mRNA was dissolved in 10 mM sodium citrate (Sigma, Korea) buffer. The ethanol containing the ionizable lipid, cholesterol, phospholipid, and lipid-PEG and the citrate buffer were mixed at a volume ratio of 1:3 through a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) at a flow rate of 12 mL / min to prepare nucleic acid-loaded lipid nanoparticles (LNPs). [Table 3] [Table 4]

[0055] <2-2> Physicochemical properties of nucleic acid-loaded lipid nanoparticles <2-2-1> Particle size measurement The size of the mRNA-carrying lipid nanoparticles synthesized in Example <2-1> above was measured. Specifically, the firefly luciferase mRNA (mFluc, sequence number 1) contained in the EW244-E-7 lipid nanoparticles synthesized in Example <2-1> above was diluted with PBS to a concentration of 1 μg / ml, and the particle size, polydispersity index (PDI), and surface charge (zeta potential) of the lipid nanoparticles were measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano (Malvern Instruments, UK). As a result, as shown in Table 5, it was confirmed that the EW244-E-7 lipid nanoparticles exhibited a particle size that was excellent for drug release, and the particles were uniform.

[0056] <2-2-2> Measurement of drug encapsulation efficiency To measure the nucleic acid drug encapsulation efficiency, Ribogreen assay was performed. Specifically, in the Ribogreen assay (Quant-iT™ RiboGreen® RNA, Invitrogen), lipid nanoparticles encapsulating nucleic acid drugs were diluted with 50 μL of 1xTE buffer to a final RNA concentration of 4–7 μg / mL in a 96-well plate. 50 μL of 1xTE buffer was added to the Triton-X-untreated group (Triton-x LNP(-)), and 50 μL of 2% Triton-X buffer was added to the Triton-X-treated group (Triton-x LNP(+)). The mixture was incubated at 37°C for 10 minutes to dissociate the lipid nanoparticles with Triton-X, releasing the encapsulated nucleic acids. 100 μL of Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNP(-) and Triton LNP(+) was measured using an Infinite® 200 PRO NanoQuant (Tecan) at wavelengths of 485 nm (excitation) and 528 nm (emission). The drug encapsulation efficiency (%) was calculated as follows.

[0057] Drug encapsulation efficiency (%) = (fluorescence intensity of Triton LNP(+) - fluorescence intensity of Triton LNP(-)) / (fluorescence intensity of Triton LNP(+)) × 100

[0058] As a result, as shown in Table 5, it was confirmed that EW244-E-7 lipid nanoparticles can encapsulate drugs with high efficiency. [Table 5]

[0059] [Experimental Example 1: Intracellular nucleic acid delivery and cytotoxicity of lipid nanoparticles] Using the mFluc-encapsulated lipid nanoparticles prepared in Example <2-1>, experiments were carried out to confirm the intracellular nucleic acid delivery efficiency and cytotoxicity. Specifically, Hela cells or HEK293 cells were treated with 20 ng of mFluc-encapsulated lipid nanoparticles, and luminescence was measured 24 hours later.

[0060] As a result, as shown in Figure 3, EW244-E-7 lipid nanoparticles showed a higher expression effect in HeLa cells than other lipid nanoparticles. Furthermore, as shown in Figure 4, high expression effects of EW221-E-7 and EW244-E-7 lipid nanoparticles were confirmed in HEK293 cells. It was also confirmed that EW221-E-7 and EW244-E-7 lipid nanoparticles have almost no toxicity.

[0061] [Experimental Example 2: Confirmation of hepatotoxicity of lipid nanoparticles] The mFluc-encapsulated lipid nanoparticles prepared in Example <2-1> were administered to mice to conduct an experiment to confirm toxicity in animals.

[0062] AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are values ​​that can measure the presence of diseases such as hepatocellular disease and hepatitis. While these enzymes are normally present in low concentrations in the blood, they can be released when liver cells are damaged, resulting in elevated blood concentrations. Therefore, they can be used as indicators of liver toxicity.

[0063] Specifically, mRNA-encapsulated lipid nanoparticles were intravenously administered to 7-week-old C57BL / 6 mice at a dose of 2 mg / kg of mRNA. Blood was collected 24 hours after administration, and blood AST and ALT concentrations were measured. As controls, SM-102, an ionized lipid used in the Moderna vaccine, and ALC-0315, an ionized lipid used in the Pfizer vaccine, were used. [Table 6]

[0064] As a result, as shown in Table 6, Figures 5 and 6, mice administered with EW244-E-7 lipid nanoparticles showed lower levels of liver toxicity compared to mice administered with Moderna's SM-102 or Pfizer's ALC-0315, suggesting that EW244-E-7 lipid nanoparticles are very safe.

