Novel ionizable lipid and lipid nanoparticle composition comprising same

Abstract

The present invention relates to a novel ionizable lipid and a lipid nanoparticle composition comprising same. Lipid nanoparticles formed from a novel ionizable lipid compound, according to the present invention, exhibit an excellent gene encapsulation rate and in vivo gene delivery rate, and moreover, exhibit an excellent gene delivery function even in a liver damage model, and thus may be usefully employed as a composition for drug delivery.

Description

Novel ionized lipid and lipid nanoparticle composition comprising the same

[0001] The present invention relates to a novel ionizable lipid and a lipid nanoparticle composition comprising the same.

[0002] This invention was researched as a project (S3368433) for "Development of a novel biodegradable ionized lipid material as a core carrier for lipid nanoparticle technology for gene delivery" of the Regional Specialized Industry Promotion+ (R&D) project hosted by the Small and Medium Business Technology Information Promotion Agency of the Ministry of SMEs and Startups.

[0003] Nucleic acid-based medicines are being used not only as therapeutic agents but also as preventative agents that protect against diseases by injecting genes capable of expressing antigens for specific diseases. Among these, gene-based vaccines are categorized as DNA vaccines, RNA vaccines, and viral vector vaccines. RNA vaccines, which inject mRNA encoding an antigen into the body, trigger antigen expression and antibody production. RNA vaccines do not have the potential risks of infection associated with viral vector-based vaccines or genetic mutation associated with DNA vaccines. Furthermore, they offer the advantage of rapid development, drawing attention as an effective response to the COVID-19 outbreak in 2019.

[0004] However, genes are easily broken down by nucleases in the human body and, as negatively charged macromolecules, are difficult to deliver into cells. Therefore, a stable and efficient method for delivering them to the desired location is needed. Various delivery methods based on materials such as lipids, polymers, dendrimers, and inorganic metals have been reported as gene delivery systems. Among these, lipid nanoparticles, as lipid delivery vehicles, were used in the development of patisiran, the first siRNA drug approved by the FDA in 2018, as well as the mRNA vaccine for COVID-19, which received emergency use authorization in 2020. Since then, lipid nanoparticle technology has been recognized for its technological value as a clinically useful delivery system for nucleic acid drugs such as siRNA and mRNA.

[0005] Current lipid nanoparticles are generally used in a form in which four components are mixed in a certain ratio: ionized lipid, phospholipid (helper lipid), cholesterol (structural maintenance lipid), and PEG-lipid. However, as the development of gene therapy for various indications is in full swing, unmet needs have been identified in various aspects of lipid nanoparticle-based carriers to date, such as enhanced gene delivery ability, maintenance of gene delivery ability in specific diseases such as liver disease, enhanced stability, improved targeting characteristics, ensuring ease of storage and distribution, mitigation of side effects, cost reduction, and response to breakthrough infections. Therefore, there is a continuous demand for the development of lipid nanoparticles using new ionized lipid carriers.

[0006] The purpose of the present invention is to provide a novel ionizable lipid compound.

[0007] Another object of the present invention is to provide a lipid nanoparticle composition comprising the compound.

[0008] Another object of the present invention is to provide a medical use of the lipid nanoparticle composition.

[0009] In order to achieve the above purpose, the present invention provides a compound selected from the compounds represented by the following chemical formula Ⅰ, pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof:

[0010] <Chemical Formula Ⅰ>

[0011]

[0012] In the above chemical formula Ⅰ, n is 0 or 1, L is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and in the above alkylene, alkenylene and alkynylene, at least one -CH2- is -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- is substituted or unsubstituted, and R 1 is one of hydrogen, (C1-C20)alkyl, (C2-C20)alkenyl and (C2-C20)alkynyl, and R 2 and R 3are each the same or different, and are one of 5 to 30 membered heteroaryls including one or more heteroatoms selected from the group consisting of (C5-C30)alkyl, (C5-C30)alkenyl, (C5-C30)alkynyl, (C3-C30)cycloalkyl, (C3-C30)cycloalkenyl, (C3-C30)cycloalkynyl, (C5-C30)aryl and N, O and S, and in the alkyl, alkenyl and alkynyl, one or more -CH2- is -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- may be substituted or unsubstituted.

[0013] The present invention provides a lipid nanoparticle composition comprising the above compound.

[0014] In addition, the present invention provides a drug delivery composition comprising the lipid nanoparticle composition described above; and a preventive or therapeutic agent.

[0015] The present invention relates to a novel ionized lipid compound and a lipid nanoparticle composition using the same. The lipid nanoparticle exhibits excellent gene encapsulation rate and in vivo gene transfer rate, and also exhibits excellent gene transfer ability in a liver damage model, and thus can be usefully utilized in the development of gene therapeutic agents for various indications.

[0016] Figure 1 illustrates a manufacturing process of compound 1 according to one embodiment of the present invention.

[0017] Figure 2 shows the manufacturing process of compound 2.

[0018] Figure 3 shows the manufacturing process of compound 3.

[0019] Figure 4 shows the manufacturing process of compound 4.

[0020] Figure 5 shows the manufacturing process of compound 5.

[0021] Figure 6 shows the manufacturing process of compound 6.

[0022] Hereinafter, the present invention will be described in detail.

[0023]

[0024] The present inventors synthesized a novel ionizable lipid compound and confirmed that a lipid nanoparticle composition prepared using the same can deliver genes into a living body with excellent efficiency, thereby completing the present invention.

[0025]

[0026] The present invention provides a compound selected from the group consisting of a compound represented by the following chemical formula Ⅰ, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof:

[0027] <Chemical Formula Ⅰ>

[0028]

[0029] In the above chemical formula Ⅰ, n is 0 or 1,

[0030] L is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and in said alkylene, alkenylene and alkynylene, one or more -CH2- is substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- or Non-substituted,

[0031] R 1is one of hydrogen, (C1-C20)alkyl, (C2-C20)alkenyl and (C2-C20)alkynyl,

[0032] R 2 and R 3 are each the same or different, and are one of 5 to 30 membered heteroaryls including one or more heteroatoms selected from the group consisting of (C5-C30)alkyl, (C5-C30)alkenyl, (C5-C30)alkynyl, (C3-C30)cycloalkyl, (C3-C30)cycloalkenyl, (C3-C30)cycloalkynyl, (C5-C30)aryl and N, O and S, and in the alkyl, alkenyl and alkynyl, one or more -CH2- is -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- may be substituted or unsubstituted.

