Lipid nanoparticle composition containing novel sterol lipid or derivative thereof

Abstract

The present invention relates to a lipid nanoparticle composition containing a novel sterol lipid or a derivative thereof. The novel sterol lipid compound can replace part or all of the cholesterol, which is essential for the manufacture of lipid nanoparticles, so that the lipid nanoparticles are efficiently delivered to tissues other than the liver, thereby being able to be advantageously used in the development of gene therapeutic agents for various indications.

Description

Lipid nanoparticle composition containing a novel sterol lipid or a derivative thereof

[0001] The present invention relates to a lipid nanoparticle composition containing a novel sterol lipid or a derivative thereof.

[0002] Nucleic acid-based medicines are utilized not only as therapeutic agents, but also as preventative agents that protect against diseases by injecting genes that can express antigens for specific diseases. Gene-based vaccines are categorized into DNA vaccines, RNA vaccines, and viral vector vaccines. Among them, RNA vaccines inject mRNA encoding an antigen into the body, causing the antigen to be expressed in the body and inducing antibody formation. RNA vaccines have the advantage of being able to be developed quickly without the potential risks of infection associated with viral vector-based vaccines or genetic mutation associated with DNA vaccines, and thus have garnered attention as an effective response to the COVID-19 outbreak in 2019.

[0003] However, genes are easily broken down by nucleases in the human body and, as negatively charged macromolecules, are not easily delivered into cells. Therefore, a method for delivering them stably and efficiently to the desired location is needed. Various delivery techniques 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 applied to the development of patisiran (ONPATTRO®), 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 as a clinically useful delivery system for nucleic acid drugs such as siRNA and mRNA.

[0004] Current lipid nanoparticles are generally used in a form in which four components are mixed in a certain ratio: ionized lipid, phospholipid (helper lipid), sterol (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 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 infection. Therefore, the development of lipid nanoparticles using new ionized lipid delivery systems is continuously required.

[0005] Furthermore, systemic administration of lipid nanoparticles can be utilized in the development of gene therapy, but delivery to specific tissues is required to optimize therapeutic efficacy. Typically, lipid nanoparticles bind to various proteins in the blood, forming a protein corona surrounding the nanoparticle, leading to a high rate of delivery to the liver. Therefore, delivery to tissues other than the liver is necessary. Previous studies have attempted to deliver drugs to tissues other than the liver using anionic phospholipids, fatty acids, and cationic lipids such as DOTAP. These substances commonly contain fatty acids, fatty alcohols, or fatty amines with long alkyl or alkenyl chains. However, delivery to tissues other than the liver is limited by these agents. Given the ongoing development of various gene therapy therapies, including treatments for rare diseases, immuno-oncology agents, and immunomodulators, there is a continuing need to develop novel lipid nanoparticle systems that deliver drugs to tissues other than the liver using various novel carriers, in addition to existing carriers.

[0006] An object of the present invention is to provide a lipid nanoparticle composition comprising a novel sterol lipid compound or a derivative thereof.

[0007] Another object of the present invention is to provide a medical use of the above lipid nanoparticles.

[0008] In order to achieve the above purpose, the present invention provides a lipid nanoparticle composition comprising a compound selected from the group consisting of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt, a tautomer or a stereoisomer thereof:

[0009] <Chemical Formula 1>

[0010]

[0011] In the above chemical formula 1, X is -NR 1 -, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -C(=S)NR 1 -, -NR 1 C(=S)-, -OC(=S)NR 1 -, -NR 1 C(=S)O-, -SC(=O)NR 1 -, -NR 1 C(=O)S-, -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)S-, -SC(=O)O-, -OC(=O)S-, -S- or -SS-, and the R 1 are each independently covalently bonded, H, C 1-6 Alkyl or C with or without branching 2-6 is selected from one of alkenyl groups having or not having a branch, and L is C 1-12 alkylene, C 2-12is selected from among alkenylene or alkynylene, and in said alkylene, alkenylene or alkynylene, at least one -CH2- is -O-, -CH(OH)-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OP(=O)(OR 2 )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)NR 2 -, -NR 2 C(=O)-, -OC(=O)NR 2 -, -NR 2 C(=O)O-, -C(=S)NR 2 -, -NR 2 C(=S)-, -OC(=S)NR 2 -, -NR 2 C(=S)O-, -SC(=O)NR 2 - or -NR 2 C(=O)S- is substituted or unsubstituted with any one of the above R 2 are each independently covalently bonded, H, C 1-6 Alkyl or C with or without branching 2-6 is selected from one of alkenyl having or not a branch, Y is selected from one of tertiary or quaternary amine, carboxylic acid or phosphoric acid, A is C 1-20 Alkyl or C with or without branching 2-20 It can be selected from alkenyl with or without a branch.

[0012] In addition, the present invention provides a drug delivery composition comprising the lipid nanoparticle composition described above; and a preventive or therapeutic agent.

[0013] Unlike the prior art, the present invention designs a new lipid structure compound including a cholesterol structure, thereby replacing part or all of the cholesterol essential for manufacturing lipid nanoparticles, thereby enabling lipid nanoparticles to be efficiently delivered to tissues other than the liver, and thus can be usefully utilized in the development of gene therapy for various indications.

[0014] As gene therapy enters the pharmaceutical market and continues to expand, the development of novel lipid nanoparticle systems capable of delivering to tissues other than the liver could further enhance gene delivery and address unmet needs for systemic or repeated administration, thereby contributing to the development of gene therapy for numerous rare diseases, as well as various mRNA vaccines and cancer vaccines.

[0015] Figure 1 shows the lung delivery amount compared to the liver through the luciferase expression amount of lipid nanoparticles containing a compound according to one embodiment of the present invention.

[0016] Figure 2 shows the amount of spleen delivery compared to the liver through the luciferase expression level of lipid nanoparticles containing a compound according to one embodiment of the present invention.

[0017] Hereinafter, the present invention will be described in detail.

[0018]

[0019] The present inventors synthesized a novel sterol lipid compound and confirmed that when lipid nanoparticles were manufactured by replacing part or all of the sterol lipids essential for manufacturing lipid nanoparticles, the lipid nanoparticles were delivered more efficiently to tissues other than the liver, thereby completing the present invention.

[0020]

[0021] The present invention provides a compound selected from the group consisting of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt, a tautomer or a stereoisomer thereof:

[0022] <Chemical Formula 1>

[0023]

[0024] In the above chemical formula 1, X is a bonding group, -NR 1 -, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -C(=S)NR 1 -, -NR 1 C(=S)-, -OC(=S)NR 1 -, -NR 1 C(=S)O-, -SC(=O)NR 1 -, -NR 1 C(=O)S-, -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)S-, -SC(=O)O-, -OC(=O)S-, -S- or -SS-, and the R 1 are each independently covalently bonded, H, C 1-6 Alkyl or C with or without branching 2-6 It can be selected from one of alkenyl groups with or without a branch.

[0025] L is the connector, C 1-12 alkylene, C 2-12 is selected from among alkenylene or alkynylene, and in said alkylene, alkenylene or alkynylene, at least one -CH2- is -O-, -CH(OH)-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OP(=O)(OR 2 )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)NR 2 -, -NR 2 C(=O)-, -OC(=O)NR 2-, -NR 2 C(=O)O-, -C(=S)NR 2 -, -NR 2 C(=S)-, -OC(=S)NR 2 -, -NR 2 C(=S)O-, -SC(=O)NR 2 - or -NR 2 C(=O)S- is substituted or unsubstituted with any one of the above R 2 are each independently covalently bonded, H, C 1-6 Alkyl or C with or without branching 2-6 It can be selected from one of alkenyl groups with or without a branch.