[0065] [Experimental Example 3: Confirmation of in vivo expression of lipid nanoparticles] <3-1> Delivery of lipid nanoparticles via intravenous injection First, we prepared lipid nanoparticles encapsulating mFluc and confirmed their physicochemical properties. As a result, as shown in Table 7, the size of the EW221-E-7 and EW244-E-7 lipid nanoparticles encapsulating mFluc was confirmed to be efficient for drug delivery, the particles were uniform, and the drug encapsulation effect was excellent. [Table 7]

[0066] Next, the mFluc-loaded lipid nanoparticles were intravenously injected into mice, and bioluminescence was observed to confirm the drug delivery efficiency in vivo. Specifically, 7-week-old C57BL / 6 mice were intravenously injected with 2 μg of EW221-E-7 and EW244-E-7 lipid nanoparticles encapsulating mFluc, and 3 hours later, luciferin was administered intraperitoneally at 0.25 mg / kg. Bioluminescence was confirmed using an IVIS (PerkinElmer, USA) device.

[0067] As a result, as shown in Figures 7 and 8, it was confirmed that the majority of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the liver. These results suggest that intravenous administration of lipid nanoparticles is possible for drug delivery in vivo.

[0068] <3-2> Delivery of lipid nanoparticles by intramuscular injection First, we prepared lipid nanoparticles encapsulating mFluc and confirmed their physicochemical properties. As a result, as shown in Table 8, the size of the EW221-E-7 and EW244-E-7 lipid nanoparticles encapsulating mFluc was confirmed to be efficient for drug delivery, the particles were uniform, and the drug encapsulation efficiency was excellent. [Table 8]

[0069] Next, the mFluc-loaded lipid nanoparticles were intramuscularly injected into mice, and bioluminescence was observed to confirm the drug delivery efficiency in vivo. Specifically, 7-week-old C57BL / 6 mice were intramuscularly injected with 2 μg of EW221-E-7 or EW244-E-7 lipid nanoparticles encapsulating mFluc, and 3 hours later, 0.25 mg / kg of luciferin was administered intraperitoneally. Bioluminescence was then confirmed using an IVIS (PerkinElmer, USA) device.

[0070] As a result, as shown in Figures 9 and 10, it was confirmed that the majority of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the injection site. These results suggest that intramuscular administration of lipid nanoparticles is possible for drug delivery in vivo.

[0071] [Experimental Example 4: Confirmation of the early immune induction ability of lipid nanoparticles] First, lipid nanoparticles encapsulating hEPO (human EPO) mRNA (SEQ ID NO: 2) were prepared, and as a control, the same mRNA was encapsulated in SM-102, an ionized lipid in the Moderna vaccine, and their physicochemical properties were compared (Table 9). [Table 9]

[0072] After administering hEPO mRNA-encapsulated nanolipid particles to mice, the blood human EPO and MCP-1 concentrations were measured to confirm early protein expression and early immune induction ability. Specifically, 7-week-old Balb / c mice were intravenously injected with 0.5 mg / kg of hEPO mRNA-encapsulated lipid nanoparticles. Six hours later, blood was collected for serum analysis, and hEPO and MCP-1 levels were quantitatively analyzed using hEPO ELISA kits and MCP-1 ELISA kits. SM-102, an ionized lipid contained in the Moderna vaccine, was used as a control.

[0073] As a result, as shown in Figures 11 and 12, when EW244-E-7 and SM-102 were administered, serum hEPO concentrations were similarly high, and the expression of MCP-1 induced by EPO was observed to be higher when EW244-E-7 was administered than when SM-102 was administered. These results suggest that EW244-E-7 lipid nanoparticles have high protein expression capacity and excellent early immune induction ability.

[0074] [Experimental Example 5: Confirmation of immune induction ability of lipid nanoparticles] <5-1> Confirmation of immunity induction ability against coronavirus First, we prepared lipid nanoparticles encapsulating COVID-19 spike mRNA (SEQ ID NO: 3) and confirmed their physicochemical properties. COVID-19 spike mRNA was provided by Seoul National University, and ALC-0315 was used as a positive control.