[0033] Preferably, in the following chemical formula Ⅰ, L is one of (C1-C6)alkylene, (C2-C6)alkenylene and (C2-C6)alkynylene, and in the alkylene, alkenylene and alkynylene, at least one -CH2- is one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- Substituted, R 1 is hydrogen or (C1-C10)alkyl, and R 2 and R3 may be the same or different, and are one of (C10-C30)alkyl, (C10-C30)alkenyl and (C10-C30)alkynyl, and in said alkyl, alkenyl and alkynyl, one or more -CH2- is one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- Can be replaced.

[0034] Preferably, in the above chemical formula Ⅰ, L is (C2-C6)alkylene, and in the above alkylene, at least one -CH2- is substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O- and -OC(=O)O-, and R 1 is hydrogen or (C1-C5)alkyl, and R 2 and R 3 may be the same or different, and are one of (C10-C30)alkyl, (C10-C30)alkenyl and (C10-C30)alkynyl, and in the alkyl, alkenyl and alkynyl, one or more -CH2- may be substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -S- and -SS-.

[0035] More preferably, in the above formula I, L is (C2-C6)alkylene, and in the alkylene, one -CH2- is replaced with -OC(=O)- or -C(=O)O-, and R 1 is hydrogen or (C1-C5)alkyl, and R 2 and R 3may be the same or different, and are (C10-C30)alkyl, and one or two -CH2- in said alkyl may be substituted with one of -OC(=O)-, -C(=O)O-, -S- and -SS-.

[0036] Specifically, the compound may be selected from the following group of compounds:

[0037] (1) (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0038] (2) bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate) [bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate)];

[0039] (3) bis(2-butyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate;

[0040] (4) bis(2-hexyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate;

[0041] (5) bis(2-hexyldecyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate;

[0042] (6) (((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis (hexane-6,1-diyl)bis(2-hexyldecanoate) [(((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis (hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0043] (7) bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate;

[0044] (8) bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate;

[0045] (9) ((3-((3-(pyridin-4-ylamino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((3-((3-(pyridin-4-ylamino)propanoyl)oxy) propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0046] (10) ((3-((3-methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((3-((3-(methyl(pyridin-4-yl)amino) propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0047] (11) ((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azanediyl) bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0048] (12) (((pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(disulfanediyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)(2-hexyldecanoate) [(((pyridin-4-ylazanediyl)bis(hexane-6,1-diyl))bis(disulfanediyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)]; and

[0049] (13) Bis(2-(2-(hexyldisulfanyl)ethyl)octyl)6,6'-(pyridin-4-ylazanediyl) dihexanoate.

[0050]

[0051] More specifically, the above (1) (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (hereinafter, compound 1) has a structure as shown in the following chemical formula 1-1, and the molecular formula is C 49 H 90 It is N2O4 and its molecular weight is 771.25:

[0052] <Chemical Formula 1-1>

[0053]

[0054]

[0055] The above (2) bis(2-butyloctyl)10,10'-(pyridin-4-ylazanedyl)bis(decanoate) (hereinafter, compound 2) has a structure as shown in the following chemical formula 1-2, and the molecular formula is C 49 H 90 It is N2O4 and its molecular weight is 771.25:

[0056] <Chemical Formula 1-2>

[0057]

[0058]

[0059] The above (3) bis(2-butyloctyl)8,8'-(pyridin-4-ylazanedyl)dioctanoate (hereinafter, compound 3) has a structure as shown in the following chemical formula 1-3, and the molecular formula is C 45 H 82 It is N2O4 and its molecular weight is 715.14:

[0060] <Chemical Formula 1-3>

[0061]

[0062]

[0063] The above (4) bis(2-hexyloctyl)8,8'-(pyridin-4-ylazanedyl)dioctanoate (hereinafter, compound 4) has a structure as shown in the following chemical formula 1-4, and the molecular formula is C 49 H 90 It is N2O4 and its molecular weight is 771.25:

[0064] <Chemical Formula 1-4>

[0065]

[0066]

[0067] The above (5) bis(2-hexyldecyl)8,8'-(pyridin-4-ylazanedyl)dioctanoate (hereinafter, compound 5) has a structure as shown in the following chemical formula 1-5, and the molecular formula is C 53 H 98 It is N2O4 and its molecular weight is 827.36:

[0068] <Chemical Formula 1-5>

[0069]

[0070]

[0071] The above (6) (((2-(pyridin-4-ylamino)ethoxy)carbonyl)azane edyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (hereinafter, compound 6) has a structure as shown in the following chemical formula 1-6, and the molecular formula is C 52 H 95 It is N3O6 and its molecular weight is 858.33:

[0072] <Chemical Formula 1-6>

[0073]

[0074]

[0075] The above (7) bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanedyl)dihexanoate (hereinafter, compound 7) has a structure as shown in the following chemical formula 1-7, and the molecular formula is C 49 H 90 It is N2O4 and its molecular weight is 771.25:

[0076] <Chemical Formula 1-7>

[0077]

[0078]

[0079] The above (8) bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanedyl)dihexanoate (hereinafter, compound 8) has a structure as shown in chemical formula 1-8, and the molecular formula is C 45 H 82 It is N2O4 and its molecular weight is 715.14:

[0080] <Chemical Formula 1-8>

[0081]

[0082]

[0083] The above (9) ((3-((3-(pyridin-4-ylamino)propanoyl)oxy)propyl)azane ethyl)bis(hexane-6,1-ethyl)bis(2-hexyldecanoate) (hereinafter, compound 9) has a structure as shown in the following chemical formula 1-9, and the molecular formula is C 55 H 101 It is N3O6 and its molecular weight is 900.41:

[0084] <Chemical Formula 1-9>

[0085]

[0086]

[0087] The above (10) ((3-((3-methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azane ethyl)bis(hexane-6,1-ethyl)bis(2-hexyldecanoate) (hereinafter, compound 10) has a structure as shown in the following chemical formula 1-10, and the molecular formula is C56 H 103 It is N3O6 and its molecular weight is 914.43:

[0088] <Chemical Formula 1-10>

[0089]

[0090]

[0091] The above (11) ((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azane ethyl)bis(hexane-6,1-ethyl)bis(2-hexyldecanoate) (hereinafter, compound 11) has a structure as shown in the following chemical formula 1-11, and the molecular formula is C 55 H 101 It is N3O6 and its molecular weight is 900.41:

[0092] <Chemical Formula 1-11>

[0093]

[0094]

[0095] The above (12) (((pyridin-4-yl azanedyl)bis(hexane-6,1-diyl)bis(disulfandyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)(2-hexyldecanoate) (hereinafter, compound 12) has a structure as shown in the following chemical formula 1-12, and the molecular formula is C 49 H 90 It is N2O4S4 and its molecular weight is 899.51:

[0096] <Chemical Formula 1-12>

[0097]

[0098]

[0099] The above (13) bis(2-(2-(hexyldisulfanyl)ethyl)octyl)6,6'-(pyridin-4-ylazanedyl)dihexanoate (hereinafter, compound 13) has a structure as shown in the following chemical formula 1-13, and the molecular formula is C 49 H 90 It is N2O4S4 and its molecular weight is 899.51:

[0100] <Chemical Formula 1-13>

[0101]

[0102]

[0103] The present invention provides a lipid nanoparticle composition comprising a compound selected from the compound represented by the above chemical formula I, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0104] The above compound can act as an ionizable lipid.

