Novel histidine amide cholesterol compounds and lipid nanoparticles for RNA delivery containing the same

Abstract

This invention relates to a novel histidineamide cholesterol compound and lipid nanoparticles for RNA delivery containing the same. According to this invention, it is possible to improve mRNA delivery efficiency and expression efficiency while maintaining biocompatibility.

Description

Technical Field

[0001] The present invention relates to a novel histidine amide cholesterol compound and lipid nanoparticles for RNA delivery comprising the same. Background Art

[0002] With the global COVID-19 pandemic, lipid nanoparticle (LNP) vaccines for mRNA delivery have been actively researched. Moderna and BioNTech / Pfizer have developed lipid nanoparticle vaccines to deliver mRNA encoding the SARS-CoV-2 viral spike glycoprotein and obtained emergency use authorization from the FDA. Since this authorization, the development of new lipid nanoparticles aiming at the treatment of other diseases including COVID-19 has been promoted worldwide.

[0003] Disease treatment via mRNA delivery has many advantages over conventional treatments using DNA. Unlike DNA, mRNA does not integrate into the host genome, so it does not cause genetic modification of cells, and does not require going through various procedures for protein production compared to DNA. In the case of conventional DNA, gene transcription does not start unless it passes through the cytoplasm and enters the nucleus, whereas in the case of mRNA, protein translation is performed in the cytoplasm without needing to enter the nucleus, which allows the desired therapeutic protein to be effectively expressed intracellularly. However, naked mRNA alone cannot achieve sufficient protein expression to obtain the desired therapeutic effect. mRNA is unstable, is easily degraded by RNases in vivo, has a large molecular weight, high negative charge density, and causes electrostatic repulsion with negatively charged cell membranes, making it difficult to pass through cell membranes. To overcome these limitations, lipid nanoparticles are used for effective and stable mRNA delivery in vivo.

[0004] Lipid nanoparticles are composed of ionizable lipids, auxiliary lipids (phospholipids), cholesterol, and PEG-lipids. Ionizable lipids play a crucial role in transporting RNA into the cytoplasm; in acidic pH environments, they become positively charged, allowing them to condense with negatively charged RNA through electrostatic attraction. On the other hand, ionizable lipids have a neutral charge at physiological pH and are non-toxic, but after absorption into cells, the low pH within endosomes causes the ionizable lipids to become positively charged, destabilizing the endosomal membrane and thereby promoting mRNA release. This allows for stable delivery of mRNA into the cytoplasm. Moderna has produced an mRNA-1273 vaccine using SM-102 as an ionizable lipid, while BioNTech / Pfizer has produced a BNT162b2 vaccine using ALC-0315 as a component. Other ionizable lipids such as DLin-MC3-DMA(MC3) and C12-200 have also been developed.

[0005] Ionizable lipids consist of a head, a linker, and a tail. The head has an ionizable portion and mainly contains amine, guanidine, and imidazole groups. Conventional MC3 and ALC-0315 have tertiary amine groups, and ionization occurs in a pH-dependent manner. By regulating this, hydrogen bonding interactions with RNA can be improved. The linker affects the stability and biodegradability of the formed lipid nanoparticles. When an ester bond is used as the linker, biodegradability increases and gene transfer efficiency increases, while in the case of a disulfide bond, degradation occurs according to the degree of intracellular reduction, enabling selective LNP degradation and gene transfer. When such a biodegradable linker is present, it is rapidly removed in vivo, which has the advantage of reducing side effects and toxicity. The tail is the hydrophobic portion and affects the pKa and fluidity of LNPs.

[0006] In lipid nanoparticles, cholesterol is known to contribute to structural stability and is involved in endosomal transport and recycling mechanisms. In this invention, while maintaining the structural stability characteristic of conventional cholesterol, we developed ionizable cholesterol by introducing an ionizable amine into it to further facilitate endosomal escape and ultimately improve protein expression efficiency. We then used this ionizable cholesterol to fabricate lipid nanoparticles, thus completing the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide histidineamide cholesterol (3β[L-histidinamide-carbamoyl]cholesterol; Hchol), a cholesterol-modified substance, and lipid nanoparticles containing the same, in order to improve mRNA delivery effect and expression efficiency while maintaining biocompatibility. [Means for solving the problem]

[0008] To achieve the above objective, the present invention provides a histidineamide cholesterol compound represented by the following formula (1), its stereoisomer, or a pharmaceutically acceptable salt thereof. [ka]

[0009] In another embodiment, the present invention provides lipid nanoparticles comprising a histidineamide cholesterol compound represented by formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0010] The lipid nanoparticles are further characterized by comprising one or more selected from the group consisting of ionizable lipids, phospholipids, cholesterol, and PEG lipids.

[0011] The ionizable lipid is characterized by being SM-102 or DLin-MC3-DMA.

[0012] The phospholipid is characterized by being DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine).

[0013] The PEG lipid is characterized by being DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000).