[0026] Y can be selected from one of a tertiary or quaternary amine, a carboxylic acid or a phosphoric acid, and A is C 1-20 Alkyl or C with or without branching 2-20 It can be selected from alkenyl with or without a branch.

[0027]

[0028] The compound may include a compound represented by the following chemical formula 1-1:

[0029] <Chemical Formula 1-1>

[0030]

[0031] In the above chemical formula 1-1, the definitions of X, L, and Y are the same as in the above chemical formula 1, and R is hydrogen, C 1-4 Alkyl or C 2-4 can be selected from alkenyl.

[0032]

[0033] Preferably, in the above chemical formula 1, X is -NR 1 -, -C(=O)NR 1 -, -NR 1 is selected from any one of C(=O)-, -O-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -S- or -SS-, and the R 1are each independently covalently bonded, H, C 1-4 Alkyl or C with or without branching 2-4 It can be selected from one of alkenyl groups with or without a branch.

[0034] L is C 1-12 alkylene, C 2-12 is selected from among alkenylene or alkynylene, and in said alkylene, alkenylene or alkynylene, at least one -CH2- is -O-, -CH(OH)-, -C(=O)-, -S-, -SS-, -C(=O)NR 2 -, or -NR 2 C(=O)- is substituted or unsubstituted with any one of the above R 2 are each independently covalently bonded, H, C 1-4 Alkyl or C with or without branching 2-4 It can be selected from one of alkenyl groups with or without a branch.

[0035]

[0036] Y can be selected from the following structures:

[0037]

[0038] R is hydrogen, C 1-2 Alkyl or C 2-3 can be selected from alkenyl.

[0039]

[0040] More preferably, the compound may be selected from the group consisting of, but not limited to:

[0041] compound (1)

[0042]

[0043] : 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethane-1-amininium[2-(((3S,8S,9S,10R,13R,1 4S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-t etradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethan-1-aminium, Molecular formula: C 32 H 56 NO2, molecular weight: 486.80]

[0044] compound (2)

[0045]

[0046] : 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-3-oxopropan-1-amininium[3-(((3S,8S,9S,10R,13R,14S,17R) S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-te tradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-3-oxopropan-1-aminium, Molecular formula: C 33 H 58 NO2, molecular weight: 500.83]

[0047] compound (3)

[0048]

[0049] : 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-aminium[4-(((3S,8S,9S,10R,13R,1 4S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-t etradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-aminium, Molecular formula: C 34 H 60 NO2, molecular weight: 514.86]

[0050] compound (4)

[0051]

[0052] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(dimethylamino)propanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(dimethylamino)propanoate, molecular formula: C 32 H 55 NO2, molecular weight: 485.80]

[0053] compound (5)

[0054]

[0055] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate, molecular formula: C 33 H 57 NO2, molecular weight: 499.82]

[0056] compound (6)

[0057]

[0058] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1H-imidazol-1-yl)butanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1H-imidazol-1-yl)butanoate, Molecular formula: C 34 H 54 N2O2, molecular weight: 522.82]

[0059] compound (7)

[0060]

[0061] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(2-methyl-1H-imidazol-1-yl)butanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(2-methyl-1H-imidazol-1-yl)butanoate, Molecular formula: C 35 H 56 N2O2, molecular weight: 536.85]

[0062] compound (8)

[0063]

[0064] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1H-imidazol-4-yl)butanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1H-imidazol-4-yl)butanoate, Molecular formula: C 34 H 54 N2O2, molecular weight: 522.82]

[0065] compound (9)

[0066]

[0067] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(piperidin-1-yl)propanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(piperidin-1-yl)propanoate, molecular formula: C 35 H 59 NO2, molecular weight: 525.86]

[0068] compound (10)

[0069]

[0070] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(4-methylpiperazin-1-yl)propanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(4-methylpiperazin-1-yl)propanoate, Molecular formula: C 35 H 60 N2O2, molecular weight: 540.88]

[0071] compound (11)

[0072]

[0073] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(Pyridin-4-yl)propanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(pyridin-4-yl)propanoate, molecular formula: C 35 H 53 NO2, molecular weight: 519.81]

[0074] Compound (12)

[0075]

[0076] : 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoic acid[4-(((3S,8S,9S,10R, 13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoic acid, Molecular formula: C 31 H 50 O4, molecular weight: 486.74]

[0077] compound (13)

[0078]

[0079] : 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid[5-(((3S,8S,9S,10R,1 3R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid, molecular formula: C 32 H 52 O4, molecular weight: 500.76]

[0080] compound (14)

[0081]

[0082] : 7-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoic acid[7-(((3S,8S,9S,10R,1 3R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoic acid, molecular formula: C 34 H 56 O4, molecular weight: 528.82]

[0083] Compound (15)

[0084]

[0085] : 10-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-10-oxodecanoic acid[10-(((3S,8S,9S,10R ,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-10-oxodecanoic acid, Molecular formula: C 37 H 62 O4, molecular weight: 570.90]

[0086] compound (16)

[0087]

[0088] : (4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutyl)phosphonic acid[(4-(((3S,8S,9S,10R,13 R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutyl)phosphonic acid, molecular formula: C 31 H 53 O5P, molecular weight: 536.73]

[0089] Compound (17)

[0090]

[0091] : (6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-6-oxohexyl)phosphonic acid[(6-(((3S,8S,9S,10R,13 R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-6-oxohexyl)phosphonic acid, molecular formula: C 33 H 57 O5P, molecular weight: 564.79]

[0092] compound (18)

[0093]

[0094] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(phosphonooxy)propanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(phosphonooxy)propanoate, molecular formula: C 30 H 51 O6P, molecular weight: 538.71]

[0095] Compound (19)

[0096]

[0097] : (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-(phosphonooxy)pentanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-(phosphonooxy)pentanoate, molecular formula: C 32 H 55 O6P, molecular weight: 566.76]

[0098] Compound (20)

[0099]

[0100] : 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-N,N,N-trimethyl-2-oxoethane-1-aminum[2-(((3S,8S,9S,10R,13R,14S,17R) S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-te tradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium, Molecular formula: C 32 H 57 N2O, molecular weight: 485.82]

[0101] Compound (21)

[0102]

[0103] : 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-N,N,N-trimethyl-3-oxopropan-1-aminum[3-(((3S,8S,9S,10R,13R,14S,17R) S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tet radecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-N,N,N-trimethyl-3-oxopropan-1-aminium, Molecular formula: C 33 H 59 N2O, molecular weight: 499.85]

[0104] Compound (22)

[0105]

[0106] : 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-5-oxopentanoic acid[5-(((3S,8S,9S,10R,1 3R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-5-oxopentanoic acid, Molecular formula: C 32 H 53 NO3, molecular weight: 499.78]

[0107] Compound (23)

[0108]

[0109] : 7-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-7-oxoheptanoic acid[7-(((3S,8S,9S,10R,1 3R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-7-oxoheptanoic acid, Molecular formula: C 34 H 57 NO3, molecular weight: 527.83]

[0110] compound (24)

[0111]

[0112] : 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethane-1-amininium[2-(((3S,8S,9S,10R,13R,14S ,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16 ,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethan-1-aminium, Molecular formula: C 34 H 60 NO2, molecular weight: 514.86]

[0113] Compound (25)

[0114]

[0115] : 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-3-oxopropan-1-amininium[3-(((3S,8S,9S,10R,13R,14S ,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16 ,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-3-oxopropan-1-aminium, Molecular formula: C 35 H 62 NO2, molecular weight: 528.89]

[0116] compound (26)

[0117]

[0118] : 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid[5-(((3S,8S,9S,10R,13 R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11, 12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid, molecular formula: C 34 H 56 O4, molecular formula: 528.82]

[0119] Compound (27)

[0120]

[0121] : 7-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoic acid[7-(((3S,8S,9S,10R,13 R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11, 12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoic acid, molecular formula: C 36 H 60 O4, molecular weight: 556.87]

[0122]

[0123] The above compound may be a compound derived from a sterol or an analog thereof.