[0105] The above lipid nanoparticle composition may further include one or more selected from the group consisting of neutral lipids, steroids, and polymerized lipids.

[0106] The above neutral lipid may be selected from phospholipids or glycolipids. Specifically, the phospholipids include dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), It may be at least one selected from the group consisting of phosphatidylethanolamine (PE) and dipalmitoylphosphatidylethanolamine, and the glycolipid may be at least one selected from the group consisting of glucosylceramide, galactosylceramide, glucosylsphingosine, galactosylsphingosine, and phosphoglycoceramide, but is not limited thereto.

[0107] The above steroid may be one or more selected from the group consisting of cholesterol, bile acid derivatives and cholic acid derivatives, but is not limited thereto.

[0108] The above polymer lipid may be a pegylated lipid having a structure in which a water-soluble polymer and a lipid are combined, and the pegylated lipid may be at least one selected from the group consisting of PEG bound to dialkyloxypropyl (PEG-DAA); PEG bound to diacylglycerol (PEG-DAG) such as PEG-c-DOMG and PEG-DMG; PEG bound to phospholipids such as phosphatidylethanolamine (PEG-DLPE, PEG-DMPE and PEG-DSPE); PEG bound to ceramide (PEG-CER); PEG bound to cholesterol or a derivative thereof; PEG-modified phosphatidic acid; PEG-modified dialkylamine; and PEG-modified dialkylglycerol, but is not limited thereto. In addition, the pegylated lipid may be a functionalized PEG in which a functional group is combined on a side that is not combined with lipid. The functional group that can be used at this time may be one or more selected from the group consisting of succinyl, carboxylic acid, maleimide, n-hydroxysuccinimide, amine, biotin, cyanuric, and folate, but is not limited thereto.

[0109] The lipid nanoparticle composition may be manufactured by including ionized lipids, neutral lipids, steroids, polymerized lipids, etc. In order to have a composition suitable for manufacturing lipid nanoparticles, the lipid composition, excluding active ingredients such as genes such as RNA and DNA, may include 20 to 65 mol% of ionized lipids, 2.5 to 30 mol% of neutral lipids, 20 to 60 mol% of steroids, and 0.5 to 5 mol% of polymerized lipids.

[0110] For example, ionized lipid: neutral lipid: steroid: polymer polymerized lipid is 60:5:33.5:1.5, 50:10:38.5:1.5, 40:15:43.5:1.5, 30:20:48.5:1.5, 25:25:48.5:1.5, 59.5:5:33.5:2.0, 49.5:10:38.5:2.0, 39.5:15:43.5:2.0, 29.5:20:48.5:2.0, 24.5:25:48.5:2.0, 59:5:33.5:2.5, 49:10:38.5:2.5, 39:15:43.5:2.5, It can be composed in one mol% ratio selected from the group consisting of 29:20:48.5:2.5, 24:25:48.5:2.5, 58.5:5:33.5:3.0, 48.5:10:38.5:3.0, 38.5:15:43.5:3.0, 28.5:20:48.5:3.0, 23.5:25:48.5:3.0, 58:5:33.5:3.5, 48:10:38.5:3.5, 38:15:43.5:3.5, 28:20:48.5:3.5, and 23:25:48.5:3.5.

[0111] The lipid nanoparticle composition may further include a preventive or therapeutic agent, and the preventive or therapeutic agent may be a gene including RNA, DNA or a mixture thereof, and specifically, may be at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA) and mixtures thereof, but is not limited thereto. It is not.

[0112] Alternatively, the active ingredient of the composition may be an anionic biopolymer-drug conjugate such as various anionic peptides, protein drugs, protein-nucleic acid structures, or hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, antibodies, etc.

[0113] The lipid nanoparticle composition including ionized lipids, neutral lipids, steroids, polymerized lipids, etc. of the present invention can be prepared as a lipid solution by dissolving in a solvent miscible with ethanol or water. Separately, the active ingredient can be prepared as an active ingredient solution by dissolving in a citric acid or acetic acid buffer having a pH of 4.0±1.0. In addition, the lipid solution and the active ingredient solution can be mixed at a volume ratio of 1:3 at a flow rate of about 10 to 15 mL / min using a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) to prepare lipid nanoparticles.

[0114]

[0115] In addition, the present invention provides a drug delivery composition comprising the lipid nanoparticle composition and a preventive or therapeutic agent.

[0116] The above preventive or therapeutic agent may be a gene including RNA, DNA or a mixture thereof, and specifically may be one or more selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA) and mixtures thereof, but is not limited thereto.

[0117] The lipid nanoparticles according to the present invention possess excellent safety and stability, and can function as delivery vehicles so that genes containing RNA, DNA, or a mixture thereof as active ingredients can effectively exert their effects within cells. Accordingly, the novel lipid nanoparticles comprising the ionizable lipids exhibit excellent gene encapsulation rates and in vivo gene delivery rates, and on the other hand, exhibit excellent gene delivery functions even in liver injury models, and thus can be usefully utilized as drug delivery compositions.

[0118] The above drug delivery composition can be administered to mammals including humans by various routes, including parenteral administration, and parenteral administration can be applied intravenously, subcutaneously, intraperitoneally, or locally, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art.

[0119] When formulating the above drug delivery composition according to an example, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, lyophilizing agents, binders, wetting agents, disintegrating agents, and surfactants.

[0120] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0121] Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0122] The drug delivery composition of the present invention can be administered while containing a pharmaceutically effective amount of a preventive or therapeutic agent. The effective dosage level of the preventive or therapeutic agent can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route and excretion rate, the duration of treatment, factors including concomitant medications, and other factors well known in the medical field. In one embodiment, the composition can be administered as an individual therapeutic agent or in combination with another therapeutic agent, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art. For example, the composition can be administered at 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 10 mg / kg.