[0014] The ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts thereof; cholesterol; and PEG lipids are characterized by being mixed in a molar ratio of 10-60:1-20:5-70:0-30:0.5-5.

[0015] In yet another aspect, the present invention provides a composition for delivering nucleic acids, proteins, or drugs, comprising the lipid nanoparticles.

[0016] The nucleic acid is characterized by being selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0017] The protein is characterized by being an antibody.

[0018] The drug is characterized in that it is a therapeutic agent or a preventive agent.

[0019] In yet another embodiment, the present invention provides a vaccine composition comprising the lipid nanoparticles. [Effects of the Invention]

[0020] According to the present invention, lipid nanoparticles produced using Hchol can improve intracellular gene transfer efficiency and protein expression efficiency because Hchol interacts with mRNA inside the nanoparticles to increase encapsulation efficiency and contributes to the stabilization of the lipid nanoparticles.

[0021] In addition, the lipid nanoparticles are biocompatible and can effectively deliver and express mRNA into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] 1 shows the synthesis procedure of histidine amide cholesterol according to an embodiment of the present invention. [Figure 2] 2 shows 1H NMR analysis data of histidine amide cholesterol according to an embodiment of the present invention. [Figure 3] 3 shows molecular weight analysis data of histidine amide cholesterol according to an embodiment of the present invention. [Figure 4] 4 shows the pKa value analysis results of lipid nanoparticles according to an embodiment of the present invention. [Figure 5] 5 shows the gene encapsulation efficiency analysis results of lipid nanoparticles according to an embodiment of the present invention. [Figure 6] 6 shows the results of analysis on the stability over time and gene encapsulation efficiency of lipid nanoparticles according to an embodiment of the present invention. [Figure 7] 7 shows the evaluation results of intracellular gene transfer efficiency of lipid nanoparticles according to an embodiment of the present invention. [Figure 8] 8 shows the evaluation results of intracellular toxicity of lipid nanoparticles according to an embodiment of the present invention. [Figure 9] 9 shows the results of analysis on intracellular delivery and protein expression efficiency of lipid nanoparticles according to an embodiment of the present invention. [Figure 10]The results of in vivo gene transfer efficiency and expression analysis of lipid nanoparticles according to one embodiment of the present invention are shown. (A) Lipid nanoparticles prepared using SM102 lipid were administered by intramuscular injection at a dose of 0.1 mg / kg. (B) Lipid nanoparticles prepared using MC3 lipid with Hchol adjusted to 50%, 75%, and 100% in the sterol ratio, respectively, were administered by intramuscular injection at a dose of 0.1 mg / kg. [Modes for carrying out the invention]

[0023] The present invention will be described in detail below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by skilled experts in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0024] In this specification, the term "lipid nanoparticle" or "LNP" refers to lipid-based particles in the submicron range that are drug delivery bodies capable of encapsulating poorly soluble substances. Lipid nanoparticles consist of ionizable lipids capable of binding to anions, phospholipids to maintain the lipid bilayer structure of the lipid nanoparticles, cholesterol to maintain the particles in a rigid and stable manner, and polyethylene glycol (PEG) to prevent aggregation between particles and ensure structural stability.

[0025] In the present invention, the lipid nanoparticles may be, but are not limited to, being used for nucleic acid, protein, or drug delivery.

[0026] In the present invention, the nucleic acid may be DNA, RNA, or PNA, and preferably mRNA.

[0027] In the present invention, the protein may be an antibody in one embodiment, or a peptide in another embodiment.

[0028] In the present invention, the drug may be a therapeutic agent or a preventive agent, and may, for example, be a compound.

[0029] Therefore, in the present invention, the lipid nanoparticles can be loaded with target-specific antibodies, proteins, fluorescent dyes, etc., depending on the disease.

[0030] In other words, the present invention relates, from another perspective, to the use of lipid nanoparticles for the delivery of nucleic acids, proteins, or drugs. Furthermore, the present invention can provide the use of said lipid nanoparticles in the manufacture of pharmaceuticals for the delivery of nucleic acids, proteins, or drugs.

[0031] In this specification, the term "ionizable lipid" refers to a main component of lipid nanoparticles that directly bind to mRNA through electrostatic attraction and determine the efficiency of mRNA delivery and release, possessing an appropriate range of acid dissociation constants (pKa) and a structure in which a tertiary amine head and a long hydrocarbon tail are linked by a linker, with the degree of ionization changing depending on the pH in the body.

[0032] In the present invention, the ionizable lipid can be characterized as a cationic lipid, but is not limited thereto.

[0033] In the present invention, the cationic lipid refers to a lipid that exhibits cationic properties at a neutral pH, and preferably SM-102 or DLin-MC3-DMA is used, but is not limited thereto.

[0034] In this specification, the term "phospholipid" includes all amphiphilic lipids having two fatty acid moieties, a hydrophilic moiety based on a phosphate structure, and are involved in maintaining the lipid bilayer structure of lipid nanoparticles and ensuring structural stability.

[0035] In the present invention, the phospholipid used is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), but is not limited thereto.