[0075] As a result, as shown in Table 10, the size of the EW244-E-7 lipid nanoparticles encapsulating COVID-19 spike mRNA was confirmed to be efficient for drug delivery, the particles were uniform, and the drug encapsulation effect was excellent. [Table 10]

[0076] Next, lipid nanoparticles encapsulating COVID-19 spike mRNA were delivered to mice, and neutralizing antibody titers and IFN-gamma levels were measured. Specifically, 7-week-old Balb / c mice were first given 0.25 mg / kg of mRNA intramuscularly. Three weeks later, a second dose was administered in the same manner. Three weeks after the second dose, serum samples were collected, and the spleens were removed. Antibody titers against SARS-CoV-2 and IFN-gamma levels were measured.

[0077] As a result, it was confirmed that EW244-E-7 lipid nanoparticles exhibited a neutralizing antibody titer equivalent to that of the positive control ALC-0315, as shown in Figure 13. Furthermore, it was confirmed that IFN-gamma secretion increased with increasing peptide concentration in EW244-E-7 lipid nanoparticles, as shown in Figure 14.

[0078] These results suggest that EW244-E-7 lipid nanoparticles can induce both humoral and cellular immune responses.

[0079] <5-2> Confirmation of the immune induction ability of a COVID-19 universal vaccine First, lipid nanoparticles encapsulating in-silico spike mRNA (SEQ ID NO: 4) were prepared. After administering the lipid nanoparticles encapsulating COVID-19 mRNA to mice, neutralizing antibody titers and IFN-gamma levels were measured.

[0080] Specifically, 7-week-old Balb / c mice were first given 0.25 mg / kg of mRNA by intramuscular injection. Three weeks later, a second dose was administered in the same manner. Three weeks after the second dose, serum was collected, and the spleens were removed. Antibody titers against SARS-CoV-2 and IFN-gamma levels were measured.

[0081] As a result, as shown in Figures 15 to 17, mice vaccinated with EW244-E-7 lipid nanoparticles were confirmed to exhibit similar neutralizing antibody titers against all COVID-19 strains, including the Wuhan strain (S), the Indian strain (Delta), and the Omicron strain (BA.5). Furthermore, as shown in Figure 18, high levels of IFN-gamma secretion were confirmed, confirming a vigorous T cell response.

[0082] These results suggest that EW244-E-7 lipid nanoparticles can induce both humoral and cellular immune responses and may be versatile against various COVID-19 strains.

[0083] <5-3> Confirmation of immune induction ability against cellular respiratory syncytial virus First, lipid nanoparticles encapsulating respiratory syncytial virus (RSV)-encoding mRNA (SEQ ID NO: 5) were prepared, and their physicochemical properties were confirmed.

[0084] As a result, as shown in Table 11, the size of the EW244-E-7 lipid nanoparticles encapsulating RSV-encoding mRNA was confirmed to be efficient for drug delivery, the particles were uniform, and the drug encapsulation efficiency was excellent. [Table 11]

[0085] Lipid nanoparticles loaded with RSV-encoding mRNA were then delivered to mice, and IgG titers were measured. Specifically, 7-week-old Balb / c mice were first administered 0.5 mg / kg of mRNA intramuscularly. Two weeks later, a second administration was administered in the same manner. One week after the second administration, serum samples were collected and RSV neutralizing antibody titers were measured. The positive control group [(+) ctrl.] received the adenovirus vector-based vaccine rAd / 3xGmFcm, and the negative control group [(-) ctrl.] received no vaccine.

[0086] As a result, as shown in FIG. 19, after the second administration, the group administered with EW244-E-7 lipid nanoparticles had a higher IgG titer than the positive control group. These results suggest that EW244-E-7 lipid nanoparticles have excellent immunity-inducing potential against cellular respiratory syncytial viruses.

Claims

1. An ionizable lipid compound represented by the following formula 1, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 [In the formula, R １ and R ２ are each independently —H, —C １－１０ Alkyl, -Y, or -C １－１０ Alkyl-NR Ａ R Ｂ is one selected from R ３ and R ４ are each independently —H, —C １－１０ Alkyl, -Y, or -C １－１０ Alkyl-NR Ａ R Ｂ or are bonded together to form a 4- to 8-membered heterocycloalkyl containing two N atoms, However, R １ ~R ４ At least one of the following is -C １－１０ Alkyl-NR Ａ R Ｂ and R Ａ and R Ｂ are each independently —H, —C １－１０ alkyl, or -Y; n is an integer from 0 to 6, at least one -Y substituent is present; Y is represented by the following formula 2: When multiple Y's are present, they may be different. 【Chemistry 2】