[0123] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0124]

[0125] <Example 1> Preparation of compound

[0126] 1-1. Preparation of compound 1

[0127] As shown in Fig. 1, compound 1-b (9.74 g, 85.3 mmol) was added to xylene (o-xylene) (100 mL), compound 1-a (10.0 g, 85.3 mmol) was added, and the mixture was stirred at 140°C for 12 hours under nitrogen gas. The mixture was diluted with petroleum ether (PE) (200 mL) and purified on silica gel (methanol (MeOH) / dichloromethane (DCM) = 0 / 100 to 10 / 90) to obtain compound 1-c as a white solid. Compound 1-c (5.00 g, 21.6 mmol) was dissolved in tetrahydrofuran (THF) (50 mL), and dimethyl sulfide borane (10 M, 8.7 mL) was added dropwise at 25°C. The mixture was stirred at 70°C for 12 h. MeOH (10 mL) was additionally added dropwise and stirred at 25°C for 0.5 h. The mixture was concentrated under reduced pressure and purified on silica gel (MeOH / DCM = 0 / 100 to 15 / 85, containing 0.5% aqueous ammonia) to obtain compound 1-d as a white solid. Compound 1-d (3.5 g, 16.1 mmol), compound 1-e (3.14 g, 20.9 mmol, hydrochloride), and N,N-diisopropylethylamine (DIEA) (10.4 g, 80.5 mmol) were added to dimethylformamide (DMF) (5 mL) and stirred at 100°C for 12 h. Saturated sodium bicarbonate solution (sat. NaHCO3) (50 mL) was added to the mixture, and the extract was washed with saturated sodium chloride solution (brine) (50 mL × 2). The extract was dried over sodium sulfate (Na2SO4), concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 10 / 90, containing 0.1% aqueous ammonia) to obtain compound 1-f as a red oil. Compound 1-f (400 mg, 1.) was added to DMF (5 mL).36 mmol) was added, compound 1-g (1.05 g, 4.08 mmol), 1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDCI) (781 mg, 4.08 mmol), and dimethylaminopyridine (DMAP) (499 mg, 4.08 mmol) were dissolved, and stirred at 25°C for 12 hours. The mixture was added sat. After adding Na2CO3 (30 mL), the extracted solution was washed with brine (50 mL x 2), dried with sodium sulfate (Na2SO4), concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97 and MeOH / DCM = 0 / 100 to 2 / 98, containing 0.1% ammonia water) to obtain compound 1 as a yellow oil, and compound 1. 1 The results of H NMR analysis were as follows.

[0128] 1 H NMR (400 MHz, CDCl3)δ8.17 (d,J= 6.4 Hz, 2H), 6.45 (d,J= 6.4 Hz, 2H), 4.08 (t,J= 6.4 Hz, 4H), 3.22 - 3.35 (m, 4H), 2.27 - 2.36 (m, 2H), 1.56 - 1.69 (m, 12H), 1.36 - 1.47 (m, 12H), 1.18 - 1.32 (m, 40H), 0.80 - 0.95 (m, 12H).

[0129]

[0130] 1-2. Preparation of Compound 2

[0131] As shown in Fig. 2, compound 2-a (11.0 g, 39.5 mmol) and compound 2-b (3.0 g, 17.9 mmol, 2.70 mL) were added to DMF (50 mL), followed by adding potassium carbonate (K2CO3) (12.4 g, 89.7 mmol) and potassium iodide (KI) (7.45 g, 44.9 mmol), and stirred at 80°C for 12 h. Purified water (500 mL) was added to the mixture, and the extracted solution was washed with brine (300 mL × 3). After drying over Na2SO4, the mixture was concentrated under reduced pressure and purified on silica gel (100% DCM) to obtain compound 2-c as a yellow oil. Compound 2-c (7.2 g, 12.8 mmol) was added to 1,2-dichloroethane (DCE) (70 mL), and 1-chloroethyl carbonochloridate (2.19 g, 15.3 mmol) and DIEA (3.30 g, 25.54 mmol) were added at 0°C, followed by stirring at 15°C for 2 hours. The mixture was concentrated under reduced pressure, dissolved in ethyl alcohol (EtOH) (100 mL), stirred at 60°C for 0.5 hours, and then concentrated. Sat. The mixture was diluted with NaHCO3 (100 mL), extracted with EA (100 mL × 2), dried over Na2SO4, concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 1 / 99) to obtain compound 2-d as a pale white solid. Compound 2-d (4.4 g, 10.6 mmol) and compound 2-e (3.99 g, 26.6 mmol hydrochloride), DavePhos (1.67 g, 4.25 mmol), tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) (1.95 g, 2.13 mmol), and t-butoxysodium (t-BuONa) (5.62 g, 58.5 mmol) were added to toluene (40 mL) and stirred at 100°C for 15 h. The mixture was added sat.NaHCO3 (100 mL) was added, and the extract was dried over Na2SO4, concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 1 / 99) to obtain compound 2-f as a red oil. Compound 2-f (800 mg, 1.63 mmol) was added to THF (6 mL), and sodium hydroxide (326 mg, 8.15 mmol) dissolved in purified water (2 mL) was added, and the mixture was stirred at 15°C for 3 hours. The mixture was concentrated, adjusted to pH 4 to 5 using 1 M hydrochloric acid solution, and the extract was dried over Na2SO4, concentrated under reduced pressure, and the red solid compound 2-g was obtained. Compound 2-g (500 mg, 1.15 mmol) and compound 2-h (643 mg, 3.45 mmol), EDCI (662 mg, 3.45 mmol), and DMAP (422 mg, 3.45 mmol) were dissolved in DMF (5 mL) and stirred at 10°C for 12 h. After adding sat. Na2CO3 (30 mL) to the mixture, the solution was extracted with EA (50 mL × 2 times), and washed with brine (50 mL × 3 times). After drying with sodium sulfate (Na2SO4), concentration under reduced pressure, purification on silica gel (MeOH / DCM = 0 / 100 to 3 / 97), and then purification by prep-HPLC (C8, 50*40 mm, 5 μm, purified water / acetonitrile (ACN) = 35 / 65 to 5 / 95, 20 min) was performed to obtain compound 2 as a yellow oil. 1 The results of H NMR analysis were as follows.

[0132] 1H NMR (400 MHz, CDCl3) δ 8.16 (d,J= 6.4 Hz, 2H), 6.43 (d,J= 6.8 Hz, 2H), 3.98 (d,J= 5.6 Hz, 4H), 3.26 (t,J= 3. 2.7 (H2), Hz, 4H), 1.53 - 1.67 (m, 10H), 1.24 - 1.35 (m, 52H), 0.81 - 0.97 (m, 12H).