[0036] In this specification, the term "cholesterol" refers to a type of sterol, which is a combination of a steroid and an alcohol, and includes all cholesterol derivatives that have a cholesterol structure. Cholesterol is a lipid present in the cell membranes of all animal cells and is an organic molecule transported via the blood. Cholesterol plays a role in making lipid nanoparticles more robust and stable, maintaining their shape, and delivering RNA to the cytoplasm, which constitutes the contents of the cell.

[0037] In this specification, the term "sterol" is an abbreviation for steroid alcohol and means steroid compounds, and can include all sterol derivatives having a sterol structure. Examples include, but are not limited to, the compound represented by formula (1) of the present invention, campesterol, sitosterol, stigmasterol, cholesterol, etc.

[0038] In this specification, the terms "polyethylene glycol lipid" or "PEG lipid" refer to lipids composed of a hydrophobic tail, a linker, and a polyethylene glycol (PEG) head, which regulate the size of lipid nanoparticles, prevent aggregation between particles, help lipid nanoparticles maintain a stable colloidal state, and enhance biosafety so that lipid nanoparticles delivered safely into cells can remain in the body for extended periods.

[0039] In the present invention, the PEG lipid may be characterized as DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), but is not limited to these.

[0040] The terms "polyethylene glycol" or "PEG" as used herein are general terms for mixtures of ethylene oxide polymers and water, and are polymers having a repeating ether unit of -CH2CH2O-.

[0041] As used herein, the term "encapsulation" refers to surrounding a delivery substance and enclosing it in order to efficiently introduce it into a living organism.

[0042] In this specification, the term "mRNA" means synthetic mRNA capable of expressing genes (in vitro transcribed mRNA).

[0043] The present invention This invention relates to novel cholesterol derivatives, which are core components of functional lipid nanoparticles for RNA delivery, and to lipid nanoparticles for RNA delivery using these derivatives.

[0044] The present invention provides a histidineamide cholesterol compound represented by the following formula (1), its stereoisomer, or a pharmaceutically acceptable salt thereof. [ka]

[0045] In another embodiment, the present invention provides lipid nanoparticles comprising a histidineamide cholesterol compound represented by formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0046] The lipid nanoparticles may further contain one or more selected from the group consisting of ionizable lipids, phospholipids, cholesterol, and PEG lipids. Preferably, when mixing the ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers, or pharmaceutically acceptable salts thereof; cholesterol; and PEG lipids, the ionizable lipids are preferably present in a ratio of 10 to 60 molars, more preferably 20 to 60, and even more preferably 30 to 60 molars, based on 1 mole of PEG lipids; the phospholipids are present in a ratio of 1 to 30, more preferably 5 to 20, and most preferably 15 molars; the histidineamide cholesterol compounds, their stereoisomers, or pharmaceutically acceptable salts thereof are present in a ratio of 5 to 70, more preferably 5 to 65, and most preferably 5 to 60 molars; the cholesterol is present in a ratio of 0 to 40, more preferably 0 to 30 molars; and the PEG lipids are present in a ratio of 0.5 to 5 molars.

[0047] The ionizable lipid may be, but is not limited to, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoic acid (DLin-MC3-DMA);

[0048] The phospholipid may be, but is not limited to, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine).

[0049] The PEG lipid may be, but is not limited to, DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000).

[0050] More specifically, the lipid nanoparticles preferably contain sterols, including histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts, and cholesterol, in a molar ratio of 38.5% of the total organic phase. In this case, the molar ratio of histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts, and cholesterol is preferably 1:4 to 1:0.

[0051] As one embodiment, the present invention may be characterized in that the molar ratio of ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts; cholesterol; and PEG lipids in the lipid nanoparticles is 50:10:7.7:30.8:1.5.

[0052] In another preferred embodiment, the molar ratio of ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts; cholesterol; and PEG lipids within the lipid nanoparticles may be 50:10:19.25:19.25:1.5.

[0053] In yet another preferred embodiment, the molar ratio of ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts; cholesterol; and PEG lipids within the lipid nanoparticles may be 50:10:26.95:11.55:1.5.

[0054] In yet another preferred embodiment, the molar ratio of ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts; cholesterol; and PEG lipids within the lipid nanoparticles may be 50:10:34.65:3.85:1.5.

[0055] In yet another preferred embodiment, the molar ratio of ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts; cholesterol; and PEG lipids within the lipid nanoparticles may be 50:10:38.5:0:1.5.

[0056] In one embodiment, the lipid nanoparticles are characterized in that the ionizable lipid in the lipid nanoparticles is SM-102, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (3β[L-histidinamide-carbamoyl]cholesterol; Hchol), the PEG lipid is DMG-PEG2000, and the SM-102:DSPC:Hchol:cholesterol:DMG-PEG2000 are present in a molar ratio of 50:10:7.7:30.8:1.5.

[0057] In another embodiment, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is SM-102, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the SM-102:DSPC:Hchol:cholesterol:DMG-PEG2000 is contained in a molar ratio of 50:10:19.25:19.25:1.5.

