Novel histidine amide cholesterol compound, and RNA delivery lipid nanoparticles comprising same

Abstract

The present invention relates to a novel histidine amide cholesterol compound and RNA delivery lipid nanoparticles comprising same. The present invention can have improved mRNA delivery efficiency and expression efficiency while having biocompatibility.

Description

Novel histidine amide cholesterol compound and lipid nanoparticle containing the same for RNA delivery

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

[0002] With the global COVID-19 pandemic, lipid nanoparticle (LNP) vaccines for mRNA delivery are being actively researched. Moderna and BioNTech / Pfizer have developed lipid nanoparticle vaccines to deliver mRNA encoding the SARS-CoV-2 viral spike glycoprotein and received emergency use authorization (EUA) from the FDA. Following this authorization, the development of novel lipid nanoparticles for the treatment of other diseases, including COVID-19, is underway worldwide.

[0003] Disease treatment via mRNA delivery offers many advantages over conventional DNA-based therapies. Unlike DNA, mRNA does not integrate into the host genome, thus avoiding genetic mutations in cells. Furthermore, unlike DNA, mRNA bypasses the multiple steps required for protein production. While conventional DNA must cross the cytoplasm and enter the nucleus to initiate gene transcription, mRNA translation occurs in the cytoplasm without entering the nucleus, enabling effective expression of therapeutic proteins within cells. However, naked mRNA alone does not achieve sufficient protein expression to achieve the desired therapeutic effect. mRNA is unstable, easily degraded by RNases in vivo, has a large molecular weight, and its high negative charge density creates electrostatic repulsion with the negatively charged cell membrane, making it difficult to cross the cell membrane. To overcome these limitations, lipid nanoparticles are used for effective and stable mRNA delivery in vivo.

[0004] Lipid nanoparticles are composed of ionizable lipids, accessory lipids (phospholipids), cholesterol, and PEG-lipids. Ionizable lipids play a crucial role in transporting RNA into the cytoplasm. In acidic pH environments, they are positively charged, allowing them to condense with negatively charged RNA through electrostatic attraction. Conversely, ionizable lipids are neutral at physiological pH and thus non-toxic. However, after intracellular uptake, the low pH in the endosomes causes the ionizable lipids to become positively charged, destabilizing the endosomal membrane and promoting mRNA release. This facilitates the stable delivery of mRNA into the cytoplasm. Moderna has developed the mRNA-1273 vaccine, which uses SM-102 as the ionizable lipid, and BioNTech / Pfizer has developed the BNT162b2 vaccine, which uses ALC-0315. Other ionizable lipids, such as DLin-MC3-DMA (MC3) and C12-200, are also in development.

[0005] Ionizable lipids are composed of a head group, a linker, and a tail. The head group contains ionizable moieties, typically amine, guanidine, or imidazole groups. Conventional lipids like MC3 and ALC-0315 possess a tertiary amine head, which undergoes pH-dependent ionization. This can be manipulated to improve hydrogen bonding interactions with RNA. The linker affects the stability and biodegradability of the formed lipid nanoparticles. Ester linkers increase biodegradability, enhancing gene delivery efficiency. Disulfide linkages degrade depending on the degree of intracellular reduction, enabling selective LNP degradation and gene delivery. These biodegradable linkers are rapidly eliminated in vivo, reducing side effects and toxicity. The tail, a hydrophobic moiety, influences the pKa and fluidity of the LNP.

[0006] The role of cholesterol in lipid nanoparticles is known to contribute to their structural stability and to be involved in endosomal transport and recycling mechanisms. In the present invention, ionizable cholesterol was developed by introducing an ionizable amine into existing cholesterol to enhance endosomal escape while maintaining the structural stability of existing cholesterol, ultimately improving protein expression efficiency. This cholesterol was used to fabricate lipid nanoparticles, completing the present invention.

[0007] The present invention aims to provide histidine amide cholesterol (3β[L-histidinamide-carbamoyl] cholesterol; Hchol), a cholesterol variant, and lipid nanoparticles containing the same to improve mRNA delivery effect and expression efficiency while maintaining in vivo compatibility.

[0008] To achieve the above purpose, the present invention provides a histidine amide cholesterol compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0009] <Chemical Formula 1>

[0010]

[0011] In another aspect, the present invention provides a lipid nanoparticle comprising a histidine amide cholesterol compound represented by the above chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0012] The lipid nanoparticle is characterized in that it further comprises at least one selected from the group consisting of ionizable lipids, phospholipids, cholesterol, and PEG lipids.

[0013] The above ionizable lipid is characterized as SM-102 or DLin-MC3-DMA.

[0014] The above phospholipid is characterized as being DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine).

[0015] The above PEG lipid is characterized as DMG-PEG2000 (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000).