[0133]

[0134] 1-3. Page 3 pages

[0135] As shown in Figure 3, compound 3-a (25.1 g, 100 mmol) and compound 3-b (7.60 g, 45.5 mmol), K2CO3 (31.4 g, 227 mmol), and sodium iodide (NaI) (17.0 g, 114 mmol) were added to DMF (150 mL) and stirred at 80°C for 12 h. The mixture was diluted with purified water (500 mL), extracted with EA (300 mL × 3 times), washed with brine (500 mL × 3 times), dried over Na2SO4, filtered, concentrated under reduced pressure, and purified on silica gel (DCM / MeOH = 100 / 0 to 97 / 23) to obtain compound 3-c as a pale yellow oil. Compound 3-c (21.5 g, 42.4 mmol) was added to DCE (220 mL), and DIEA (11.0 g, 84.7 mmol) and chloroethyl chloroformate (7.27 g, 50.8 mmol) were added at 0°C, followed by stirring at 20°C for 2 hours. The mixture was concentrated under reduced pressure, dissolved in EtOH (200 mL), stirred at 60°C for 0.5 hours, and concentrated under reduced pressure. Diluted with EA (100 mL), saturated sodium bicarbonate solution (sat. NaHCO3) (200 mL) was added, and the extract was dried over Na2SO4, filtered, concentrated under reduced pressure, and purified on silica gel (EA / PE = 0 / 100 to 6 / 94) to obtain yellow solid compound 3-d. Compound 3-d (10.9 g, 30.4 mmol) and compound 3-e (11.4 g, 75.9 mmol hydrochloride), DavePhos (4.78 g, 12.1 mmol), Pd2(dba)3 (5.56 g, 6.07 mmol), and t-BuONa (17.5 g, 182 mmol) were dissolved in toluene (120 mL) and stirred at 100°C under nitrogen gas for 15 h. Sat. NaHCO3 (250 mL) was added to the mixture, filtered, and extracted with EA (200 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure.After purification twice on silica gel (MeOH / DCM = 0 / 100 to 5 / 95), dissolved in EA (100 mL), washed with 10% (2R)-2-acetamido-3-sulfanyl propanoic acid solution ((2R)-2-acetamido-3-sulfanyl propanoic acid in water) (80 mL x 2), filtered, washed with sat. NaHCO3 (50 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 3-f as an orange oil. Compound 3-f was added to THF (15 mL), MeOH (5 mL), and purified water (5 mL), sodium hydroxide (870 mg, 21.7 mmol) was added, and the mixture was stirred at 20°C for 2 hours. The mixture was concentrated, diluted with DCM (20 mL) and purified water (10 mL), adjusted to pH 4 with 2 M hydrochloric acid solution, extracted with DCM / MeOH (10 / 1, 30 mL × 3 times), dried over Na2SO4, and concentrated under reduced pressure to obtain compound 3-g as a yellow solid. Compound 3-g (350 mg, 0.925 mmol), compound 3-h (517 mg, 2.77 mmol), EDCI (532 mg, 2.77 mmol), and DMAP (339 mg, 2.77 mmol) were dissolved in DMF (10 mL) and stirred at 30°C for 12 h. Saturated sat. Na2CO3 (40 mL) was added to the mixture, extracted with EA (40 mL × 3 times), and washed with brine (50 mL × 2 times). After drying over Na2SO4, filtering, and concentrating under reduced pressure, the residue was purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97), then purified by prep-HPLC (C4, 150*19 mm, 5 μm, purified water (fluoroacid) / ACN = 90 / 10 to 20 / 80, 22 min) and lyophilized. The dried product was diluted with DCM (30 mL) and evaporated.After washing with Na2CO3 (30 mL) and brine (30 mL), the DCM layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 3 as a yellow oil. 1 The results of H NMR analysis were as follows.

[0136] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d,J= 6.8 Hz, 2H), 6.42 (d,J= 6.4 Hz, 2H), 3.98 (d,J= 6.0 Hz, 4H), 3.21 - 3.32 (m, 4H), 2.31 (t,J= 7.6) Hz, 4H), 1.54 - 1.68 (m, 10H), 1.25 - 1.37 (m, 44H), 0.85 - 0.94 (m, 12H).

[0137]

[0138] 1-4. Preparation of compound 4

[0139] As shown in Figure 4, compound 4-a (380 mg, 1.00 mmol) and compound 4-b (646 mg, 3.01 mmol) were added to DMF (10 mL), followed by adding EDCI (578 mg, 3.01 mmol) and DMAP (368 mg, 3.01 mmol), and stirred at 30°C for 12 h. Saturated sat. NaHCO3 (50 mL) was added to the mixture, and the extracted solution was washed with brine (50 mL × 2 times). Drying over Na2SO4, concentration under reduced pressure, and purification on silica gel (MeOH / DCM = 0 / 100 to 3 / 97) were followed by prep-HPLC (C4, 150*19 mm, 5 μm, purified water (fluoroacid) / ACN = 90 / 10 to 20 / 80, 22 min). After adding sat. Na2CO3 (30 mL) to the purified water, the mixture was extracted with DCM (30 mL x 2), washed with brine (30 mL), dried with Na2SO4, and concentrated under reduced pressure to obtain compound 4 as a yellow oil.1 The results of H NMR analysis were as follows.

[0140] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d,J= 6.4 Hz, 2H), 6.42 (d,J= 6.4 Hz, 2H), 3.98 (d,J= 5.6 Hz, 4H), 3.21 - 3.29 (m, 4H), 2.31 (t,J= 7.6 Hz, 4H), 1.54 - 1.67 (m, 10H), 1.24 - 1.36 (m, 52H), 0.89 (t,J= 6.4 Hz, 12H).

[0141]

[0142] 1-5. Preparation of compound 5

[0143] As shown in Figure 5, compound 5-a (350 mg, 0.925 mmol) and compound 5-b (673 mg, 2.77 mmol) were added to DMF (10 mL), followed by addition of EDCI (532 mg, 2.77 mmol) and DMAP (339 mg, 2.77 mmol), and stirred at 30°C for 14 h. The mixture was washed with saturated sat. After diluting with NaHCO3 (100 mL), the solution extracted with EA (80 mL x 2) was washed with brine (120 mL x 4), dried with Na2SO4, filtered, concentrated under reduced pressure, purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97), and then purified by prep-HPLC (C4, 150*19 mm, 5 μm, purified water (fluoro acid) / ACN = 90 / 10 to 20 / 80, 22 min). The product was diluted with DCM (30 mL), washed with sat. NaHCO3 (40 mL), extracted with DCM (30 mL), washed with brine (40 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 5 as a pale yellow oil. 1 The results of H NMR analysis were as follows.

[0144] 1H NMR (400 MHz, CDCl3) δ 8.17 (d,J= 6.4 Hz, 2H), 6.35 - 6.47 (m, 2H), 3.97 (d,J= 6.0 Hz, 4H), 3.26 (d,J= 8.0 Hz, 4H), 2.31 (d,J= 7.6 Hz, 4H), 1.49 - 1.70 (m, 10H), 1.20 - 1.38 (m, 60 H), 0.75 - 1.01 (m, 12H).