[0058] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is SM-102, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the SM-102:DSPC:Hchol:cholesterol:DMG-PEG2000 is contained in a molar ratio of 50:10:26.95:11.55:1.5.

[0059] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is SM-102, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the SM-102:DSPC:Hchol:cholesterol:DMG-PEG2000 is contained in a molar ratio of 50:10:34.65:3.85:1.5.

[0060] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is SM-102, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the SM-102:DSPC:Hchol:cholesterol:DMG-PEG2000 is contained in a molar ratio of 50:10:38.5:0:1.5.

[0061] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is DLin-MC3-DMA, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA:DSPC:Hchol:cholesterol:DMG-PEG2000 are present in a molar ratio of 50:10:7.7:30.8:1.5.

[0062] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is DLin-MC3-DMA, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA:DSPC:Hchol:cholesterol:DMG-PEG2000 are present in a molar ratio of 50:10:19.25:19.25:1.5.

[0063] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is DLin-MC3-DMA, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA:DSPC:Hchol:cholesterol:DMG-PEG2000 are present in a molar ratio of 50:10:26.95:11.55:1.5.

[0064] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is DLin-MC3-DMA, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA:DSPC:Hchol:cholesterol:DMG-PEG2000 are present in a molar ratio of 50:10:34.65:3.85:1.5.

[0065] In yet another form, the lipid nanoparticles may be characterized in that the ionizable lipid within the lipid nanoparticles is DLin-MC3-DMA, the phospholipid is DSPC, the histidineamide cholesterol compound, its stereoisomer, or its pharmaceutically acceptable salt is the compound represented by formula (1) (Hchol), the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA:DSPC:Hchol:cholesterol:DMG-PEG2000 are present in a molar ratio of 50:10:38.5:0:1.5.

[0066] The higher the ratio of histidineamide cholesterol compounds, their stereoisomers, or their pharmaceutically acceptable salts, the greater the effect on mRNA encapsulation efficiency, intracellular gene transfer efficiency, and in vivo protein expression rate.

[0067] In another embodiment, the present invention provides a composition for delivering nucleic acids, proteins, or drugs, comprising the lipid nanoparticles.

[0068] The nucleic acid can be selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof, and is preferably mRNA.

[0069] The protein is preferably an antibody.

[0070] The drug is preferably a therapeutic or prophylactic agent.

[0071] In another embodiment, the present invention provides a vaccine composition comprising the lipid nanoparticles.

[0072] In the present invention, the lipid nanoparticles may be administered via a route selected from the group consisting of intravenous, intramuscular, intravitreous, intrathecal, intratumoral, intranasal, pulmonary, and subcutaneous administration, but are not limited to these.

[0073] In the present invention, the lipid nanoparticles may be produced by mixing them with a buffer solution in a volume ratio of 1:1 to 1:5, preferably 1:2 to 1:4, but are not limited thereto.

[0074] In this case, the buffering agent can be selected from the group consisting of saline solution, phosphate-buffered saline solution, and Ringer's lactate solution, but is not limited to these.

[0075] In the present invention, the lipid nanoparticles can be produced by hydrating the ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts thereof; cholesterol; and PEG lipids in a buffer containing nucleic acids (e.g., mRNA), proteins, or other drugs.

[0076] In the present invention, the lipid nanoparticles can be produced by dissolving the ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts; cholesterol; and PEG lipids in a hydrateable organic solvent; then preparing a buffer containing nucleic acids (e.g., mRNA), proteins, or other drugs; mixing this solution using a filtration membrane, filter, mixer, or stirrer; and finally removing the ethanol.

[0077] If necessary, additional steps for sterilization or preservation may be added during the production of the lipid nanoparticles.

[0078] In the present invention, the lipid nanoparticles can be mixed by a microfluidic mixing device or by in-tube pipetting.

[0079] In the present invention, the lipid nanoparticles can be formulated to be administered intranasally, and in this case, they can be formulated in powder form to minimize water content.

[0080] For example, in the case of powder formulations, solid lipid nanoparticles can be produced via a spray dryer, and by extending the residence time in the nasal mucosa, potential local and systemic immune responses can be increased.

[0081] The present invention has the advantage of improved loading efficiency compared to conventional lipid nanoparticles, particularly in terms of mRNA loading.

[0082] Therefore, in another view, the present invention relates to a vaccine composition comprising an mRNA vaccine and the lipid nanoparticles.

[0083] In the present invention, the vaccine composition may be administered via a route selected from the group consisting of intravenous, intramuscular, intravitreous, intrathecal, intratumoral, intranasal, pulmonary, and subcutaneous administration, but is not limited to these.

[0084] The vaccine composition according to the present invention can be formulated in the administration methods described herein, such as other topical, intranasal, intratracheal, or injectable forms (e.g., intravenous, intraocular, intravitreous, intramuscular, intracardiac, intraperitoneal, subcutaneous).