[0016] The present invention provides a lipid nanoparticle characterized in that the mixture of the above ionizable lipid; phospholipid; histidine amide cholesterol compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid is mixed in a molar ratio of 10-60:1-20:5-70:0-30:0.5-5.

[0017] In another aspect, the present invention provides a composition for delivery of a nucleic acid, protein or drug comprising the lipid nanoparticle.

[0018] The nucleic acid is characterized in that it is selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0019] The above protein is characterized as being an antibody.

[0020] The above drug is characterized as being a therapeutic or preventive agent.

[0021] In addition, in another aspect, the present invention provides a vaccine composition comprising the lipid nanoparticles.

[0022] According to the present invention, lipid nanoparticles manufactured using Hchol can improve intracellular gene delivery efficiency and protein expression efficiency by increasing encapsulation efficiency and contributing to stabilization of lipid nanoparticles by Hchol interacting with mRNA inside the particles.

[0023] Additionally, it has biocompatibility and can effectively deliver and express mRNA into cells.

[0024] Figure 1 illustrates a synthesis process of histidine amide cholesterol according to one embodiment of the present invention.

[0025] Figure 2 is a diagram of histidine amide cholesterol according to one embodiment of the present invention. 1 This shows the H NMR analysis data.

[0026] Figure 3 shows molecular weight analysis data of histidine amide cholesterol according to one embodiment of the present invention.

[0027] Figure 4 shows the results of pKa value analysis of lipid nanoparticles according to one embodiment of the present invention.

[0028] Figure 5 shows the results of analysis of gene encapsulation efficiency of lipid nanoparticles according to one embodiment of the present invention.

[0029] Figure 6 shows the results of analysis of stability and gene encapsulation efficiency over time of lipid nanoparticles according to one embodiment of the present invention.

[0030] Figure 7 shows the results of evaluating the intracellular gene delivery efficiency of lipid nanoparticles according to one embodiment of the present invention.

[0031] Figure 8 shows the results of an intracellular toxicity evaluation of lipid nanoparticles according to one embodiment of the present invention.

[0032] Figure 9 shows the results of analysis of intracellular introduction and protein expression efficiency of lipid nanoparticles according to one embodiment of the present invention.

[0033] Figure 10 illustrates the results of in vivo gene delivery efficiency and expression analysis of lipid nanoparticles according to one embodiment of the present invention. (A) Lipid nanoparticles manufactured using SM102 lipid were injected intramuscularly at 0.1 mg / kg. (B) Lipid nanoparticles manufactured using MC3 lipid, with Hchol adjusted to 50%, 75%, and 100% of the sterol ratio, were injected intramuscularly at 0.1 mg / kg.

[0034] Hereinafter, the present invention will be described in detail.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0036] The term "lipid nanoparticle" or "LNP" as used herein refers to submicron lipid particles that are drug delivery systems capable of encapsulating poorly soluble substances. Lipid nanoparticles are composed of ionizable lipids capable of binding anions, phospholipids to maintain the lipid bilayer structure of the lipid nanoparticles, cholesterol to keep the particles firm and stable, and polyethylene glycol (PEG) to prevent particle aggregation and ensure structural stability.

[0037] In the present invention, the lipid nanoparticle may be characterized as being for nucleic acid, protein or drug delivery, but is not limited thereto.

[0038] In the present invention, the nucleic acid may be DNA, RNA or PNA, and preferably mRNA.

[0039] In the present invention, the protein may be an antibody in one embodiment and a peptide in another embodiment.

[0040] In the present invention, the drug may be a therapeutic agent or a preventive agent, and may be, for example, a compound.

[0041] Therefore, in the present invention, the lipid nanoparticles can be loaded with target-specific antibodies, proteins, fluorescent dyes, etc. depending on the disease.

[0042] That is, the present invention relates, from another perspective, to the use of lipid nanoparticles for the delivery of nucleic acids, proteins, or drugs. The present invention can also provide the use of such lipid nanoparticles in the manufacture of pharmaceuticals for the delivery of nucleic acids, proteins, or drugs.

[0043] The term “ionizable lipid” as used herein refers to a main component of lipid nanoparticles that directly binds to mRNA through electrostatic attraction and determines the delivery and release efficiency of mRNA, has an acid dissociation constant (pKa) of an appropriate range, and has a structure in which a tertiary amine head group whose degree of ionization varies depending on the body pH and a long hydrocarbon tail are connected by a linker.

[0044] In the present invention, the ionizable lipid may be characterized as being a cationic lipid, but is not limited thereto.

[0045] In the present invention, the cationic lipid means a lipid having a cationic property at neutral pH, and preferably, SM-102 or DLin-MC3-DMA can be used, but is not limited thereto.