[0145]

[0146] 1-6. 화합물 6 제조

[0147] As shown in Fig. 6, compound 6-b (500 mg, 2.99 mmol) and compound 6-a (3.14 g, 7.48 mmol), K2CO3 (1.24 g, 8.97 mmol), cesium carbonate (Cs2CO3) (974 mg, 2.99 mmol), and NaI (89.6 mg, 598 μmol) were added to ACN (30 mL) and stirred at 80°C for 12 h under nitrogen gas. Purified water (100 mL) was added to the mixture, and the extracted solution was washed with brine (100 mL × 2). After drying over Na2SO4, the mixture was filtered, concentrated under reduced pressure, and purified on silica gel (DCM / MeOH = 0 / 100 to 5 / 95) to obtain compound 6-c as a yellow oil. Compound 6-c (2.20 g, 2.61 mmol) was added to DCE (20 mL), and DIEA (674 mg, 5.21 mmol) and compound 6-d (447 mg, 3.13 mmol) were added at 0°C, followed by stirring at 25°C for 5 h. The mixture was concentrated under reduced pressure, dissolved in MeOH (20 mL), stirred at 60°C for 0.5 h, concentrated, and then sat. NaHCO3 (30 mL) was added and extracted with EA (50 mL × 2 times). The extracted solution was washed with brine (50 mL × 2 times), dried over Na2SO4, concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97, containing 0.5% ammonia water) to obtain compound 6-e as a white oil. Compound 6-g (10.0 g, 88.1 mmol) was added to 2-aminoethanol (100 mL), and the mixture was purged three times with nitrogen gas to remove the gas. The mixture was stirred at 110°C for 3 hours under nitrogen gas. The mixture was purified on silica gel (DCM / MeOH = 0 / 100 to 10 / 90) to obtain compound 6-h as a white solid. Compound 6-h (3.60 g, 26.1 mmol) and di-tert-butyldicarbonate (Boc2O) (11.4 g, 52.1 mmol) was added and stirred at 25°C for 12 hours. The mixture was concentrated and purified on silica gel (MeOH / DCM = 2 / 98 to 3 / 97, containing 0.5% ammonia water) to obtain compound 6-f as a yellow oil. Compound 6-f (1.17 g, 4.90 mmol), compound 6-g (1.09 g, 5.39 mmol), and triethylamine (Et3N) (991 mg, 9.80 mmol) were added to 2-methyltetrahydrofuran (2-MeTHF) (20 mL) and stirred at 20°C for 5 hours, then compound 6-e (1.70 g, 2.45 mmol) was added and stirred at 20°C for 12 hours. The mixture was diluted with purified water (100 mL) and extracted with EA (100 mL × 2) to obtain sat. After washing with Na2CO3 (100 mL × 2 times) and brine (200 mL), dried with Na2SO4, filtered, concentrated under reduced pressure, and purified on silica gel (EA / PE = 0 / 100 to 25 / 75), compound 6-h was obtained as a white oil. Compound 6-h was added to a mixture of trifluoroacetic acid (TFA) (3 mL) and DCM (3 mL), stirred at 20°C for 12 h, adjusted to pH 9 with sat. Na2CO3 (20 mL), extracted with DCM (20 mL × 2 times), dried with Na2SO4, filtered, and concentrated under reduced pressure. The oil obtained by purification on silica gel (MeOH / DCM = 0 / 100 to 2 / 98, containing 0.5% ammonia water) and prep-HPLC (C4, 150*19 mm, 5 μm, purified water (hydrofluoric acid) / ACN = 80 / 20 to 20 / 80, 22 min) was dissolved in DCM (20 mL), washed with sat. Na2CO3 (10 mL), dried over Na2SO4, filtered, concentrated, and lyophilized to obtain compound 6 as a white oil. Compound 6. 1 The results of H NMR analysis were as follows.

[0148] 1H NMR (400 MHz, CDCl3) δ 8.19 (d,J= 6.0 Hz, 2H), 6.47 (d,J= 6.4 Hz, 2H), 4.95 (s, 1H), 4.33 (t,J= 5.2 Hz, 2H), 4.06 (t,J= 6.8 Hz, 4H), 3.42 (q,J= 5.2 Hz, 2H), 3.07 - 3.28 (m, 4H), 2.25 - 2.39 (m, 2H), 1.55 - 1.64 (m, 8H), 1.39 - 1.48 (m, 6H), 1.20 - 1.38 (m, 50H), 0.88 (t,J=6.4 Hz, 12H).

[0149]

[0150] <Example 2> Manufacturing of lipid nanoparticles

[0151] Lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNA Gene, Korea) were prepared using the compound (ionized lipid) prepared in Example 1 above, dioleoylphosphatidylethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and PEG 2000-DMG (polymerized lipid).

[0152] Ionized lipids, DOPE / DSPC, cholesterol, and PEG 2000-DMG were mixed in a molar ratio of 49:10:38.5:2.5 and dissolved in ethanol at a concentration of 4.8 mg / mL to prepare a lipid solution. Separately, Firefly Luciferase mRNA was dissolved in a citric acid buffer solution (pH 4.0±1.0) or an acetic acid buffer solution (pH 5.0±1.0) at a weight ratio of 1:16 for mRNA: ionized lipid to prepare an active ingredient solution. Then, lipid nanoparticles were prepared by passing the mixture through a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) at a flow rate of approximately 15 mL / min so that the lipid solution: active ingredient solution had a volume ratio of 1:3. The manufactured lipid nanoparticles were diluted with Tris buffer containing 8.7% sucrose and dialyzed using a dialysis centrifuge tube to ensure that the ethanol content was less than 1%, and the final concentration was manufactured to be 0.2 mg / mL based on mRNA.

[0153] For comparison, lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNA Gene, Korea) were prepared using MC3 ionized lipid, SM-102 ionized lipid, distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and DMG-PEG2000 (polymer polymerized lipid). MC3, DSPC, cholesterol, and DMG-PEG2000 or SM-102, DSPC, cholesterol, and DMG-PEG2000 were prepared in a molar % ratio of 50:10:38.5:1.5 and used as lipid nanoparticles for comparison.

[0154]

[0155] <Example 3> Confirmation of lipid nanoparticle size and polydispersity

[0156] The size and surface charge of lipid nanoparticles prepared according to the ionized lipid in Example 1 were measured. To measure the size of lipid nanoparticles, each lipid nanoparticle was diluted with PBS to a concentration of 1 μg / mL, and the diameter and polydispersity index (PDI) of LNPs were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0157] As a result, as shown in Table 1 below, all lipid nanoparticles exhibited a size of approximately 120 nm or less and a good polydispersity of less than 0.2.