[0085] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may include, but are not limited to, water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbita fatty acid esters sorbitan, and mixtures thereof, as commonly used in the industry. In certain embodiments for parenteral administration, the composition may be mixed with solubilizers such as cremofor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0086] Injectable formulations can be sterilized, for example, by filtration using a bacterial-retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in a sterile number or other sterile injectable medium before use.

[0087] If it is preferable to delay the absorption of the active ingredient from subcutaneous or intramuscular injection in order to extend the effect of the active ingredient, this can be achieved by using a liquid suspension of a crystalline or amorphous substance that is poorly water-soluble.

[0088] In yet another aspect, the present invention relates to the use of compositions comprising mRNA vaccines and lipid nanoparticles for the prevention or treatment of infectious diseases.

[0089] In yet another aspect, the present invention relates to the use of compositions comprising mRNA vaccines and lipid nanoparticles for the manufacture of pharmaceuticals (or drugs) for the prevention or treatment of infectious diseases.

[0090] In yet another aspect, the present invention relates to a method for preventing or treating infectious diseases, comprising the step of administering a composition containing the mRNA vaccine and the lipid nanoparticles to an individual in need.

[0091] The present invention will be described in more detail below through specific examples. The following examples are merely illustrative to aid in understanding the present invention, and the scope of the present invention is not limited thereto.

[0092] Example 1. Synthesis of histidineamide cholesterol (Hchol) Linkamide MBHAR resin (500 mg, 400 μmol) was dissolved in DCM, swollen, and drained. Piperidine and DMF solution were added to the reactor and stirred to remove the Fmoc protecting group. Fmoc-His(trt)-OH was added to the reactor containing the resin from which the protecting group had been removed by dissolving in DMF, HOBt / DIC was added, and the mixture was stirred to complete the synthesis. Next, piperidine and DMF were added to the reactor containing the resin and stirred to remove the Fmoc protecting group on histidine. Cholesteryl chloroformate was dissolved in DMF and DCM and added to the reactor containing the resin, DIPEA (N,N-diisopropylethylamine) was added, and the mixture was stirred overnight. The resin was then washed with DMF and DCM. Finally, to remove the trityl protecting group attached to histidine, the mixture was added to a peptide dried with trifluoroacetic acid and stirred. The filtered product was then slowly added to cold ether to precipitate crystals, and the final product was obtained by freeze-drying (see Figure 1).

[0093] Hchol was also synthesized by the following method: Histidineamide dihydrochloride dissolved in DMF was reacted with cholesteryl chloroformate dissolved in dichloromethane (DCM) in a ratio of 1 equivalent (DMF / DCM = 4 / 1, v / v). Six equivalents of DIPEA were added to the reaction solution and the reaction was carried out at 50°C. After the reaction, the mixture was precipitated by adding it little by little to 40 mL of distilled water. The reaction mixture was filtered and washed several times with distilled water. The product was then recovered with DCM and purified by preparative TLC (DCM:diethyl ether:ethyl acetate:ammonium hydroxide = 5:1:0.5:0.5:0.1).

[0094] The compound is dissolved in methanol-d4 at 600 MHz. 1 We analyzed the 1H nuclear magnetic resonance (Figure 2) and molecular weight (Figure 3) to confirm that the synthesis was successful.

[0095] Example 2. Production of lipid nanoparticles (LNPs) Lipid nanoparticles (LNPs) were prepared by mixing an organic phase contained in ethanol and nucleic acids contained in a citrate buffer solution (pH 4) in a 1:2 ratio using a microfluidic mixing apparatus (Spark, Precision Nanosystems). The organic phase consisted of ionizable lipids (DLin-MC3-DMA lipids (MC3) or SM102 lipids), phospholipids (DSPC), sterols (cholesterol or histidineamide cholesterol), and PEG lipids (DMG-PEG2000) mixed in ethanol in a molar ratio of 50:10:38.5:1.5. The aqueous phase contained nucleic acids, which were diluted using firefly luciferase mRNA or EGFP mRNA to a final citrate concentration of 50 mM. The amounts of cholesterol and histidineamide cholesterol (Hchol) were adjusted so that the Hchol ratio ranged from 0% to 100% within the total lipid ratio, and LNPs were produced accordingly.

[0096] To remove ethanol from the generated LNPs and adjust the pH in the body, the LNPs were dialyzed with 1X PBS for 16 hours using an MWCO 3,500Da Slide-A-Lyzer® dialysis cassette (Thermo Scientific) or an MWCO 3,500Da dialysis membrane. After dialysis, the lipid nanoparticle solution was filtered through a 0.22 μm filter to obtain lipid nanoparticles (LNPs) containing nucleic acids.

[0097] The ratio of N / P to 5.5 or 6.5 was used, based on the number of amines (N) in ionizable lipids and the number of phosphate groups (P) in mRNA.

[0098] The size, polydispersity (PDI), and zeta potential of lipid nanoparticles containing mFluc were measured using Zetasizer Nano ZS (Malvern, UK).