[0046] The term “phospholipid” in this specification includes all amphipathic lipids having a hydrophilic portion based on a phosphate structure and two hydrophobic fatty acid moieties, and are involved in maintaining the lipid bilayer structure of lipid nanoparticles and thus maintaining the structure stably.

[0047] In the present invention, the phospholipid may be DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), but is not limited thereto.

[0048] The term "cholesterol" in this specification refers to a type of sterol, a combination of a steroid and an alcohol, and encompasses all cholesterol derivatives with a cholesterol structure. Cholesterol is a lipid present in the cell membranes of all animal cells and is an organic molecule transported through the bloodstream. Cholesterol helps to form and maintain the shape of lipid nanoparticles, making them more rigid and stable, and it also plays a role in transporting RNA into the cytoplasm, which constitutes the intracellular contents.

[0049] In this specification, the term “sterol” is an abbreviation for steroid alcohol, and refers to a steroid series compound, and may include all sterol derivatives having a sterol structure, and examples thereof include, but are not limited to, a compound represented by the chemical formula 1 of the present invention, campesterol, sitosterol, stigmasterol, cholesterol, and the like.

[0050] The term “polyethylene glycol lipid” or “PEG lipid” described herein refers to a lipid composed of a hydrophobic tail, a linker, and a polyethylene glycol (PEG) head, which controls the size of lipid nanoparticles, prevents aggregation between particles, helps lipid nanoparticles maintain a stable colloidal state, and increases stability in the body so that lipid nanoparticles safely delivered into cells can remain in the body for a long period of time.

[0051] In the present invention, the PEG lipid may be characterized as being DMG-PEG2000 (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), but is not limited thereto.

[0052] The term “polyethylene glycol” or “PEG” as described herein is a general term for a mixture of ethylene oxide polymer and water, which is a polymer having an ether repeating unit of -CH2CH2O-.

[0053] The term “encapsulation” as described herein means encapsulating a delivery substance to surround it and efficiently introduce it into a living body.

[0054] The term “mRNA” in this specification refers to synthetic mRNA (in vitro transcribed mRNA) capable of expressing a gene.

[0055] The present invention relates to a novel derivative of cholesterol, which is a key component of RNA delivery functional lipid nanoparticles, and a lipid nanoparticle for RNA delivery using the same.

[0056] The present invention provides a histidine amide cholesterol compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0057] <Chemical Formula 1>

[0058]

[0059] In another aspect, the present invention provides a lipid nanoparticle comprising a histidine amide cholesterol compound represented by the above chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0060] The lipid nanoparticle may further include at least one selected from the group consisting of ionizable lipids, phospholipids, cholesterol, and PEG lipids, and preferably, when mixing the ionizable lipids; phospholipids; histidine amide cholesterol compounds, stereoisomers thereof, or pharmaceutically acceptable salts thereof; cholesterol; and PEG lipids, it is preferable that the ionizable lipids are included in a molar ratio of 10 to 60, more preferably 20 to 60, and even more preferably 30 to 60, based on 1 mol of PEG lipids. The phospholipid is contained in a molar ratio of 1 to 30, more preferably 5 to 20, most preferably 15, the histidine amide cholesterol compound, its stereoisomer or its pharmaceutically acceptable salt is contained in a molar ratio of 5 to 70, more preferably 5 to 65, most preferably 5 to 60, the cholesterol is contained in a molar ratio of 0 to 40, more preferably 0 to 30, and the PEG lipid is contained in a molar ratio of 0.5 to 5.

[0061]

[0062] The ionizable lipid may be, but is not limited to, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (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)butanoate (DLin-MC3-DMA).

[0063] The above phospholipid may be, but is not limited to, DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine).

[0064] The above PEG lipid may be, but is not limited to, DMG-PEG2000 (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000).

[0065] More specifically, the lipid nanoparticle preferably comprises a sterol comprising a histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and cholesterol; in a molar ratio of the total organic phase, at 38.5%, and in this case, the molar ratio of the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; and cholesterol is preferably 1:4 to 1:0.

[0066] As one embodiment, the present invention may be characterized in that the molar ratio of the ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid in the lipid nanoparticle is 50:10:7.7:30.8:1.5.

[0067] In another preferred embodiment, the molar ratio of the ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid in the lipid nanoparticle may be 50:10:19.25:19.25:1.5.

[0068] In another preferred embodiment, the molar ratio of the ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid in the lipid nanoparticle may be 50:10:26.95:11.55:1.5.

[0069] In another preferred embodiment, the molar ratio of the ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid in the lipid nanoparticle may be 50:10:34.65:3.85:1.5.

[0070] In another preferred embodiment, the molar ratio of the ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid in the lipid nanoparticle may be 50:10:38.5:0:1.5.