[0124] The above sterol or analogue thereof is 7-ketocholesterol, 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol (27-HC), 24(S)-hydroxycholesterol (24S-HC), 22-hydroxycholesterol, 20(S)-hydroxycholesterol, 7-dehydrocholesterol, 24-dehydrocholesterol (Desmosterol), 5α-saturated sterol (Lathosterol), cholesterol amine, May include, but are not limited to, β-sitosterol, Campesterol, Stigmasterol, Avenasterol, Brassicasterol, Fucosterol, Sitostanol, Lanosterol, Tomatidine, Ursolic acid, alpha-tocopherol, etc.

[0125]

[0126] The above compound may include a pharmaceutically acceptable salt form exhibiting the same or similar activity.

[0127] As used herein, “pharmaceutically acceptable” means that the compound or salt thereof is not toxic to cells or humans exposed to it, and thus has a safety and efficacy profile suitable for administration to humans.

[0128] The above salt may be used in the form of either a pharmaceutically acceptable basic salt or an acid salt. The basic salt may be used in the form of either an organic basic salt or an inorganic basic salt, and may be selected from the group consisting of sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt.

[0129] Acid salts are useful as acid addition salts formed by free acids. Inorganic acids and organic acids can be used as free acids, and inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, diphosphoric acid, nitric acid, etc., and organic acids include citric acid, acetic acid, maleic acid, malic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, stearic acid, etc., but are not limited thereto, and all salts formed using various inorganic acids and organic acids commonly used in the art can be included.

[0130] In addition, the compound may include not only the above salts, but also all salts, hydrates, solvates, derivatives, etc. that can be prepared by conventional methods. Addition salts can be prepared by conventional methods, and can be prepared by dissolving in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, adding an excess amount of organic base, or adding an aqueous base solution of an inorganic base, and then precipitating or crystallizing. Alternatively, the addition salt can be obtained by evaporating the solvent or excess base from the mixture and then drying, or by suction filtration of the precipitated salt.

[0131]

[0132] The present invention provides a lipid nanoparticle composition comprising the above compound.

[0133] Preferably, the compound may be a sterol lipid.

[0134]

[0135] The lipid nanoparticle composition according to the present invention refers to a composition for drug delivery, which comprises the novel sterol compound described above and forms nanoparticles having an average diameter of 1 μm or less, preferably 500 nm or less, and more preferably 250 nm or less in an aqueous phase. The lipid nanoparticle composition may further comprise, in addition to the compound, at least one selected from the group consisting of ionized lipids, neutral lipids, sterol lipids, and polymerized lipids. In addition, the lipid nanoparticle composition may further comprise at least one polymer or polysaccharide composed of repeating monomers.

[0136] The above ionizable lipid is ((2-(6-aminopyridin-3-yl)ethyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((2-(6-aminopyridin-3-yl)ethyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate); ionizable lipid 1], (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); Ionized lipid 2], di((Z)-decenylyl)4,4'-((3-((4-(Z)-4-decenyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl)4,4'-((3-((4- ((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate; Ionized lipid 3], di((Z)-dec-4-en-1-yl) 4-(3-(((Z)-dec-4-en-1-yl)oxy)-3-oxopropyl)-4-(3-(piperidin-1-yl)propanamido)heptanedioate; Ionized lipid 4], 2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester [2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester; ALC-0315], 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester [8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester;SM-102], 4-(dimethylamino)butanoic acid, (10Z,13Z)-1-[(9Z,12Z)-9,12-octadecadien-1-yl]-10,13-nonadecadien-1-yl ester [-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl; DLin-MC3-DMA], and 1,1′-[[2-[4-[2-[2-[bis(2-hydroxydodecyl)amino]ethylamino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, 1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol; C12-200], but is not limited thereto.;

[0137] The above neutral lipid acts as a helper lipid to surround and protect the core formed by the interaction between ionized lipids and drugs in lipid nanoparticles, and may be selected from phospholipids or glycolipids that can promote the fusion of lipid nanoparticles.

[0138] Preferably, the phospholipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylglycerol (DPPG), and mixtures thereof. It may be, but is not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), more preferably.

[0139] Preferably, the glycolipid may be selected from the group consisting of glucosylceramide, galactosylceramide, glucosylsphingosine, galactosyl sphingosine, phosphoglycoceramide, and mixtures thereof, but is not limited thereto.

[0140] The above sterol lipid is a structural lipid that can function as a structural lipid that provides morphological rigidity to lipid filling within lipid nanoparticles and improves the stability of the nanoparticles, and can be at least one selected from the group consisting of cholesterol, bile acid, cholic acid, and derivatives thereof. For example, 7-ketocholesterol, 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol (27-HC), 24(S)-hydroxycholesterol (24S-HC), 22-hydroxycholesterol, 20(S)-hydroxycholesterol, 7-dehydrocholesterol, 24-dehydrocholesterol (Desmosterol), 5α-saturated sterol (Lathosterol), cholesteryl amine, May include, but are not limited to, β-sitosterol, Campesterol, Stigmasterol, Avenasterol, Brassicasterol, Fucosterol, Sitostanol, Lanosterol, Tomatidine, Ursolic acid, alpha-tocopherol, etc.

[0141] The above polymer-polymerized lipid is a pegylated lipid, which is a structure in which a water-soluble polymer and a lipid are combined, and contributes to the particle stability of the nanoparticle within the lipid nanoparticle. For example, the PEG-modified lipid may be selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkyl glycerol, and mixtures thereof, and preferably, PEG-modified dimyristoyl glycerol (DMG) (DMG-PEG), but is not limited thereto.

[0142] In addition, the PEGylated lipid may be a functionalized PEG in which a functional group is bonded to the side that is not bonded to the lipid. In this case, the usable functional group may be at least one selected from the group consisting of a succinyl group, a carboxylic acid, a maleimide, an n-hydroxysuccinimide, an amine group, biotin, a cyanuric group, and a folate, but is not limited thereto.

[0143] In addition, the composition may additionally include polymers, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic-co-glycolic acid) and poly(lactide-co-glycolide), and homopolymers comprising glycolic acid units and homopolymers comprising lactic acid units, PEGylated polymers and copolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA, and derivatives thereof). Additionally, acrylic polymers may include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, amino alkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic acid) polyacrylamide, amino alkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylate, and the like. In addition, cationic polymers such as poly(lysine), polyethylene imine (PEI), and poly(amidoamine) may also be included.

[0144] In addition, the composition may additionally include polysaccharides, such as chitosan, gelatin, collagen, mannan, dextran sulfate, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, fructooligosaccharide, isomaltooligosaccharide, inulin, hyaluronic acid, alginate, glycogen, amylose, carboxymethyldextran, beta-glucan, hydroxyethylcellulose, carboxymethylcellulose, fucoidan, or chondroitin.

[0145] The composition may comprise ionized lipids, neutral lipids, sterol lipids and polymerized lipids, preferably, the composition may comprise 0 to 65 mol% of ionized lipids, 2.5 to 70 mol% of neutral lipids, 25 to 75 mol% of sterol lipids and 0.5 to 5 mol% of polymerized lipids, more preferably, the composition may comprise 0 to 65 mol% of ionized lipids, 2.5 to 30 mol% of neutral lipids, 25 to 75 mol% of sterol lipids and 0.5 to 5 mol% of polymerized lipids, but is not limited thereto.

