Modified peptide-based novel lipid, lipid nanoparticles comprising same, and use thereof

Abstract

The present invention relates to a peptide-based novel modified lipid compound, a lipid nanoparticle composition comprising same, a vaccine composition comprising the lipid nanoparticle composition, and a preparation method therefor. According to the present invention, the compound may replace existing PEGylated lipids to form lipid nanoparticles, and may inhibit aggregation between particles to give excellent colloidal stability. In addition, the lipid nanoparticle composition may effectively encapsulate a therapeutic or prophylactic agent and thus may be usefully employed as a carrier for nucleic acids.

Description

Novel lipids based on modified peptides, lipid nanoparticles containing the same, and uses thereof

[0001] The present invention relates to a novel lipid compound based on a modified peptide, a lipid nanoparticle composition containing the same, a vaccine composition, and a method for preparing the same.

[0002] Nucleic acid-based drugs, which began over 40 years ago with the method of injecting plasmid DNA into the human body to aid in the production of deficient proteins, have since seen the reporting of various types that inhibit gene transcription and translation, including antigenes, decoys, antisense, interfering RNA (siRNA), and microRNA (miRNA). Nucleic acid-based drugs have garnered attention as personalized treatments because they target DNA or RNA rather than proteins, forming complementary bindings with specific DNA or RNA sequences. Nucleic acid-based drugs are utilized not only as therapeutic agents but also as preventive agents to defend against diseases by injecting genes capable of expressing antigens for specific illnesses. Gene-based vaccines are classified into DNA vaccines, RNA vaccines, and viral vector vaccines. Among these, RNA vaccines involve injecting messenger RNA (mRNA) encoding an antigen into the body to induce antigen expression and antibody formation; they possess the advantages of rapid development and eliminate potential risks associated with viral vector-based vaccines, such as concerns about infection, or DNA vaccines, such as genetic mutations.

[0003] Currently, lipid nanoparticles (LNPs) are generally used in the form of a mixture of four components in specific proportions: ionized lipids, phospholipids (helper lipids), cholesterol (structure-maintaining lipids), and PEGylated lipids. In particular, PEGylated lipids are widely used in lipid nanoparticle formulations for anticancer drugs as well as in messenger RNA vaccines; however, since hypersensitivity reactions including anaphylaxis and unexpected immune responses have been reported in association with many PEG-containing formulations, there is an urgent need to develop a substitute for PEGylated lipids that can maintain stability in the body with fewer side effects.

[0004] Meanwhile, since PEGylated lipids in lipid nanoparticles play a role in providing colloidal stability to the particle surface and inhibiting aggregation between particles, simply controlling hydrophilicity or hydrophobicity is not sufficient to replace them; a structure is required that simultaneously satisfies stable particle formation, nucleic acid encapsulation, dispersion stability in physiological environments, and delivery efficiency within the lipid nanoparticle composition. Therefore, there is a need for novel modified lipid compounds and compositions containing them that can replace existing PEGylated lipids while maintaining or improving the stability and delivery characteristics of lipid nanoparticles.

[0005] The technical problem that the present invention aims to solve is to provide a peptide-based modified lipid compound that can replace PEGylated lipids.

[0006] Another technical objective of the present invention is to provide a lipid nanoparticle composition comprising the above compound.

[0007] Another technical objective of the present invention is to provide a vaccine composition comprising the lipid nanoparticle composition.

[0008] Another technical objective of the present invention is to provide a method for manufacturing lipid nanoparticles using the above compound.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0010] To solve the above problem, the present invention provides a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof:

[0011]

[0012] In the above chemical formula 1,

[0013] The above R n It is hydrogen or -(C=O)-R 1 And,

[0014] The above R c is -NH2 or And,

[0015] The above R 1 It is a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms, and

[0016] The above X is a polymer represented by [EEZ]5, and

[0017] The above E is And,

[0018] The above Z is

[0019] and It is one of the selected ones.

[0020] (where R n In the case of this hydrogen, R c Is and R n This -(C=O)-R 1 In the case of R c is -NH2)

[0021] According to one side, the above R 1 It can be an alkyl group having 6 to 17 carbon atoms.

[0022] According to one side, the above E may be a glutamic acid residue that forms a main chain through a γ-carboxyl group.