[0071] In one embodiment, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is SM-102, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound represented by the chemical formula 1 (3β[L-histidinamide-carbamoyl] cholesterol; Hchol), and the PEG lipid is DMG-PEG2000, and the SM-102: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:7.7:30.8:1.5.

[0072] In another aspect, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is SM-102, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the chemical formula 1, and the PEG lipid is DMG-PEG2000, and the SM-102: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:19.25:19.25:1.5.

[0073] In another embodiment, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is SM-102, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the chemical formula 1, and the PEG lipid is DMG-PEG2000, and the SM-102: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:26.95:11.55:1.5.

[0074] In another embodiment, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is SM-102, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the above chemical formula 1, and the PEG lipid is DMG-PEG2000, and the SM-102: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:34.65:3.85:1.5.

[0075] In another embodiment, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is SM-102, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the chemical formula 1, and the PEG lipid is DMG-PEG2000, and the SM-102: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:38.5:0:1.5.

[0076] In another aspect, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is DLin-MC3-DMA, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the above chemical formula 1, and the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:7.7:30.8:1.5.

[0077] In another aspect, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is DLin-MC3-DMA, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the above chemical formula 1, and the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:19.25:19.25:1.5.

[0078] In another embodiment, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is DLin-MC3-DMA, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the above chemical formula 1, and the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:26.95:11.55:1.5.

[0079] In another aspect, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is DLin-MC3-DMA, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the above chemical formula 1, and the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:34.65:3.85:1.5.

[0080] In another embodiment, in the lipid nanoparticle, the ionizable lipid in the lipid nanoparticle is DLin-MC3-DMA, the phospholipid is DSPC, the histidine amide cholesterol compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is a compound (Hchol) represented by the above chemical formula 1, and the PEG lipid is DMG-PEG2000, and the DLin-MC3-DMA: DSPC: Hchol: cholesterol: DMG-PEG2000 may be included in a molar ratio of 50:10:38.5:0:1.5.

[0081] As the ratio of histidine amide cholesterol compound, its stereoisomer or its pharmaceutically acceptable salt increases, the mRNA encapsulation efficiency, intracellular gene delivery efficiency and in vivo protein expression rate increase.

[0082] In another aspect, the present invention provides a composition for delivery of a nucleic acid, protein or drug comprising the lipid nanoparticle.

[0083] The nucleic acid may be selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof, and preferably may be mRNA.

[0084] It is preferred that the above protein is an antibody.

[0085] It is preferable that the above drug be a therapeutic or preventive agent.

[0086] In addition, in another aspect, the present invention provides a vaccine composition comprising the lipid nanoparticles.

[0087] In the present invention, the lipid nanoparticles may be administered by a route selected from the group consisting of intravenous, intramuscular, intravitreal, intrathecal, intratumoral, intranasal, pulmonary, and subcutaneous, but are not limited thereto.

[0088] In the present invention, the lipid nanoparticles may be characterized in that they are manufactured by mixing 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.

[0089] In this case, the buffer may be selected from the group consisting of saline solution, phosphate buffered saline solution, and lactated Ringer's lactate, but is not limited thereto.

[0090] In the present invention, the lipid nanoparticle can be prepared by hydrating the ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid with a buffer containing a nucleic acid (e.g., mRNA), protein or other drug.

[0091] In the present invention, the lipid nanoparticles can be prepared by dissolving the ionizable lipid; phospholipid; histidine amide cholesterol compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid; in a hydratable organic solvent, then preparing a buffer solution containing a nucleic acid (e.g., mRNA), a protein or other drug, mixing the solution using a filter, a mixer or a stirrer, and then removing the ethanol.

[0092] If necessary, a step for sterilization or storage may be added during the manufacture of the lipid nanoparticles.

[0093] In the present invention, the lipid nanoparticles can be mixed using a microfluidic mixing device or in vitro pipetting.

[0094] 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.

[0095] For example, in powder formulations, solid lipid nanoparticles can be manufactured using a spray dryer to extend the residence time in the nasal mucosa, thereby increasing potential local and systemic immune responses.

[0096] The present invention has the advantage of improved loading efficiency compared to existing lipid nanoparticles, particularly in loading mRNA.

[0097] Accordingly, the present invention relates to a vaccine composition comprising an mRNA vaccine and the lipid nanoparticles from another aspect.

[0098] In the present invention, the vaccine composition may be characterized in that it is administered by a route selected from the group consisting of intravenous, intramuscular, intravitreal, intrathecal, intratumoral, intranasal, pulmonary, and subcutaneous, but is not limited thereto.

[0099] The vaccine composition according to the present invention may be formulated into dosage forms described herein, such as other topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous) forms.