[0146] For example, the composition may have a ratio of ionized lipid: neutral lipid: sterol lipid: polymer polymer lipid of 60:5:33.5:1.5, 50:10:38.5:1.5, 40:15:43.5:1.5, 30:20:48.5:1.5, 20:25:53.5:1.5, 10:25:63.5:1.5, 0:25:73.5:1.5, 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, 19.5:25:53.5:2.0, 9.5:25:63.5:2.0, 0:25:73:2.0, 59:5:33.5:2.5, 49:10:38.5:2.5, 39:15:43.5:2.5, 29:20:48.5:2.5, 19:25:53.5:2.5, 9:25:63.5:2.5, 0:25:72.5:2.5, 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, 18.5:25:53.5:3.0, 8.5:25:63.5:3.0, It can be composed in one mol% ratio selected from the group consisting of 0:25:72:3.0, 58:5:33.5:3.5, 48:10:38.5:3.5, 38:15:43.5:3.5, 28:20:48.5:3.5, 18:25:53.5:3.5, 8:25:63.5:3.5, and 0:25:71.5:3.5, but is not limited thereto.

[0147] In addition, the composition is ionized lipid: neutral lipid: sterol lipid: polymer polymerized lipid in the ratio of 35:25:38.5:1.5, 25:35:38.5:1.5, 20:40:38.5:1.5, 15:55:28.5:1.5, 10:60:28.5:1.5, 0:65:33.5:1.5, 35:25:37.5:2.5, 25:35:37.5:2.5, 20:40:37.5:2.5, 15:55:27.5:2.5, 10:60:27.5:2.5, 0:65:32.5:2.5, 35:25:35:5, It can be composed in one mol% ratio selected from the group consisting of 25:35:35:5, 20:40:35:5, 15:55:25:5, 10:60:25:5, and 0:65:30:5, but is not limited thereto.

[0148] Preferably, some or all of the total sterol lipid ratio of the lipid nanoparticles may be replaced with the novel sterol lipid compound of the present invention. For example, the existing sterol lipid: novel sterol lipid may be composed in a mol% ratio selected from the group consisting of, but not limited to, 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10.

[0149]

[0150] The composition may further comprise a prophylactic or therapeutic agent.

[0151] The therapeutic or prophylactic agent may be one or more selected from the group consisting of, but not limited to, nucleic acids, peptides, proteins and protein-nucleic acid structures.

[0152] Specifically, the preventive or therapeutic agent 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.

[0153] The lipid nanoparticles according to the present invention can have a delivery rate of 10% or more, preferably 30% or more, and more preferably 50% or more to tissues other than the liver, such as the lungs or spleen. Conventional general lipid nanoparticles have the characteristic of being delivered to the liver by being surrounded by a protein coat in the blood, and therefore have a very low delivery rate to tissues other than the liver, such as the spleen or lungs, which is generally less than 5%. However, the novel lipid nanoparticles containing sterol lipids according to the present invention have the characteristic of being delivered to the spleen or lungs in addition to the liver, and therefore can be usefully utilized in the development of immunomodulators and anticancer agents using genes.

[0154] In addition, the lipid nanoparticle composition may also exhibit characteristics that facilitate absorption in acidic environments. While the general physiological environment is pH 7.4, the tumor microenvironment forms a slightly acidic condition with a pH below 7. Therefore, lipid nanoparticles that facilitate absorption in acidic environments can be utilized for cancer tissue delivery in vitro and in vivo.

[0155]

[0156] The lipid nanoparticle composition may be prepared as a lipid solution by dissolving it in a solvent miscible with ethanol or water. Separately, the active ingredient may be prepared as an active ingredient solution by dissolving it in a citric acid or acetic acid buffer having a pH of 4.0±1.5. In addition, the lipid solution and the active ingredient solution may 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 Nanoassembly, Precision Nanosystems) to prepare lipid nanoparticles.

[0157]

[0158] In addition, the present invention provides a drug delivery composition comprising the lipid nanoparticle composition described above; and a preventive or therapeutic agent.

[0159] The lipid nanoparticle composition comprising the above compound has excellent safety and stability, and can function as a delivery vehicle so that a gene including RNA, DNA, or a mixture thereof as an active ingredient can effectively exhibit its effect within a cell. Accordingly, the lipid nanoparticles manufactured by including the novel sterol lipid compound exhibit excellent gene encapsulation rate and in vivo gene delivery rate, and on the other hand, exhibit excellent gene delivery function even in delivery to tissues other than the liver, such as the spleen and lungs, and thus can be usefully utilized as a drug delivery composition.

[0160] Corresponding features can be substituted for the above-mentioned parts.

[0161]

[0162] 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.

[0163] 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.

[0164] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can 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.

[0165] The above drug delivery composition may be administered containing a pharmaceutically effective amount of a therapeutic or prophylactic agent. The effective dosage level of the therapeutic or prophylactic agent may 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 may be administered as an individual therapeutic agent or in combination with another therapeutic agent, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art. For example, the composition may be administered at 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 10 mg / kg.

[0166] 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.

[0167]

[0168] <Example 1> Preparation of compound

[0169] 1-1. Preparation of compound 1

[0170] Compound 1 was prepared as shown in the following reaction scheme 1.

[0171] [Reaction Formula 1]

[0172] [Correction pursuant to Rule 91, September 2025]

[0173] Specifically, compound 1-a (516 mg, 2.72 mmol) was dissolved in toluene (15 mL), thionyl chloride (862 mg, 7.24 mmol) was added at 30°C, stirred at 80°C for 3 hours, and then concentrated under reduced pressure. After diluting with dichloromethane (DCM) (20 mL), cholesterol (700 mg, 1.81 mmol), pyridine (143 mg, 1.81 mmol), and dimethylaminopyridine (DMAP) (221 mg, 1.81 mmol) were added, and stirred at 50°C for 8 hours. After completion of the reaction, the reaction was quenched with purified water (2 mL), concentrated under reduced pressure, and then diluted with dimethylformamide (DMF) (18 mL). The mixture was stirred for 15 minutes, filtered, washed with dimethylformamide (2 mL), and purified (prep-HPLC) to obtain compound 1 (yield: 13%).

[0174] 1H NMR (400 MHz, d6-DMSO) δ 5.39 (s, 1H), 4.55 - 4.75 (m, 1H), 4.45 (s, 2 H), 3.23 (s, 9H), 2.34 (d,J= 6.0 Hz, 2H), 1.72 - 2.01 (m, 5H), 1.23 - 1.65 (m, 11H), 1.05 - 1.22 (m, 7H), 0.99 (s, 6H), 0.90 (d,J= 5.2 Hz, 3H), 0.84 (d,J= 6.0 Hz, 6H), 0.65 (s, 3H).

[0175]

[0176] 1-2. Preparation of compound 2

[0177] Compound 2 was prepared as shown in the following reaction scheme 2.

[0178] [Reaction Formula 2]

[0179] [Correction pursuant to Rule 91, September 2025]

[0180] Specifically, compound 2-a (554 mg, 2.72 mmol) was dissolved in toluene (15 mL), and then thionyl chloride (862 mg, 7.24 mmol) was added at 30°C, stirred at 80°C for 3 hours, and then concentrated under reduced pressure. After diluting with dichloromethane (20 mL), cholesterol (700 mg, 1.81 mmol), pyridine (143 mg, 1.81 mmol), and dimethylaminopyridine (221 mg, 1.81 mmol) were added, and stirred at 50°C for 8 hours. After completion of the reaction, the reaction was quenched with purified water (2 mL), concentrated under reduced pressure, and then diluted with dimethylformamide (18 mL). The mixture was stirred for 15 minutes, filtered, washed with dimethylformamide (2 mL), and purified (prep-HPLC) to obtain compound 2 (yield: 15%).