[0099] In the experimental examples of the present invention, HeLa (human cervical adenocarcinoma cell line), HepG2 (human hepatocellular carcinoma cell line), and NIH3T3 (mouse embryonic fibroblast cell line) were cultured in an incubator maintained at 37°C, 5% CO2, and 95% humidity using DMEM containing 10% FBS and antibiotics / antifungal agents.

[0100] Experimental Example 1. pKa Analysis of Lipid Nanoparticles TNS experiments were performed to measure the pKa of lipid nanoparticles. Lipid nanoparticles were diluted in PBS to 1 mM, and TNS was prepared in DMSO to 300 μM. Buffers used in the TNS experiments were prepared using sodium phosphate, sodium borate, sodium citrate, and sodium chloride to achieve pH levels from 3.5 to 10. 94 μL of pH buffer was added to a black 96-well plate, 4 μL of lipid nanoparticles were added to each buffer, and 2 μL of TNS solution was added and thoroughly mixed. The plates were placed in a fluorescence plate reader, and TNS fluorescence was measured at excitation emission wavelengths of 330 nm and 435 nm. The pKa of each lipid nanoparticle was determined as half of the maximum fluorescence value measured. The results are shown in Figure 4.

[0101] pKa is the acid dissociation constant and is commonly used as an indicator of the acidity of a substance. The pKa value of lipid nanoparticles is important in terms of their in vivo stability and drug release. Anionic TNSs (transions of nitrates) increase in fluorescence intensity when they come into close proximity with positively charged lipid nanoparticles, as the interaction between their hydrophobic parts is strengthened. As the pH value approaches the pKa value of each LNP, the interaction between the hydrophobic parts weakens rapidly, and more water molecules quench the TNS fluorescence, reducing it to about 50% of the maximum fluorescence value. Subsequently, as the pH continues to increase, the fluorescence intensity gradually decreases, and a graph showing the fluorescence value according to pH could be created. Using these results, the pKa value of lipid nanoparticles could be measured. In particular, numerous studies have reported that lipid nanoparticles with a pKa of 6.0 to 7.0 interact efficiently with endosomal membranes and can easily escape endosomes when acidified, resulting in superior efficiency of gene transfer and expression in vivo.

[0102] As can be seen in Figure 4, the lipid nanoparticles of the present invention were confirmed to exhibit a pKa range of 6.0 to 7.0, which is excellent for in vivo stability and drug release.

[0103] Experimental Example 2: Analysis of gene inclusion efficiency and stability Lipid nanoparticles were formed using luciferase mRNA with ionizable lipids:DSPC:sterols:DMG-PEG2000 (50:10:38.5:1.5 molar ratio). Two types of ionizable lipids, MC3 lipid and SM102 lipid, were used, and 100% cholesterol (Chol) or histidineamide cholesterol (Hchol), a cholesterol-modified substance, was added to the sterol portion to produce lipid nanoparticles (LNPs).

[0104] Analysis of gene inclusion efficiency To measure the luciferase mRNA encapsulation efficiency of lipid nanoparticles, the Quant-it® RiboGreen RNA Assay Kit (Invitrogen) was used. LNPs were treated with 1X TE buffer or 2% Triton X-100, incubated at 37°C for 10 minutes, and RiboGreen fluorescence (excitation wavelength 485 nm, emission wavelength 538 nm) was measured. The mRNA encapsulation efficiency of the nanoparticles was calculated using the following formula. <Number 1> mRNA encapsulation efficiency (%) = [(Total mRNA concentration - Unencapsulated mRNA concentration) / Total mRNA concentration] × 100 (%)

[0105] As shown in Figure 5, the nucleic acid encapsulation efficiencies of MC3-Chol LNP and MC3-Hchol LNP, prepared with an N / P ratio adjusted to 6.7, were 72% and 91%, respectively. The nucleic acid encapsulation efficiencies of SM102-Chol LNP and SM102-Hchol LNP, prepared with an N / P ratio adjusted to 5.6, were 78% and 87%, respectively. This confirmed that histidineamide cholesterol, produced by modifying cholesterol, exhibits superior mRNA encapsulation efficiency compared to cholesterol.

[0106] Analysis of stability over time and mRNA inclusion efficiency. The manufactured lipid nanoparticles were stored at 4°C, and their particle size and polydispersity index (PDI) values ​​were measured at regular intervals using a dynamic light scattering instrument. The change in mRNA encapsulation efficiency of the lipid nanoparticles was also measured at each of these intervals.

[0107] As a result (Figure 6), it was found that all lipid nanoparticles according to the present invention maintained a stable state even over time, and it was confirmed that the mRNA encapsulation efficiency was also maintained.

[0108] Experimental Example 3. Measurement of lipid nanoparticle size and zeta potential Lipid nanoparticles prepared in the same manner as in Experimental Example 1 were obtained by passing them through a filter, then diluted in 1X PBS at a neutral pH, and the particle size and zeta potential were measured. [Table 1]

[0109] As shown in Table 1 above, all lipid nanoparticles, including the control group, were approximately 120 nm in size, and their PDI values ​​were generally measured at 0.2 or less, indicating that uniform and appropriately sized lipid nanoparticles were formed. The zeta potential was confirmed to be slightly negative, near 0. Therefore, it was confirmed that lipid nanoparticles of a consistent size were formed.