[0100] Liquid dosage forms for parenteral administration may 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 forms may contain inert diluents commonly used in the art, including but 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 (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and the fatty acid ester sorbitan of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, the composition may be mixed with a solubilizing agent, such as cremophor, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0101] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0102] In order to prolong the effect of an active ingredient, if it is desirable to slow down its absorption from subcutaneous or intramuscular injection, this can be achieved by the use of liquid suspensions of crystalline or amorphous substances with poor water solubility.

[0103] In another aspect, the present invention relates to the use of an mRNA vaccine and a composition comprising the lipid nanoparticles for preventing or treating infectious diseases.

[0104] In another aspect, the present invention relates to the use of an mRNA vaccine and a composition comprising the lipid nanoparticles for the manufacture of a medicament (or drug) for preventing or treating an infectious disease.

[0105] In another aspect, the present invention relates to a method for preventing or treating an infectious disease, comprising a step of administering a composition comprising the mRNA vaccine and the lipid nanoparticles to a subject in need thereof.

[0106]

[0107] Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely illustrative examples to aid understanding of the present invention and are not intended to limit the scope of the present invention.

[0108]

[0109] Example 1. Synthesis of Histidinamide Cholesterol (Hchol)

[0110] Add Rink amide MBHA resin (500 mg, 400 μmol) to DCM, allow to swell, and drain. Piperidine and DMF solutions are added to the reactor, stirred, and the Fmoc protecting group is removed. Fmoc-His(trt)-OH, dissolved in DMF, is added to the reactor containing the deprotected resin, and HOBt / DIC is added and stirred to proceed with the synthesis process. Piperidine and DMF are then added to the reactor containing the resin, stirred, and the Fmoc protecting group on histidine is removed. Cholesteryl chloroformate is dissolved in DMF and DCM, added to the reactor containing the resin, and DIPEA (N,N-diisopropylethylamine) is added, stirred overnight, and the resin is washed with DMF and DCM. Finally, to remove the trityl protecting group attached to histidine, it is added to the peptide dried with trifluoroacetic acid and stirred. Afterwards, the filtered composite was slowly added to cold Ether to precipitate crystals, and the composite was finally obtained by freeze-drying (see Fig. 1).

[0111] Hchol was also synthesized by the following method. Histidine amide dihydrochloride dissolved in DMF was reacted with 1 equivalent (DMF / DCM = 4 / 1, v / v) of cholesteryl chloroformate dissolved in dichloromethane (DCM). 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 small portions of 40 mL of distilled water. The reaction mixture was filtered and washed several times with distilled water. The product was then collected in DCM and purified by preparative TLC (DCM: diethyl ether: ethyl acetate: ammonium hydroxide = 5:1:0.5:0.5:0.1).

[0112] The composite was dissolved in Methanol-d4 and measured at 600 MHz. 1 H nuclear magnetic resonance (Fig. 2) and molecular weight (Fig. 3) were analyzed, and it was confirmed that the synthesis was successful.

[0113]

[0114] Example 2. Preparation of lipid nanoparticles (LNPs)

[0115] Lipid nanoparticles (LNPs) were fabricated by adding the organic phase in ethanol and the nucleic acids in a citric acid buffer solution (pH 4) at a molar ratio of 1:2 into a microfluidic mixing device (Spark, Precision Nanosystems). The organic phase contained ionizable lipids (DLin-MC3-DMA lipid (MC3) or SM102 lipid), phospholipids (DSPC), sterols (cholesterol or histidinamide cholesterol), and PEG lipids (DMG-PEG2000) in ethanol at a molar ratio of 50:10:38.5:1.5, while the aqueous phase contained nucleic acids, which were diluted to a final citric acid concentration of 50 mM using firefly luciferase mRNA or EGFP mRNA. LNPs were fabricated by adjusting the amounts of cholesterol and histidinamide cholesterol (Hchol) at a molar ratio of 38.5% of the total lipid content to allow the Hchol content to range from 0% to 100%.

[0116] To remove ethanol from the generated LNPs and adjust the pH to the body, they were dialyzed for 16 h against 1X PBS using MWCO 3,500 Da Slide-A-Lyzer™ Dialysis Cassettes (Thermo Scientific) or MWCO 3,500 Da Dialysis membranes. After dialysis, the lipid nanoparticle solution was passed through a 0.22 μm filter to obtain lipid nanoparticles (LNPs) loaded with nucleic acids.

[0117] The ratio of N / P of 5.5 or 6.5 was used based on the number of amines (N) in ionizable lipids: the number of phosphate groups (P) in mRNA.

[0118] The size, polydiversity (PDI), and zeta potential of lipid nanoparticles containing mFluc were measured using a Zetasizer Nano ZS (Malvern, UK).

[0119]

[0120] In an experimental example of the present invention, HeLa (Human cervix adenocarcinoma cell line), HepG2 (Human hepatocellular carcinoma cell line), and NIH3T3 (Mouse embryonic fibroblast cell line) cell lines were cultured in an incubator maintained at 37°C, 5% CO2, and 95% humidity using DMEM containing 10% FBS and antibiotic-antimycotic reagent.