[0181] 1H NMR (400 MHz, d6-DMSO) δ 5.39 (s, 1H), 4.55 - 4.75 (m, 1H), 3.63 (t,J= 6.0 Hz, 2H), 3.23 (s, 9H), 2.69 (t,J= 6.0 Hz, 2H), 2.34 (d,J= 6.0 Hz, 2H), 1.72 - 2.01 (m, 5H), 1.23 - 1.65 (m, 11H), 1.05 - 1.22 (m, 7H), 0.99 (s, 6H), 0.90 (d,J= 5.2 Hz, 3H), 0.84 (d,J= 6.0 Hz, 6H), 0.65 (s, 3H).

[0182]

[0183] 1-3. Preparation of compound 6

[0184] Compound 6 was prepared as shown in the following reaction scheme 3.

[0185] [Reaction Formula 3]

[0186] [Correction pursuant to Rule 91, September 2025]

[0187] Specifically, cholesterol (700 mg, 1.81 mmol), compound 6-a (335 mg, 2.17 mmol), 4-dimethylaminopyridine (442 mg, 3.62 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (521 mg, 2.72 mmol) were added to dichloromethane (20 mL) and stirred at 30°C for 12 hours. Purified water (50 mL) was added to the mixture to terminate the reaction, and the extracted solution was washed with saturated sodium chloride solution (brine) (50 mL*3 times), dried over sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure. The filtrate was purified by column (DCM:MeOH = 10:0 → 98:2) and purified (prep-HPLC) to obtain compound 6 (yield: 14%).

[0188] 1H NMR (400 MHz, CD3OD) δ 7.65 (s, 1H), 7.13 (s, 1H), 6.98 (s, 1H), 5.39 (d,J= 4.4 Hz, 1 H), 4.48 - 4.60 (m, 1H), 4.08 (t,J= 7.2 Hz, 2H), 2.25 - 2.35 (m, 4H), 2.03 - 2.13 (m, 3H), 1.80 - 2.02 (m, 4H), 1.47 - 1.68 (m, 7H), 1.27 - 1.44 (m, 4H), 1.08 - 1.23 (m, 7H), 0.97 - 1.07 (m, 6H), 0.95 (d,J= 6.4 Hz, 3H), 0.86 - 0.90 (m, 6H), 0.73 (s, 3H).

[0189]

[0190] 1-4. Preparation of compound 13

[0191] Compound 13 was prepared as shown in the following reaction scheme 4.

[0192] [Reaction Formula 4]

[0193] [Correction pursuant to Rule 91, September 2025]

[0194] Specifically, cholesterol (1.00 g, 2.59 mmol), compound 13-a (590 mg, 5.17 mmol), and triethylamine (TEA) (523 mg, 5.17 mmol) were added to acetone (20 mL) and stirred at 56°C for 26 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, dissolved in petroleum ether (PE) (20 mL), and purified (prep-HPLC) to obtain compound 13 (yield: 27%).

[0195] 1H NMR (400 MHz, CDCl3) δ 5.38 (d,J= 4.0 Hz, 1H), 4.55 - 4.70 (m, 1H), 2.44 (t,J= 7.2 Hz, 2H), 2.38 (t,J= 7.2 Hz, 2H), 2.32 (d,J= 7.6 Hz, 2H), 1.91 - 2.06 (m, 4H), 1.77 - 1.91 (m, 3H), 1.41 - 1.67 (m, 7H), 1.23 - 1.40 (m, 4H), 1.05 - 1.22 (m, 7H), 0.94 - 1.04 (m, 6H), 0.92 (d,J= 6.4 Hz, 3H), 0.85 - 0.90 (m, 6H), 0.68 (s, 3H).

[0196]

[0197] 1-5. Preparation of compound 14

[0198] Compound 14 was prepared as shown in the following reaction scheme 5.

[0199] [Reaction Formula 5]

[0200] [Correction pursuant to Rule 91, September 2025]

[0201] Specifically, cholesterol (387 mg, 1.00 mmol), compound 14-a (160 mg, 1.00 mmol), and dimethylaminopyridine (6.1 mg, 0.050 mmol) were dissolved in dichloromethane (10 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (192 mg, 1.00 mmol, hereinafter referred to as EDC-HCl) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in ethanol, and purified by column chromatography (C18) (water: acetonitrile = 9:1 → 5:5) to obtain compound 14 (yield: 55%).

[0202] 1H NMR (400 MHz, CDCl3) δ 5.38 - 5.37 (m, 1H), 4.65 - 4.57 (m, 1H), 2.36 (t,J= 7.51 Hz, 2H), 2.31 - 2.30 (m, 2H), 2.29 (t,J= 7.51 Hz, 2H), 2.03 - 1.93 (m, 2H), 1.87 - 1.78 (m, 3H), 1.70 - 0.94 (m, 26H), 1.02 (s, 3H), 0.91 (d,J= 6.52 Hz, 3H), 0.86 (dd,J= 6.64 Hz & 1.80 Hz, 6H), 0.68 (s, 3H).

[0203]

[0204] 1-6. Preparation of compound 15

[0205] Compound 15 was prepared as shown in the following reaction scheme 6.

[0206] [Reaction Formula 6]

[0207] [Correction pursuant to Rule 91, September 2025]

[0208] Specifically, cholesterol (387 mg, 1.00 mmol), compound 15-a (202 mg, 1.00 mmol), and dimethylaminopyridine (6.1 mg, 0.050 mmol) were dissolved in dichloromethane (10 mL), EDC-HCl (192 mg, 1.00 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, dissolved in ethanol, and column purified (C18) (water: acetonitrile = 9:1 → 5:5) to obtain compound 15 (yield: 31%).

[0209] 1H NMR (400 MHz, CDCl3) δ 5.38 - 5.37 (m, 1H), 4.65 - 4.57 (m, 1H), 2.35 (t,J= 7.51 Hz, 2H), 2.33 - 2.30 (m, 2H), 2.26 (t,J= 7.51 Hz, 2H), 2.03 - 1.94 (m, 2H), 1.87 - 1.78 (m, 3H), 1.65 - 0.94 (m, 34H), 1.02 (s, 3H), 0.91 (d,J= 6.52 Hz, 3H), 0.86 (dd,J= 6.64 Hz & 1.80 Hz, 6H), 0.68 (s, 3H).

[0210]

[0211] <Example 2> Preparation of lipid nanoparticles

[0212] Lipid nanoparticles (LNPs) encapsulating Firefly Luciferase mRNA (ENAGENE, Korea) were prepared using the compound (sterol lipid) prepared in Example 1, ionized lipid, dioleoylphosphatidylethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (sterol lipid), and PEG 2000-DMG (polymer lipid). However, compound 12 was purchased from Sigma-Aldrich and used.

[0213] Ionized lipids, DOPE / DSPC, cholesterol and PEG 2000-DMG were prepared in a molar % ratio of 50:10:38.5:1.5 (composition indicated as A) or 49:10:38.5:2.5 (composition indicated as B) 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 of pH 4.0±1.0 or an acetic acid buffer solution of pH 5.0±1.0 (composition indicated as *) at a weight ratio of mRNA:ionized lipid of 1:16 to prepare an active ingredient solution. At this time, the new sterols (compound 1, compound 2) and cholesterol were used in the mol% ratios of 50:50, 75:25, and 100:0 as total sterol lipids, or the new sterols (compound 6) and cholesterol were used in the mol% ratios of 10:90, 25:75, and 50:50, or the new sterols (compound 12, compound 13, compound 14, and compound 15) and cholesterol were used in the mol% ratios of 10:90, 20:80, and 30:70. However, for the formulations using the new sterols (compounds 12, 13, and 14), formulations were also manufactured in which the molar percentage of other additives was reduced to 95% or 90% and instead 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was added to the molar percentage of 5% or 10%.