[0110] Experimental Example 4. Evaluation of intracellular gene transfer efficiency To evaluate the mRNA delivery efficiency of LNPs prepared using luciferase mRNA, experiments were conducted using HeLa cells and HepG2 cell lines. 24 hours prior to sample administration, cells were aliquoted into 96-well plates at a rate of 15,000 cells per well and cultured at 37°C, 5% CO2, and 95% humidity. Lipid nanoparticles were administered to each well at a rate of 100 ng / well to evaluate gene transfer efficiency at the cellular level. 24 hours after sample injection, the cell culture medium was removed and washed with DPBS. Next, 1X Reporter Lysis Buffer (Promega) was added, and the cells were completely lysed in a shaking incubator at room temperature for 30 minutes. The cells were then harvested and centrifuged at 13,200 rpm for 10 minutes. Only the supernatant containing protein was obtained, and luciferase activity was confirmed using luciferin reagent. The amount of protein produced was calculated using Micro BCA® Protein Assay, and quantified relative luminescence intensity (RLU) values ​​were obtained.

[0111] As a result, as shown in Figure 7, when the same amount of mRNA was injected, LNPs using histidineamide cholesterol (Hchol) lipids showed higher luciferase protein expression than LNPs using cholesterol (Chol) lipids. In HeLa cell lines, SM102-Hchol LNPs showed 9.6 times higher expression efficiency than SM102-Chol LNPs (Figure 7(A)), and in HepG2 cell lines, they showed 16.6 times higher expression efficiency (Figure 7(B)). Furthermore, when Hchol was separated into different ratios using MC3 as an ionizable lipid and lipid nanoparticles were produced to evaluate gene transfer efficiency, it was confirmed that higher Hchol ratios resulted in higher protein expression (Figure 7(C)).

[0112] Experimental Example 5. Evaluation of Intracellular Toxicity A WST assay was performed to evaluate the toxicity of the prepared LNPs. Experiments were conducted using HepG2 (hepatocellular carcinoma) cell lines and NIH3T3 (normal cell) cell lines. Cells were dispensed into 96-well plates at a rate of 13,000 cells per well 24 hours before sample administration. Cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity. Cells were treated with LNPs at a concentration of 100 ng based on mRNA concentration, and after 24 hours, they were treated with Ez-Cytox reagent and WST-8 reagent, and reacted in the incubation for 2 hours. The absorbance of WST formazan was measured at 450 nm using a VERASmax microplate reader.

[0113] As a result, neither SM102-chol LNP nor SM102-Hchol LNP showed toxicity in normal NIH3T3 cells (Figure 8A). Furthermore, it was confirmed that no toxicity was observed in any of the samples in cancer cells, HepG2 cells (Figure 8B).

[0114] Cytotoxicity was evaluated by adding a reagent and measuring the amount of WST formazan by absorbance to assess cell viability. Each sample showed a cell viability close to 100%. Therefore, it was confirmed that lipid nanoparticles formed from histidineamide cholesterol (Hchol) are not toxic to cells.

[0115] Experimental Example 6. Analysis of intracellular introduction of lipid nanoparticles and protein expression efficiency. Using a confocal microscope, we confirmed the extent to which lipid nanoparticles flowed into cells over time and the extent to which mRNA delivered into cells via lipid nanoparticles was expressed as a protein.

[0116] HeLa cell lines were placed in u-slide 8 wells in a 1.2 × 10⁶ arrangement. 4 The cells were dispensed into wells and incubated for 24 hours at 37°C, 5% CO2, and 95% humidity. LNPs containing 100 ng / well of cyanine-5 fluorescently labeled EGFP mRNA were added to each well 24, 12, and 4 hours prior to fluorescence microscopy observation. Endosomes and nuclei of the cells were stained using Lysotracker Red and Hoechest 33342, respectively. The stained cells were observed using a confocal microscope (Carl Zeisss).

[0117] As a result, when comparing the fluorescence of LNPs prepared using the control group Chol in HeLa cell lines with that of the experimental group Hchol LNPs, no significant difference was observed at 4 hours. However, when Hchol LNPs were administered, high EGFP (green) fluorescence signals were observed at 12 and 24 hours, and the Cy5 fluorescence signal was also observed to be higher with Hchol LNPs than with Chol LNPs (Figure 9).

[0118] Experimental Example 7. Analysis of in vivo gene transfer efficiency and expression. The animal experiments were conducted with the approval of the Animal Experiment Ethics Committee of the Korea Institute of Biotechnology (Approval Number: KRIBB-AEC-22165).