2. In Formula 1, R １ and R ２ are each independently —H, —C １－６ Alkyl, -Y, or -C １－６ Alkyl-NR Ａ R Ｂ is one selected from R ３ and R ４ are each independently —H, —C １－６ Alkyl, -Y, or -C １－６ Alkyl-NR Ａ R Ｂ or are bonded together to form a 4- to 8-membered heterocycloalkyl containing two N atoms, However, R １ ~R ４ At least one of the following is -C １－６ Alkyl-NR Ａ R Ｂ and R Ａ and R Ｂ are each independently —H, —C １－６ alkyl, or -Y; n is an integer from 0 to 4, there are 2 to 6 -Y substituents; Y is represented by the following formula 2: each Y can be different; 【Transformation 3】 o and p are each independently an integer from 1 to 9; q is an integer from 1 to 5; 10. The ionizable lipid compound of claim 1, or a pharmaceutically acceptable salt thereof.

3. In Formula 1, R １ and R ２ are each independently —H, —C １－４ Alkyl, -Y, or -C １－４ Alkyl-NR Ａ R Ｂ is one selected from R ３ and R ４ are each independently —H, —C １－４ Alkyl, -Y, or -C １－４ Alkyl-NR Ａ R Ｂ or are bonded to each other to form a 5- to 6-membered heterocycloalkyl containing two N atoms, However, R １ From R ４ At least one of them is -C １－４ Alkyl-NR Ａ R Ｂ and R Ａ and R Ｂ are each independently —H, —C １－４ alkyl, or -Y; n is an integer from 1 to 2, there are 3 to 6 -Y substituents; Y is represented by the following formula 2: each Y can be different; 【Chemistry 4】 o and p are each independently an integer from 1 to 9; q is an integer from 1 to 5; 10. The ionizable lipid compound of claim 1, or a pharmaceutically acceptable salt thereof.

4. In Formula 1, R １ and R ２ are each independently —H, —C １－４ Alkyl, -Y, or -C １－４ Alkyl-NR Ａ R Ｂ is one selected from R ３ and R ４ are each independently —H, —C １－４ Alkyl, -Y, or -C １－４ Alkyl-NR Ａ R Ｂ or are bonded to each other to form a 5- to 6-membered heterocycloalkyl containing two N atoms, However, R １ From R ４ At least one of the following is -C １－４ Alkyl-NR Ａ R Ｂ and R Ａ and R Ｂ are each independently —H, —C １－４ alkyl, or -Y; n is an integer from 1 to 2, there are 3 to 6 -Y substituents; Y is represented by the following formula 2: each Y can be different; 【Transformation 5】 o and p each independently represent an integer from 3 to 9; q is an integer from 1 to 3; 10. The ionizable lipid compound of claim 1, or a pharmaceutically acceptable salt thereof.

5. The compound is selected from the following compounds: 【Transformation 6】 10. The ionizable lipid compound of claim 1, or a pharmaceutically acceptable salt thereof.

6. A lipid nanoparticle comprising the ionizable lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5.

7. The lipid nanoparticle of claim 6, further comprising a phospholipid, cholesterol, or lipid-PEG (polyethylene glycol).

8. The lipid nanoparticle of claim 7, comprising an ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate in a molar ratio of 15-35:15-35:40-60:01-5.

9. The phospholipid is at least one selected from the group consisting of DOPE, DSPC, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, POPE, POPC, DOPS, and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]. The lipid nanoparticle according to claim 8.

10. The lipid nanoparticle of claim 8, wherein the lipid in the lipid-PEG conjugate is at least one selected from the group consisting of ceramide, dimyristoylglycerol (DMG), succinoyldiacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and cholesterol.

11. Ingredients: (1) The lipid nanoparticles according to claim 6; and (2) Anionic drugs, nucleic acids, or combinations thereof A drug delivery composition comprising:

12. The drug delivery composition according to claim 11, wherein the anionic drug is at least one selected from the group consisting of a peptide, a protein, a protein-nucleic acid construct, and an anionic biopolymer-drug conjugate.

13. The drug delivery composition according to claim 11, wherein the nucleic acid is at least one selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), guide ribonucleic acid (gRNA), single-stranded guide ribonucleic acid (sgRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

14. The drug delivery composition of claim 11, wherein the anionic drug, nucleic acid, or a combination thereof is encapsulated within a lipid nanoparticle.

15. The drug delivery composition of claim 11 , which is administered systemically or locally.

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