[0214] Thereafter, lipid nanoparticles were manufactured by passing the lipid solution:active ingredient solution through a microfluidic mixing device (Benchtop Nanoassembly, Precision Nanosystems) at a flow rate of approximately 15 mL / min so that the volume ratio was 1:3. The manufactured lipid nanoparticles were diluted with Tris buffer containing 8.7% sucrose and dialyzed using a dialysis centrifuge tube so that the ethanol content was less than 1%, and the final concentration was manufactured to be 0.2 mg / mL based on mRNA.

[0215] For comparison, lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNA Gene, Korea) were prepared using SM-102 ionizable lipid, distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (sterol lipid), and DMG-PEG 2000 (polymer lipid). SM-102, DSPC, cholesterol, and DMG-PEG 2000 were prepared in a molar % ratio of 50:10:38.5:1.5, which is the same as that used in SpikeVax, and were used as lipid nanoparticles for comparison.

[0216] Lipid Nanoparticle (LNP) Number Sterol Lipid Ionized Lipid Mole % of New Sterol in Total Sterol Lipid (DOTAP Mole %) Compound 1A Compound 1 Ionized Lipid 450 Compound 1B Compound 1 Ionized Lipid 475 Compound 1C Compound 1 Ionized Lipid 4100 Compound 1D Compound 1 Ionized Lipid 350 Compound 1E Compound 1 Ionized Lipid 375 Compound 1F Compound 1 Ionized Lipid 3100 Compound 1G Compound 1 SM-10275 Compound 2A Compound 2 Ionized Lipid 450 Compound 2B Compound 2 Ionized Lipid 475 Compound 2C Compound 2 Ionized Lipid 4100 Compound 6A Compound 6 Ionized Lipid 310 Compound 6B Compound 6 Ionized Lipid 325 Compound 6C Compound 6 Ionized Lipid 350 Compound 12A Compound 12 Ionized Lipid 430 Compound 12B Compound 12 Ionized Lipid 430 (5) Compound 13A Compound 13 Ionized Lipid 410 Compound 13B Compound 13 Ionized Lipid 420 Compound 13C Compound 13 Ionized Lipid 430 Compound 13D Compound 13 Ionized Lipid 430 (5) Compound 13E Compound 13 Ionized Lipid 430 (10) Compound 13F Compound 13SM-10230 Compound Compound 13G Compound 13SM-10230 (5) Compound 14A Compound 14 Ionized Lipid 430 Compound 14B Compound 14 Ionized Lipid 430 (5) Compound 15A Compound 15 Ionized Lipid 410 Compound 15B Compound 15 Ionized Lipid 420 Compound 15C Compound 15 Ionized Lipid 430 Compound 15D Compound 15 Ionized Lipid 430 (5) Compound 15E Compound 15 Ionized Lipid 430 (10) Compound 15F Compound 15SM-10230 Compound 15G Compound 15SM-10230 (5) Control 1-Ionized Lipid 40 Control 2-Ionized lipid 30-unit 3-SM-1020

[0217]

[0218] <Analysis Example 1> Confirmation of lipid nanoparticle size and polydispersity

[0219] The size and surface charge of the lipid nanoparticles of Example 2, which were prepared by including the compound of Example 1, were measured. To measure the size of the lipid nanoparticles, the concentration of mRNA contained in each lipid nanoparticle was diluted with PBS to 1 μg / mL, and the diameter and polydispersity index (PDI) of the lipid nanoparticles (LNPs) were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0220] As a result, as shown in Table 2 below, all lipid nanoparticles exhibited a size of approximately 140 nm or less and a good polydispersity of less than 0.2.

[0221] LNP Number Particle Size (nm) PDI Compound 1A1000.15 Compound 1B1070.26 Compound 1C790.16 Compound 1D880.07 Compound 1E1230.11 Compound 1F670.08 Compound 1G850.12 Compound 2A940.17 Compound 2B920.15 Compound 2C830.13 Compound 6A1000.03 Compound 6B1050.04 Compound 6C1050.03 Compound 12A730.08 Compound 12B610.09 Compound 13A780.07 Compound 13B710.08 Compound 13C670.08 Compound Compound 13D620.10 Compound 13E730.08 Compound 13F750.12 Compound 13G640.09 Compound 14A790.12 Compound 14B700.09 Compound 15A860.15 Compound 15B910.16 Compound 15C950.18 Compound 15D1040.11 Compound 15E880.08 Compound 15F900.14 Compound 15G1060.12 Control 1790.04 Control 21070.03 Control 3750.09

[0222]

[0223] <Analysis Example 2> Confirming Gene Encapsulation Rate

[0224] To confirm the gene encapsulation rate of lipid nanoparticles including the compound of Example 1 above, Quant-iT TM Ribogreen TM RNA Reagent and Kit were used. 5 μL of ribosomal RNA standard (100 μg / mL in TE buffer) was taken and diluted with 245 μL TE buffer or 0.4% Triton-TE buffer to prepare a stock solution for the 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, and then added to Quant-iTTM Ribogreen. TM After adding 100 μL of RNA Reagent and mixing, the fluorescence intensity (excitation 475 nm and emission 500-550 nm) was measured to create a calibration curve. 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 TE buffer or 2% Triton-TE buffer was added to 50 μL of the diluted nanoparticle solution, and incubated at 37°C for about 10 minutes. 40 μL of each culture medium was then placed in a microplate, and 60 μL of TE buffer and Quant-iT were added. TM Ribogreen TM 100 μL of RNA Reagent was sequentially added, mixed, and the fluorescence intensity was measured. The measured fluorescence intensity was applied to the calibration curve to calculate the gene amount in TE buffer (TO) and in 2% Triton-TE buffer (T2), and then the gene inclusion rate was calculated using Equation 1 below.

[0225] [Formula 1]

[0226]

[0227]

[0228] Table 3 below shows the gene encapsulation rate calculated according to the above formula 1, and it was confirmed that all compounds showed a good gene encapsulation rate of 85% or more.

[0229] LNP number Gene inclusion rate (%) Compound 1A93.0 Compound 1B94.4 Compound 1C96.1 Compound 1D81.6 Compound 1E89.5 Compound 1F92.8 Compound 1G92.2 Compound 2A90.8 Compound 2B91.2 Compound 2C93.4 Compound 6A93.6 Compound 6B71.0 Compound 6C68.1 Compound 12A90.7 Compound 12B93.7 Compound 13A84.6 Compound 13B87.9 Compound 13C85.5 Compound 13D94.1 Compound 13E93.9 Compound 13F87.9 Compound 13G93.7 Compound Compound 14A89.8 Compound 14B90.4 Compound 15A93.5 Compound 15B93.7 Compound 15C92.0 Compound 15D89.8 Compound 15E91.6 Compound 15F90.7 Compound 15G89.1 Control 191.7 Control 294.0 Control 393.4

[0230]

[0231] <Analysis Example 3> Confirmation of lipid nanoparticle gene delivery efficiency

[0232] 3-1. Confirmation of intracellular gene transfer efficiency 1

[0233] In order to confirm the intracellular gene delivery efficiency of lipid nanoparticles including the compound of Example 1, the reporter gene FLuc mRNA was encapsulated in the lipid nanoparticles and its intracellular expression efficiency was evaluated.

[0234] Since lipid nanoparticles mediate the LDL receptor (low-density lipoprotein receptor), a cell surface receptor, as a mechanism for intracellular uptake, HeLa cell line (Korea Cell Line Bank, Seoul), a human cervical cancer cell line with high expression of LDL receptor on the cell surface, was used. Cell culture was performed using MEM (minimum essential medium) medium (Gibco) containing 10% fetal bovine serum (Gibco, NY, USA) and 1% penicillin / streptomycin (Gibco) at 37°C and 5% CO2. Before lipid nanoparticle treatment, HeLa cell line was seeded at 2 × 10 per well. 4 Cells were cultured in 96-well plates, and after stabilization for 16 to 24 hours, lipid nanoparticles at a concentration of 0.25 μg / mL based on the encapsulated mRNA were diluted in cell culture medium and treated to the cells.