[0158] Compound NumberParticle Size (nm)PDI1770.062770.123830.114840.145850.1361150.09

[0159]

[0160] <Example 4> Confirmation of gene inclusion rate

[0161] To determine the gene encapsulation rate of lipid nanoparticles, Quant-iTTM RibogreenTM RNA Reagent and Kit were used. 5 μL of ribosomal RNA standard (100 μg / mL in TE buffer) was diluted with 245 μL TE buffer or 0.4% Triton-TE buffer to prepare a stock solution for calibration curve. 2, 5, 10, 25, and 50 μL of the stock solution were diluted with TE buffer or 0.4% Triton-TE buffer to make a total volume of 100 μL. 100 μL of Quant-iTTM RibogreenTM RNA Reagent was added, mixed, and the fluorescence intensity (excitation 475 nm and emission 500–550 nm) was measured to prepare a calibration curve.

[0162] 5 μL of the nanoparticle solution prepared in Example 2 was diluted with 245 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5 in DEPC-treated water). 50 μL of the diluted nanoparticle solution was added with 50 μL of TE buffer or 2% Triton-TE buffer, and incubated at 37°C for approximately 10 minutes. 40 μL of each culture medium was then placed in a microplate, followed by sequential addition of 60 μL of TE buffer and 100 μL of Quant-iT™ Ribogreen™ RNA Reagent, mixing, and measuring the fluorescence intensity. The measured fluorescence intensity was applied to the calibration curve to calculate the gene content (TO) of the TE buffer and the gene content (T2) of the 2% Triton-TE buffer, and then the gene inclusion rate was calculated using the following mathematical equation 1. The gene inclusion rate was confirmed and is shown in Table 2. As a result, all showed good gene inclusion rates.

[0163] [Mathematical Formula 1]

[0164]

[0165] As a result, as shown in Table 2 below, it was confirmed that all compounds showed a good gene encapsulation rate of 85% or more.

[0166] Compound number Gene inclusion rate (%) 189.9293.3394.4494.7597.1697.6

[0167]

[0168] <Example 5> Confirmation of lipid nanoparticle gene delivery efficiency

[0169] 5-1. Confirmation of in vivo gene transfer efficiency 1

[0170] To confirm the in vivo gene delivery efficiency of lipid nanoparticles, a single intravenous injection of 1 mg / kg based on the luciferase gene encapsulated in lipid nanoparticles was performed into 7-week-old C57BL / 6 mice, and the mice were sacrificed after 4 hours and their livers were removed.

[0171] To determine the level of luciferase expression in tissue samples, the weight of the liver tissue slices was measured and transferred to a tube containing 3 mm metal beads. 500 μl of Glo-Lysis Buffer (Promega, WI, USA) per 50 mg was added, and the tissue was homogenized using a bead homogenizer. The supernatant of the centrifuged homogenate was then diluted with Glo-Lysis Buffer at an appropriate ratio. 50 μl of the diluted tissue homogenate and Steady-Glo Luciferase Assay solution were each transferred to a 96-well white plate at a 1:1 ratio and incubated at room temperature. After 5 minutes, the luminescence value was detected using a GloMax Discover microplate reader (Promega). A calibration curve was generated using recombinant luciferase (Promega) as a standard, and the level of luciferase expression in the sample (ng / g tissue) was derived.

[0172] As a result, as shown in Table 3, it was confirmed that the lipid nanoparticles using the ionized lipid of the present invention exhibited an excellent gene transfer effect through systemic administration by showing a good level of luciferase expression in liver tissue when injected intravenously.

[0173] Compound number: Luciferase expression in the liver after intravenous injection (Mean ± SD, ng / g liver) 196,246 ± 27,097 221,597 ± 11,480 38,248 ± 1,030 429,027 ± 853 546,333 ± 32,091 MC 313,845 ± 3,787

[0174]

[0175] 5-2. Confirmation of in vivo gene transfer efficiency 2

[0176] To determine the in vivo gene transfer efficiency of repeated administration of lipid nanoparticles, lipid nanoparticles loaded with EPO mRNA (TriLink BioTechnologies, CA, USA) were intravenously administered to 7-week-old C57BL / 6 mice at a dose of 0.5 mg / kg per gene. To determine the in vivo EPO expression level, blood samples were collected 6 hours after administration, and the collected blood was anticoagulated to separate plasma.

[0177] To determine the concentration of EPO protein in plasma, an EPO enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, MN, USA) was used, and the analysis was performed according to the manufacturer's method. More specifically, the plasma sample was diluted 1:3000 using the specimen diluent included in the kit, and 100 μL of each (1:1 ratio) was added to the microwell and incubated at room temperature for 2 hours. After removing all wells, 200 μL of EPO conjugate was added and incubated for another 2 hours at room temperature. After washing four times with 300 μL of washing buffer, 200 μL of substrate solution was added and incubated for 20 minutes. To terminate the reaction, 100 μL of stop solution was added, and the absorbance was measured at wavelengths of 450 nm and 600 nm using a microplate (Promega). A calibration curve was derived using standard materials, and the EPO protein concentration (ng / mL) of each sample was derived within the defined range of the standard materials.

[0178] As a result, as shown in Table 4, the lipid nanoparticles using the ionized lipid of the present invention showed a good level of EPO expression in the blood compared to the lipid nanoparticles using the ionized lipids MC3 and SM-102 used for comparison when injected intravenously, confirming that they exhibit an excellent gene transfer effect through systemic administration.

[0179] Compound number EPO expression level in blood after intravenous injection (Mean ± SD, ng / mL) 14,424 ± 588 MC 3 3 84 ± 73 SM-10 2 1,154 ± 364

[0180]

[0181] 5-3. Confirmation of in vivo gene transfer efficiency 3

[0182] To confirm the gene delivery ability of lipid nanoparticles in liver damage disease, liver damage was induced in 6-week-old C57BL / 6 mice by intraperitoneal administration of CCl4 diluted to 20% in olive oil at 2 mL / kg three times a week for 2 weeks. The plasma ALT and AST levels were checked the day after the last CCl4 administration. Compared to the plasma ALT of 2,592 U / L and AST of 5,652 U / L in the olive oil vehicle group, liver damage was confirmed to have been induced. Mice with induced liver damage (liver damage model) and mice administered olive oil as a vehicle (normal model) were intravenously injected once at a dose of 1 mg / kg based on the loaded luciferase gene of each lipid nanoparticle. Six hours after administration, the mice were sacrificed and their livers were removed.