[0121]

[0122] Experimental Example 1. pKa Analysis of Lipid Nanoparticles

[0123] To measure the pKa of lipid nanoparticles, a TNS experiment was conducted. Lipid nanoparticles were diluted in PBS to a 1 mM concentration, and TNS was dissolved in DMSO to a 300 μM concentration. The buffer solutions used for the TNS experiment were prepared from pH 3.5 to 10 using sodium phosphate, sodium borate, sodium citrate, and sodium chloride. Ninety-four 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. The plates were placed in a fluorescence plate reader, and TNS fluorescence was measured at excitation and emission wavelengths of 330 nm and 435 nm. The pKa of each lipid nanoparticle was determined at the half-maximum point of the measured fluorescence value. The results are shown in Figure 4.

[0124] pKa, the acid dissociation constant, is a commonly used indicator of the acid strength of a target substance. The pKa value of lipid nanoparticles is crucial for their in vivo stability and drug release. Anionic TNS interact with positively charged lipid nanoparticles, strengthening interactions between their hydrophobic moieties and increasing fluorescence. As the pH approaches the pKa value of each LNP, the interaction between the hydrophobic moieties rapidly weakens, allowing more water molecules to quench the TNS fluorescence, decreasing the fluorescence to approximately 50% of the maximum. As the pH continues to increase, the fluorescence intensity continues to decrease, allowing for a graph showing the pH-dependent fluorescence. These results can be used to measure the pKa value of lipid nanoparticles. In particular, numerous studies have reported that lipid nanoparticles with a pKa of 6.0 to 7.0 interact efficiently with the endosomal membrane and readily escape endosomes upon acidification, resulting in superior in vivo gene delivery and expression efficiency.

[0125] As confirmed in Fig. 4, it was confirmed that the lipid nanoparticles of the present invention exhibit excellent in vivo stability and drug release in the pKa range of 6.0 to 7.0.

[0126]

[0127] Experimental Example 2. Analysis of gene encapsulation efficiency and stability

[0128] Lipid nanoparticles were formed using luciferase mRNA with ionizable lipid:DSPC:Sterol:DMG-PEG2000 (50:10:38.5:1.5 molar ratio). Two types of ionizable lipids, MC3 lipid and SM102 lipid, were used, and lipid nanoparticles (LNPs) were produced by adding 100% cholesterol (Chol) or histidine amide cholesterol (Hchol), a cholesterol variant, to the sterol site.

[0129] Analysis of gene encapsulation efficiency

[0130] 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% TritonX-100, incubated at 37°C for 10 minutes, and RiboGreen (Excitation 485 nm, Emission 538 nm) fluorescence was measured. The mRNA encapsulation efficiency of the nanoparticles was calculated using the following equation.

[0131] mRNA encapsulation efficiency (%) = [(total mRNA concentration - unencapsulated mRNA concentration) / total mRNA concentration] × 100 (%)

[0132] As a result, referring to Fig. 5, the nucleic acid encapsulation efficiencies of MC3-Chol LNP and MC3-Hchol LNP, which were manufactured with an N / P ratio of 6.7, were 72% and 91%, respectively, and the nucleic acid encapsulation efficiencies of SM102-Chol LNP and SM102-Hchol LNP, which were manufactured with an N / P ratio of 5.6, were 78% and 87%, respectively. This confirmed that histidine amide cholesterol, which was manufactured by modifying cholesterol, had superior mRNA encapsulation efficiency compared to cholesterol.

[0133] Analysis of stability and mRNA encapsulation efficiency over time

[0134] The manufactured lipid nanoparticles were stored at 4°C, and the particle size and polydispersity index (PDI) values ​​were measured at each time point using dynamic light scattering equipment. In addition, changes in the mRNA encapsulation efficiency of the lipid nanoparticles were measured at each time point.

[0135] As a result (Fig. 6), it was confirmed that all lipid nanoparticles according to the present invention maintained a stable state over time, and that the mRNA encapsulation efficiency and level were also maintained.

[0136]

[0137] Experimental Example 3. Measurement of the size and zeta potential of lipid nanoparticles

[0138] Lipid nanoparticles manufactured in the same manner as in Experimental Example 1 above were obtained through a filter, diluted in 1X PBS with a neutral pH, and the particle size and zeta potential were measured.

[0139] MC3-CholMC3-HcholSM102-CholSM102-HcholSize (nm)124.6121.291.02115.9PDI0.1640.1470.1640.19Zeta potential (mV)-8.32-1.86-3.42-1.84

[0140] As shown in Table 1 above, all lipid nanoparticles produced, including the control group, had a size of approximately 120 nm, and the PDI values ​​were generally measured to be below 0.2, indicating that lipid nanoparticles of uniform and appropriate size were formed. The zeta potential was confirmed to have a slightly negative value near 0. Therefore, it was confirmed that lipid nanoparticles were formed as nanoparticles of a consistent size.