[0235] To confirm the expression of intracellular luciferase, 6 hours after lipid nanoparticle treatment, the culture medium in the wells was removed, washed with PBS, and 100 μL of Glo-Lysis Buffer (Promega, WI, USA) was added to lyse the cells at room temperature for 5 minutes. Next, 50 μL of each cell lysate and Steady-Glo™ Luciferase Assay solution (Promega) were added at a 1:1 ratio to a 96-well white plate, and after incubation at room temperature for 5 minutes, the luminescence value was detected using a GloMax Discover microplate reader (Promega). A calibration curve was created using recombinant luciferase (Promega) as a standard, and the luciferase protein concentration (pg / mL) of the cell lysate was derived from this.

[0236] As a result, as shown in Table 4 below, it was confirmed that the lipid nanoparticles including the compound of Example 1 exhibited good gene delivery efficacy into cells.

[0237] LNP number luciferase expression level (Mean ± SD, pg / mL), HeLa cell Compound 1D3 69,682 ± 44,489 Compound 1F3 28,767 ± 27,824 Compound 6A3 49,509 ± 22,373 Compound 6B9 2,820 ± 7,269 Compound 6C30,707 ± 1,892 Compound 12A1 08,403 ± 3,095 Compound 12B2 82,458 ± 16,107 Compound 13C2 8,145 ± 1,205 Compound 13D2 17,795 ± 4,149 Compound 13E4 15,744 ± 35,397 Compound 15D20,248 ± 1,012 Contrast 3168,526 ± 8,317

[0238]

[0239] 3-2. Confirmation of intracellular gene transfer efficiency 2

[0240] To confirm the pH-dependent intracellular gene delivery efficiency of lipid nanoparticles including the compound of Example 1, lipid nanoparticles encapsulating the FLuc mRNA gene were used to analyze the intracellular expression efficiency of mRNA according to pH changes.

[0241] Specifically, the pH of the cell culture medium treating lipid nanoparticles was adjusted to pH 7.4, pH 6.5, and pH 6.0, respectively, and the intracellular luciferase expression levels were compared under the same conditions. The cell lines used in this experiment are HeLa (Korea Cell Line Bank) used in the above Analysis Example 3-1, and the normal cell lines HEK293 (human embryonic kidney 293) and NIH3T3 (mouse embryonic fibroblast) cell lines (Korea Cell Line Bank). The HeLa and HEK293 cell lines were cultured using MEM medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Gibco), and the NIH3T3 cell line was cultured using DMEM (Dulbecco's Modified Eagle Medium) medium (Gibco) containing the above supplements in the same composition at 37°C and 5% CO2 conditions. Before lipid nanoparticle treatment, HeLa cell lines were seeded at 2 × 10 per well. 4 Cell number, HEK293 cell line, 2.5 × 10 per well 4 Cell count and NIH3T3 cell line were 1.5 × 10 per well. 4 Cells were cultured in 96-well plates to reach 80–90% confluence upon cell stabilization. After 16–24 hours of stabilization, lipid nanoparticles at a concentration of 0.25 μg / mL based on the encapsulated mRNA were diluted in cell culture media adjusted to different pHs and treated to the cells.

[0242] To confirm the expression of luciferase in cells, the same conditions as the analysis method presented in Analysis Example 3-1 were used, and the luciferase protein concentration (pg / mL) value of the cell lysate was derived.

[0243] As a result, as shown in Table 5 below, it was confirmed that lipid nanoparticles including the compound of Example 1 exhibited limited absorption at pH 7.4 and relatively high intracellular gene delivery efficacy under weakly acidic conditions of pH 6.5 or pH 6.0. While the general physiological environment is pH 7.4, the tumor microenvironment forms weakly acidic conditions below pH 7, and thus can be utilized for in vitro and in vivo cancer tissue delivery.

[0244] LNP number cell line, pH luciferase expression level (Mean ± SD, pg / mL) Compound 15D HeLa, pH 7.4 21,522 ± 419 HeLa, pH 6.5 82,084 ± 1,935 HeLa, pH 6.0 119,819 ± 17,150 HEK293, pH 7.47,126 ± 2,033 HEK293, pH 6.5 94,708 ± 2,929 HEK293, pH 6.0 77,844 ± 1,880 NIH3T3, pH 7.4 14,978 ± 645 NIH3T3, pH 6.5 99,913 ± 8,440 NIH3T3, pH 6.0 369 ± 87

[0245]

[0246] 3-3. Confirmation of in vivo gene transfer efficiency

[0247] In order to confirm the in vivo gene transfer efficiency of lipid nanoparticles including the compound of Example 1, lipid nanoparticles were intravenously injected into 7-week-old C57BL / 6 strain mice at a dose of 0.5 to 1 mg / kg based on the luciferase gene loaded therein, and after 4 hours, the mice were sacrificed and tissues were extracted, or after intramuscular injection, the mice were sacrificed and tissues were extracted after 6 hours.

[0248] To determine the level of luciferase expression in liver, spleen, and lung tissue samples, the excised tissues were transferred to tubes containing 3 mm metal beads, 500 μL of Glo-Lysis Buffer (Promega) per 50 mg was added, and the tissues were homogenized using a bead homogenizer. The homogenate was centrifuged at 1,000 g for 10 minutes, and the supernatant was diluted to an appropriate ratio using Glo-Lysis Buffer. The diluted tissue homogenate and Steady-Glo Luciferase Assay (Promega) solution were each transferred to a 96-well white plate at a 1:1 ratio, and incubated at room temperature for 5 minutes. Next, the luminescence value was detected using a GloMax Discover microplate reader (Promega), and a calibration curve was generated using recombinant luciferase (Promega) as a standard, through which the luciferase expression level of the sample (ng / g tissue) was derived.

[0249] As a result, as shown in Table 6 below, the lipid nanoparticles containing the compound of Example 1 exhibited a high level of luciferase expression in lung tissue upon intravenous injection, and through this, compared to the general LNPs, which have a very low lung delivery rate of less than 3%, the LNPs of the present invention exhibited a function as a gene delivery vehicle with excellent lung tissue delivery efficacy.

[0250] Compound number Intravenous injection luciferase expression level (Mean ± SD, ng / g tissue) Liver tissue Spleen tissue Lung tissue Compound 1A1,112 ± 3111,036 ± 2651,419 ± 327 Compound 1B5,12 ± 72698 ± 1622,115 ± 517 Compound 1C1,95 ± 21528 ± 54477 ± 165

[0251]

[0252] As shown in Table 7 below, the lipid nanoparticles containing the compound of Example 1 exhibited a high level of luciferase expression in spleen tissue upon intravenous injection, thereby demonstrating that the LNP of the present invention functions as a gene delivery vehicle with excellent spleen tissue delivery efficacy, compared to the very low spleen delivery rate of less than 5% in general LNPs.

[0253] Compound number Intravenous injection luciferase expression level (Mean ± SD, ng / g tissue) Liver tissue Spleen tissue Compound 12A8 90 ± 590 1,703 ± 163 Compound 12B2 901 ± 1,489 1,965 ± 288 Compound 13A5 4,637 ± 23,963 998 ± 169 Compound 13B2 114 ± 2,654 1,849 ± 324 Compound 13C1 76 ± 1,281,296 ± 437 Compound 13D1 812 ± 787 2,272 ± 490 Compound 13E3 708 ± 1,117 1,499 ± 167 Compound 15D2 854 ± 1,3531,757 ± 151

[0254]

[0255] As shown in Table 8 below, it was confirmed that the lipid nanoparticles containing the compound of Example 1 can exhibit another characteristic of increasing the amount of luciferase expression in muscle tissue upon intramuscular injection.