[0183] To confirm the luciferase expression level in tissue samples, the weight of the liver tissue slices was measured and transferred to a tube containing 3 mm metal beads. 500 μL of Glo-Lysis Buffer (Promega, WI, USA) was added per 50 mg, and the tissue was homogenized using a bead homogenizer. The supernatant of the centrifuged homogenate was then diluted with Glo-Lysis Buffer at an appropriate ratio. 50 μL of each of the diluted tissue homogenate and Steady-Glo Luciferase Assay solution were transferred to a 96-well white plate at a 1:1 ratio and incubated at room temperature. After 5 minutes, the luminescence value was detected using a GloMax Discover microplate reader (Promega). A calibration curve was generated using recombinant luciferase (Promega) as a standard, and the luciferase expression level (ng / g tissue) of the sample was derived.

[0184] As a result, as shown in Table 5, the lipid nanoparticles using the ionized lipid of the present invention exhibited the characteristic of maintaining excellent gene transfer ability without difference from a normal animal model when intravenously injected into a liver injury animal model. In contrast, the lipid nanoparticles using SM-102, used as a comparison, exhibited the characteristic of reduced gene transfer ability in a liver injury animal model.

[0185] Compound number: Expression of luciferase in the liver after intravenous injection (Mean ± SD, ng / g liver) Normal model Liver damage model 165,704 ± 12,00386,676 ± 8,499 SM-102 21,152 ± 1974,040 ± 1,438

[0186]

[0187] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound selected from the group consisting of a compound represented by the following chemical formula Ⅰ, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof: <Chemical Formula Ⅰ> In the above chemical formula Ⅰ, n is 0 or 1, L is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and in said alkylene, alkenylene and alkynylene, one or more -CH2- is substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- or Non-substituted, R 1 is one of hydrogen, (C1-C20)alkyl, (C2-C20)alkenyl and (C2-C20)alkynyl, R 2 and R 3 are each the same or different, and are one of 5 to 30 membered heteroaryls including one or more heteroatoms selected from the group consisting of (C5-C30)alkyl, (C5-C30)alkenyl, (C5-C30)alkynyl, (C3-C30)cycloalkyl, (C3-C30)cycloalkenyl, (C3-C30)cycloalkynyl, (C5-C30)aryl and N, O and S, and in the alkyl, alkenyl and alkynyl, one or more -CH2- is -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- are substituted or unsubstituted.

2. In claim 1, In the above chemical formula Ⅰ, L is one of (C1-C6)alkylene, (C2-C6)alkenylene and (C2-C6)alkynylene, and in the alkylene, alkenylene and alkynylene, at least one -CH2- is substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O-, R 1 is hydrogen or (C1-C10)alkyl, R 2 and R 3 may be the same or different, and are one of (C10-C30)alkyl, (C10-C30)alkenyl and (C10-C30)alkynyl, and in said alkyl, alkenyl and alkynyl, one or more -CH2- is one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and -NHC(=O)O- A compound selected from a compound, a pharmaceutically acceptable salt, a tautomer, a prodrug or a stereoisomer thereof, characterized in that it is substituted.

3. In claim 1, In the above chemical formula Ⅰ, L is (C2-C6)alkylene, and in said alkylene, at least one -CH2- is substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O- and -OC(=O)O-, R 1 is hydrogen or (C1-C5)alkyl, R 2 and R 3 A compound, a pharmaceutically acceptable salt, a tautomer, a prodrug or a stereoisomer thereof, characterized in that each of which may be the same or different, is one of (C10-C30)alkyl, (C10-C30)alkenyl and (C10-C30)alkynyl, and wherein at least one -CH2- in said alkyl, alkenyl and alkynyl is substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -S- and -SS-.

4. In claim 1, In the above chemical formula I, L is (C2-C6)alkylene, and in said alkylene, one -CH2- is replaced by -OC(=O)- or -C(=O)O-, R 1 is hydrogen or (C1-C5)alkyl, R 2 and R 3 A compound selected from a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein each of which may be the same or different, is (C10-C30)alkyl, and one or two -CH2- in said alkyl are substituted with one of -OC(=O)-, -C(=O)O-, -S- and -SS-.

5. In claim 1, A compound selected from the group of compounds below, a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, characterized in that the compound is selected from the group of compounds below: (1) (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (2) bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate) [bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate)]; (3) bis(2-butyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate; (4) bis(2-hexyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate; (5) bis(2-hexyldecyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate; (6) (((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (7) bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate; (8) bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate; (9) ((3-((3-(pyridin-4-ylamino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((3-((3-(pyridin-4-ylamino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (10) ((3-((3-methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((3-((3-(methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (11) ((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (12) (((pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(disulfanediyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)(2-hexyldecanoate) [(((pyridin-4-ylazanediyl)bis(hexane-6,1-diyl))bis(disulfanediyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)]; and (13) Bis(2-(2-(hexyldisulfanyl)ethyl)octyl)6,6'-(pyridin-4-ylazanediyl) dihexanoate.

6. A lipid nanoparticle composition comprising a compound according to claim 1.

7. In claim 6, A lipid nanoparticle composition, characterized in that the compound is an ionized lipid.

8. In claim 6, A lipid nanoparticle composition, characterized in that the lipid nanoparticle composition further comprises at least one selected from the group consisting of neutral lipids, steroids, and polymerized lipids.

9. In claim 8, A lipid nanoparticle composition, characterized in that the neutral lipid is a phospholipid or a glycolipid.

10. In claim 8, A lipid nanoparticle composition, characterized in that the steroid is at least one selected from the group consisting of cholesterol, bile acid derivatives, and cholic acid derivatives.

11. In claim 8, A lipid nanoparticle composition, characterized in that the polymer lipid is a PEGylated lipid, and is at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

12. In claim 8, A lipid nanoparticle composition, characterized in that the lipid nanoparticle composition comprises 20 to 65 mol% of ionized lipid, 2.5 to 30 mol% of neutral lipid, 25 to 60 mol% of steroid, and 0.5 to 5 mol% of polymerized lipid.

13. In claim 6, A lipid nanoparticle composition, characterized in that the composition further comprises a preventive or therapeutic agent.

14. In claim 13, A lipid nanoparticle composition, characterized in that the preventive or therapeutic agent is at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA), and mixtures thereof.

15. A lipid nanoparticle composition according to claim 6; and a drug delivery composition comprising a preventive or therapeutic agent.

16. In claim 15, A drug delivery composition, characterized in that the preventive or therapeutic agent is at least one selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), micro ribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribozyme (DNAzyme), guide ribonucleic acid for gene correction (sgRNA), and mixtures thereof.

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