[0119] To evaluate the expression efficiency of proteins expressed in vivo from mRNA contained in LNPs, depending on the type of cholesterol constituting the LNPs, fLuc mRNA-LNP (0.1 mg / kg) was administered to C57BL / 6 mice (6-8 weeks old, female; Orient Bio) by intramuscular injection or tail vein infusion. To confirm the expression efficiency of luciferase protein at 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after injection, 200 μL (15 mg / mL) of luciferin was administered by intraperitoneal injection 10 minutes before each imaging time point, followed by respiratory anesthesia under isoflurane and 97% medical oxygen for 3% of the mice. Next, luminescence images were acquired using an in vivo imaging system (IVIS; PerkinElmer) and analyzed with Living Image Software (version 4.7, PerkinElmer).

[0120] As a result, it was confirmed that both Chol LNP and Hchol LNP showed maximum luminescence at 6 hours, after which it gradually decreased. Compared to the control group SM102-chol LNP, SM102-Hchol LNP showed higher luciferase activity in the body and its expression was sustained for a longer period (Figure 8(A)). In addition, LNPs prepared using MC3 lipids showed a similar effect, but the fluorescence gradually increased at 6 hours as the proportion of Hchol increased, confirming that increasing the proportion of Hchol instead of Chol increased gene transfer efficiency and protein expression (Figure 10(B)).

[0121] Statistical processing Statistical analysis of the experimental data was performed using Student's t-test or ANOVA analysis with the Prism5 program from GraphPad. p-values ​​less than 0.05 indicate statistical significance, as follows: (*, p<0.05; **, p<0.01; ***, p<0.001). ns shown in the data indicates a statistically insignificant value.

[0122] In summary, lipid nanoparticles (LNPs) produced using Hchol were found to increase intracellular gene transfer efficiency and in vivo protein expression efficiency. The results at the cellular level tended to be consistent with those at the in vivo level, and cytotoxicity was not observed in either cancer cells or normal cells. Furthermore, it was confirmed that higher Hchol ratios increased mRNA encapsulation efficiency, intracellular gene transfer efficiency, and in vivo protein expression.

[0123] As a result, Hchol is present and influences lipid nanoparticles both on the surface and inside the particles. By binding histidine amide to cholesterol, it is expected that the number of amines in lipids that can be ionized in an acidic environment will increase, facilitating intracellular introduction and simultaneously aiding escape from endosomes. Furthermore, it was found that Hchol interacts with mRNA inside the particles to enhance encapsulation efficiency and contribute to the stabilization of lipid nanoparticles.

[0124] Finally, evaluation of lipid nanoparticle toxicity in HepG2 and NIH3T3 cells confirmed that the survival rate of LNP samples was over 90% in both cancer and normal cells. This confirmed that lipid nanoparticles utilizing Hchol lipids can effectively deliver and express mRNA into cells while maintaining in vivo compatibility.

[0125] The above description is merely illustrative, and a person with ordinary skill in the art to which the present invention pertains could make various modifications without departing from the essential characteristics of the present invention. Accordingly, the examples disclosed herein are for illustrative purposes only, not to limit the present invention, and these examples do not limit the spirit or scope of the present invention. The scope of protection of the present invention should be interpreted by the following claims, and all technologies within an equivalent scope should be interpreted as being included within the scope of the present invention. [Industrial applicability]

[0126] As described above, the present invention relates to a histidineamide cholesterol compound represented by formula (1), its stereoisomer or pharmaceutically acceptable salt thereof, and a composition for the delivery of nucleic acids, proteins or drugs containing the same, which can be effectively used as a delivery body when included in pharmaceuticals, vaccines, etc.

Claims

1. A histidineamide cholesterol compound represented by the following formula (1), its stereoisomer, or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. Lipid nanoparticles comprising the histidineamide cholesterol compound described in claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

3. The lipid nanoparticles according to claim 2, characterized in that the lipid nanoparticles further comprise one or more selected from the group consisting of ionizable lipids, phospholipids, cholesterol, and PEG lipids.

4. The lipid nanoparticle according to claim 3, characterized in that the ionizable lipid is SM-102 or DLin-MC3-DMA.

5. The lipid nanoparticle according to claim 3, characterized in that the phospholipid is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine).

6. The lipid nanoparticle according to claim 3, characterized in that the PEG lipid is DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000).

7. Lipid nanoparticles according to claim 3, characterized in that the mixture of ionizable lipids; phospholipids; histidineamide cholesterol compounds, their stereoisomers or pharmaceutically acceptable salts thereof; cholesterol; and PEG lipids is in a molar ratio of 10-60:1-20:5-70:0-30:0.5-5.

8. A composition for delivering nucleic acids, proteins, or drugs, comprising lipid nanoparticles according to any one of claims 2 to 7.

9. The nucleic acid, protein, or drug delivery composition according to claim 8, characterized in that the nucleic acid is selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

10. The nucleic acid, protein, or drug delivery composition according to claim 8, characterized in that the protein is an antibody.

11. The nucleic acid, protein, or drug delivery composition according to claim 8, characterized in that the drug is a therapeutic agent or a preventive agent.

12. A vaccine composition comprising lipid nanoparticles according to any one of claims 2 to 7.

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