[0141] Experimental Example 4. Evaluation of Intracellular Gene Transfer Efficiency

[0142] To evaluate the mRNA delivery efficiency of LNPs manufactured using luciferase mRNA, experiments were conducted in HeLa and HepG2 cell lines. Cells were seeded at 15,000 cells per well in a 96-well plate 24 hours prior to sample administration and cultured at 37°C, 5% CO2, and 95% humidity. Lipid nanoparticles were administered to each well at a concentration of 100 ng / well to evaluate gene delivery efficiency at the cellular level. Twenty-four hours after sample injection, the cell culture medium was removed and washed with DPBS. After adding 1X Reporter Lysis Buffer (Promega), the cells were completely lysed in a shaking incubator at room temperature for 30 minutes. The cells were then collected and centrifuged at 13,200 rpm for 10 minutes. Only the supernatant containing protein was obtained, and the activity of luciferase was confirmed using a luciferin agent. The produced protein was calculated using Micro BCA™ Protein Assay to obtain a quantified relative light of unit (RLU) value.

[0143] As a result, as shown in Fig. 7, when the same amount of mRNA was injected, it was confirmed that LNPs using histidine amide cholesterol (Hchol) lipids showed higher Luciferase protein expression than LNPs using cholesterol (Chol) lipids. In the HeLa cell line, SM102-Hchol LNPs showed about 9.6 times higher expression efficiency than SM102-Chol LNPs (Fig. 7A), and in the HepG2 cell line, they showed about 16.6 times higher expression efficiency (Fig. 7B). In addition, when lipid nanoparticles were prepared by dividing Hchol by ratio using MC3 as an ionizable lipid and evaluating the gene delivery efficiency, it was confirmed that as the ratio of Hchol increased, higher protein expression was shown (Fig. 7C).

[0144]

[0145] Experimental Example 5. Intracellular toxicity evaluation

[0146] To evaluate the toxicity of the manufactured LNPs, a WST assay was performed. Experiments were conducted on HepG2 (liver cancer) cell lines and NIH3T3 (normal cell) cell lines. Cells were seeded at 13,000 cells per well in a 96-well plate 24 hours prior to sample administration. Cells were cultured in an incubator at 37°C, 5% CO2, and 95% humidity. Cells were treated with 100 ng of LNP (based on mRNA concentration) and, 24 hours later, treated with Ez-Cytox reagent WST-8, followed by incubation for 2 hours. WST formazan was measured for absorbance at 450 nm using a VERASmax microplate reader.

[0147] As a result, neither SM102-chol LNP nor SM102-Hchol LNP showed toxicity in normal NIH3T3 cells (Fig. 8A). In addition, it was confirmed that no toxicity was observed in any sample in HepG2 cells, a cancer cell line (Fig. 8B).

[0148] Cytotoxicity was assessed by adding a reagent and measuring the amount of WST formazan as absorbance to assess cell viability. Each sample showed a cell viability close to 100%. Therefore, it was confirmed that lipid nanoparticles formed from histidine amide cholesterol (Hchol) are not toxic to cells.

[0149]

[0150] Experimental Example 6. Analysis of intracellular introduction of lipid nanoparticles and protein expression efficiency.

[0151] Confocal microscopy was used to determine the extent to which lipid nanoparticles were introduced over time and the extent to which mRNA delivered into cells through lipid nanoparticles was expressed into protein.

[0152] HeLa cell line was seeded at 1.2×10 in 8-well u-slide 4 / well and cultured in an incubator at 37°C, 5% CO2, and 95% humidity for 24 h. LNPs containing 100 ng / well of Cyanine 5 fluorescently tagged EGFP mRNA were treated 24, 12, and 4 h before fluorescence microscopy observation. Endosomes and nuclei of cells were stained with Lysotracker Red and Hoechest 33342, respectively. The stained cells were observed using a confocal microscope (Carl Zeiss).

[0153] As a result, when comparing the fluorescence of LNPs produced using Chol as a control group and Hchol LNP as an experimental group in a HeLa cell line, there was no significant difference at 4 hours, but when Hchol LNP was administered, a high fluorescence signal of EGFP (green) was observed at 12 and 24 hours, and the fluorescence signal of Cy5 in Hchol LNP was also observed to be higher than that in Chol LNP (Fig. 9).

[0154]

[0155] Experimental Example 7. In vivo gene transfer efficiency and expression analysis

[0156] Animal experiments were conducted with approval from the Animal Experiment Ethics Committee of the Korea Research Institute of Bioscience and Biotechnology (Approval Number: KRIBB-AEC-22165).