[0256] Compound number Muscle injection Fluorescent luciferase expression (Mean ± SD, ng / g tissue) Muscle tissue Compound 6A 1 1,616 ± 7,148 Compound 6B 1 2,751 ± 5,412 Control 28,604 ± 3,263

[0257]

[0258] In summary, as shown in FIG. 1, the control LNP (control 1) without a compound according to an embodiment of the present invention had a lung delivery amount of less than 1% compared to the liver, whereas the LNP including a compound according to an embodiment of the present invention (e.g., compound 1A, compound 1B, etc.) exhibited excellent tissue targeting characteristics, delivering more than 80% to the lung.

[0259] In addition, as shown in FIG. 2, it can be confirmed that the control LNPs (control 1, control 3) without a compound according to an embodiment of the present invention have an amount of 1 to 2% of spleen delivery compared to the liver, whereas the LNPs including a compound according to an embodiment of the present invention (e.g., compound 13C, compound 13D, etc.) have excellent tissue targeting characteristics of delivering 60 to 90% or more to the spleen.

[0260]

[0261] In conclusion, LNPs containing a novel sterol compound according to one embodiment of the present invention exhibited high cell delivery ability in a pH-sensitive manner and excellent tissue-specific delivery properties to organs other than the liver, such as the lungs or spleen.

[0262]

[0263] 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

A lipid nanoparticle composition comprising a compound selected from the group consisting of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt, a tautomer, or a stereoisomer thereof: <Chemical Formula 1> In the above chemical formula 1, X is -NR 1 -, -C(=O)NR 1 -, -NR 1 C(=O)-, -OC(=O)NR 1 -, -NR 1 C(=O)O-, -C(=S)NR 1 -, -NR 1 C(=S)-, -OC(=S)NR 1 -, -NR 1 C(=S)O-, -SC(=O)NR 1 -, -NR 1 C(=O)S-, -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)S-, -SC(=O)O-, -OC(=O)S-, -S- or -SS-, and the R 1 are each independently covalently bonded, H, C 1-6 Alkyl or C with or without branching 2-6 is selected from one of the alkenyl groups having or not having a branch, L is C 1-12 alkylene, C 2-12 is selected from among alkenylene or alkynylene, and in said alkylene, alkenylene or alkynylene, at least one -CH2- is -O-, -CH(OH)-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OP(=O)(OR 2 )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)NR 2 -, -NR 2 C(=O)-, -OC(=O)NR 2 -, -NR 2 C(=O)O-, -C(=S)NR 2 -, -NR 2 C(=S)-, -OC(=S)NR 2 -, -NR 2 C(=S)O-, -SC(=O)NR 2 - or -NR 2 C(=O)S- is substituted or unsubstituted with any one of the above R 2 are each independently covalently bonded, H, C 1-6 Alkyl or C with or without branching 2-6 is selected from one of the alkenyl groups having or not having a branch, Y is selected from one of a tertiary or quaternary amine, a carboxylic acid or a phosphoric acid, A is C 1-20 Alkyl or C with or without branching 2-20 Selected from alkenyl with or without branching. In the first paragraph, The above compound is, A lipid nanoparticle composition characterized by comprising a compound represented by the following chemical formula 1-1: <Chemical Formula 1-1> In the above chemical formula 1-1, X, L and Y are the same as X, L and Y in the above chemical formula 1, R is hydrogen, C 1-4 Alkyl or C 2-4 Selected from alkenyl. In the first paragraph, The above Y is, A lipid nanoparticle composition characterized by being selected from the following structures: In the first paragraph, The above compound is, A lipid nanoparticle composition characterized by being selected from the group consisting of the following compounds: (1) 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethane-1-amininium; (2) 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-3-oxopropan-1-amininium; (3) 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-4-oxobutan-1-amininium; (4) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(dimethylamino)propanoate; (5) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate; (6) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1H-imidazol-1-yl)butanoate; (7) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(2-methyl-1H-imidazol-1-yl)butanoate; (8) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1H-imidazol-4-yl)butanoate; (9) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(piperidin-1-yl)propanoate; (10) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(4-methylpiperazin-1-yl)propanoate; (11) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(pyridin-4-yl)propanoate; (12) 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutanoic acid; (13) 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid; (14) 7-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoic acid; (15) 10-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-10-oxodecanoic acid; (16) (4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-oxobutyl)phosphonic acid; (17) (6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-6-oxohexyl)phosphonic acid; (18) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(phosphonooxy)formanoate; (19) (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-(phosphonooxy)pentanoate; (20) 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-N,N,N-trimethyl-2-oxoethane-1-aminum; (21) 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-N,N,N-trimethyl-3-oxopropan-1-aminum; (22) 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-5-oxopentanoic acid; (23) 7-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)amino)-7-oxoheptanoic acid; (24) 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-2-oxoethane-1-amininium; (25) 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-N,N,N-trimethyl-3-oxopropan-1-amininium; (26) 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-5-oxopentanoic acid; and (27) 7-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5S)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-7-oxoheptanoic acid. In the first paragraph, The above compound is, A lipid nanoparticle composition characterized by being a sterol lipid. In the first paragraph, The above composition, A lipid nanoparticle composition characterized in that it further comprises at least one selected from the group consisting of ionized lipids, neutral lipids, sterol lipids, and polymerized lipids. In paragraph 6, The above ionized lipids are, ((2-(6-aminopyridin-3-yl)ethyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((2-(6-aminopyridin-3-yl)ethyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate); ionizable lipid 1], (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); Ionized lipid 2], di((Z)-decenylyl)4,4'-((3-((4-(Z)-4-decenyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate [di((Z)-dec-4-en-1-yl)4,4'-((3-((4- ((Z)-dec-4-en-1-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)azanediyl)dibutanoate; Ionized lipid 3], di((Z)-dec-4-en-1-yl) 4-(3-(((Z)-dec-4-en-1-yl)oxy)-3-oxopropyl)-4-(3-(piperidin-1-yl)propanamido)heptanedioate; Ionized lipid 4], 2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester [2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexanediyl] ester; ALC-0315], 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester [8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester;SM-102], 4-(dimethylamino)butanoic acid, (10Z,13Z)-1-[(9Z,12Z)-9,12-octadecadien-1-yl]-10,13-nonadecadien-1-yl ester [-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl; A lipid nanoparticle composition characterized by at least one selected from the group consisting of DLin-MC3-DMA], and 1,1′-[[2-[4-[2-[2-[bis(2-hydroxydodecyl)amino]ethylamino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol, 1,1′-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol; C12-200]. In paragraph 6, The above neutral lipids are, A lipid nanoparticle composition characterized by being selected from phospholipids or glycolipids. In paragraph 6, The above sterol lipids are, A lipid nanoparticle composition characterized by comprising at least one selected from the group consisting of cholesterol, bile acid, cholic acid and derivatives thereof. In paragraph 6, The above polymer polymerized lipid is, A lipid nanoparticle composition characterized by being selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In paragraph 6, The above composition, A lipid nanoparticle composition comprising 0 to 65 mol% of ionized lipid, 2.5 to 70 mol% of neutral lipid, 25 to 75 mol% of sterol lipid, and 0.5 to 5 mol% of polymerized lipid. In the first paragraph, The above composition, A lipid nanoparticle composition characterized by having a delivery power of 30% or more to the lungs or spleen as a tissue other than the liver. In the first paragraph, The above composition, A lipid nanoparticle composition characterized in that it further comprises a preventive or therapeutic agent. A lipid nanoparticle composition according to claim 1; and a drug delivery composition comprising a preventive or therapeutic agent. In paragraph 14, The above preventive or therapeutic agent, A drug delivery composition characterized by comprising 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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