[0157] To evaluate the expression efficiency of proteins expressed in vivo from mRNA contained in LNPs depending on the type of cholesterol constituting the manufactured LNPs, fLuc mRNA-LNPs (0.1 mg / kg) were administered to C57BL / 6 mice (6-8 weeks old, female; Orient Bio) via intramuscular or tail vein injection. 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, and then the mice were anesthetized by inhalation under 3% isoflurane and 97% medical oxygen. Subsequently, luminescence images were acquired using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA) and analyzed using Living Image Software (version 4.7, PerkinElmer).

[0158] As a result, both Chol LNP and Hchol LNP showed maximum luminescence at 6 hours and then gradually decreased. Compared to the control SM102-chol LNP, SM102-Hchol LNP showed more luciferase activity in the body and the expression lasted longer (Fig. 8A). In addition, LNPs made using MC3 lipid also showed the same effect, and as the ratio of Hchol increased, the fluorescence gradually increased at 6 hours, confirming that increasing the ratio of Hchol instead of Chol increases gene delivery efficiency and protein expression (Fig. 10B).

[0159]

[0160] statistical processing

[0161] Statistical analysis of experimental data was performed using the Prism 5 program from GraphPad, Inc., using Student's t test or ANOVA analysis. A p value less than 0.05 indicates statistical significance (*, p<0.05; **, p<0.01; ***, p<0.001). The ns indicated in the data indicates statistically insignificant.

[0162]

[0163] In summary, lipid nanoparticles (LNPs) prepared using Hchol were confirmed to increase intracellular gene delivery efficiency and in vivo protein expression efficiency. The experimental results at the cellular level tended to be consistent with the in vivo experimental results, and cytotoxicity was shown to be non-toxic to both cancer cells and normal cells. Furthermore, it was confirmed that as the ratio of Hchol increased, mRNA encapsulation efficiency, intracellular gene delivery efficiency, and in vivo protein expression increased.

[0164] Consequently, Hchol exerts its influence both on the surface and throughout the interior of lipid nanoparticles. By binding histidine amide to cholesterol, it is expected to increase the number of amines in lipids that are ionizable in acidic environments, facilitating their entry into cells while simultaneously aiding their escape from endosomes. Furthermore, it is known that Hchol interacts with mRNA within the particle, enhancing encapsulation efficiency and contributing to the stabilization of lipid nanoparticles.

[0165] Finally, the toxicity of lipid nanoparticles was evaluated in HepG2 cells and NIH3T3 cells. The viability of LNP samples in both cancer cells and normal cells was over 90%, confirming that lipid nanoparticles utilizing Hchol lipids are biocompatible and effectively deliver and express mRNA into cells.

[0166]

[0167] The above description is merely an illustrative description of the present invention, and those skilled in the art will appreciate that various modifications may be made without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in this specification are intended to illustrate, rather than limit, the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all techniques within a scope equivalent thereto should be interpreted as being included within the scope of the present invention.

[0168]

[0169] As described above, the present invention relates to a histidine amide cholesterol compound represented by Chemical Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a composition for delivering a nucleic acid, protein or drug comprising the same, which can be usefully used as a delivery vehicle by being included in a pharmaceutical product, vaccine, etc.

Claims

1. A histidine amide cholesterol compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof <Chemical Formula 1> 2. Lipid nanoparticles comprising a histidine amide cholesterol compound according to paragraph 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

3. In the second paragraph, the lipid nanoparticle is characterized in that it further comprises at least one selected from the group consisting of ionizable lipids, phospholipids, cholesterol, and PEG lipids.

4. Lipid nanoparticles characterized in that the ionizable lipid in the third paragraph is SM-102 or DLin-MC3-DMA.

5. Lipid nanoparticles characterized in that the phospholipid in the third paragraph is DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine).

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

7. A lipid nanoparticle characterized in that the mixture of ionizable lipid; phospholipid; histidine amide cholesterol compound, stereoisomer thereof or pharmaceutically acceptable salt thereof; cholesterol; and PEG lipid in the molar ratio of 10-60:1-20:5-70:0-30:0.5-5 in the third paragraph.

8. A composition for delivery of a nucleic acid, protein or drug comprising a lipid nanoparticle according to any one of claims 2 to 7.

9. A composition for delivery of a nucleic acid, protein or drug, characterized in that the nucleic acid in paragraph 8 is selected from the group consisting of DNA, RNA, PNA, interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

10. A composition for delivery of a nucleic acid, protein or drug, characterized in that the protein in claim 8 is an antibody.

11. A composition for delivery of a nucleic acid, protein or drug, characterized in that the drug in paragraph 8 is a therapeutic or preventive agent.

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

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