[0023] According to one aspect, the compound may be any one or more selected from the group consisting of the following chemical formulas 1-1 to 1-15:

[0024] [Chemical Formula 1-1]

[0025]

[0026] [Chemical Formula 1-2]

[0027]

[0028] [Chemical Formula 1-3]

[0029]

[0030] [Chemical Formula 1-4]

[0031]

[0032] [Chemical Formula 1-5]

[0033]

[0034] [Chemical Formula 1-6]

[0035]

[0036] [Chemical Formula 1-7]

[0037]

[0038] [Chemical Formula 1-8]

[0039]

[0040] [Chemical Formula 1-9]

[0041]

[0042] [Chemical Formula 1-10]

[0043]

[0044] [Chemical Formula 1-11]

[0045]

[0046] [Chemical Formula 1-12]

[0047]

[0048] [Chemical Formula 1-13]

[0049]

[0050] [Chemical Formula 1-14]

[0051]

[0052] [Chemical Formula 1-15]

[0053]

[0054] According to another embodiment of the present invention, a lipid nanoparticle composition comprising any one of the above compounds, an ionized lipid, a helper lipid, and a structure-maintaining lipid is provided.

[0055] According to one side, the compound may replace PEGylated lipids to increase colloidal stability and prevent aggregation between particles.

[0056] According to one aspect, the helper lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinoreyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), It may be one or more selected from the group consisting of dipalmitoyl-phosphatidylethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0057] According to one aspect, the structure-maintaining lipid may be one or more selected from the group consisting of cholesterol and its derivatives, bile acid derivatives including alkyl lithocholates, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.

[0058] According to one aspect, the composition may comprise 8 to 12 mol% of the compound, 45 to 55 mol% of ionized lipids, 8 to 12 mol% of helper lipids, and 28 to 32 mol% of structure-maintaining lipids.

[0059] According to one aspect, the composition may comprise 10 mol% of the compound, 50 mol% of ionized lipids, 10 mol% of helper lipids, and 30 mol% of structure-maintaining lipids.

[0060] According to one side, the lipid nanoparticle composition described above may contain a therapeutic or preventive agent therein.

[0061] According to one aspect, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (DsiRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0062] According to one aspect, the therapeutic or prophylactic agent is messenger RNA (mRNA), and the lipid nanoparticle may have a diameter of 110 to 250 nm and a zeta potential of -45 to -30 mV.

[0063] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided.

[0064] According to another embodiment of the present invention, a method for manufacturing lipid nanoparticles is provided, said manufacturing method may include the steps of: preparing an organic phase comprising any one of an ionized lipid, a helper lipid, a structure-maintaining lipid, and said compound; contacting the organic phase with an aqueous phase to form lipid nanoparticles; and recovering said lipid nanoparticles.

[0065] According to one side, the above-mentioned award may include a therapeutic or prophylactic agent.

[0066] According to one aspect, the therapeutic or prophylactic agent may be one or more selected from messenger RNA (mRNA), interfering RNA (siRNA), microRNA (miRNA), self-amplifying RNA (saRNA), single guide RNA (sgRNA), guide RNA, plasmid DNA (pDNA), or antisense oligonucleotide.

[0067] The present invention provides a peptide-based modified lipid compound capable of replacing PEGylated lipids, thereby ensuring colloidal stability of lipid nanoparticles while suppressing aggregation between particles. Furthermore, lipid nanoparticles containing the compound of the present invention can efficiently encapsulate therapeutic or prophylactic agents such as nucleic acids, maintain an appropriate particle size and zeta potential, and can be usefully utilized as vaccine compositions or nucleic acid delivery vehicles. Moreover, according to the manufacturing method of the present invention, lipid nanoparticles containing the compound can be efficiently manufactured.

[0068] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by a person skilled in the art from the description below.

[0069] Figure 1 shows the results of measuring the in vitro intracellular transfection efficiency of lipid nanoparticles containing novel modified peptide lipid compounds of the present invention, where Figure 1a is a graph of fluorescence (FITC) expression levels of one experimental group including OGA093, etc., and Figure 1b is a graph of fluorescence expression levels of another experimental group including a control group (Con_LNP) and OGA129, etc.

[0070] Figure 2 is a graph showing the relative in vitro transfection efficiency (relative fluorescence ratio) of lipid nanoparticles containing novel modified peptide lipids (OGA085, OGA093, etc.) of the present invention compared to a reference substance (OGA050).

[0071] Figure 3 shows the results of an in vivo biodistribution evaluation of the lipid nanoparticles of the present invention, where Figure 3a is a graph showing the change in average spleen / liver radiance compared to the precursor material, Figure 3b is the in vivo imaging result of major organs extracted after administration of each lipid compound, and Figure 3c is a graph quantifying the signal distribution (spleen / liver ratio) of the spleen versus the liver based on the imaging results.

[0072] The inventors, noting that PEGylated lipids, which are one of the components of lipid nanoparticles, increase colloidal stability and prevent aggregation between particles to improve stability in the body but have the disadvantage of potentially causing allergic reactions and anaphylaxis, intend to provide a novel modified lipid compound based on peptides to replace it, a lipid nanoparticle composition containing the same, a vaccine composition containing the same, and a method for manufacturing the same.

[0073] The inventors provide a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof:

[0074]

[0075] In the above chemical formula 1,

[0076] The above R n It is hydrogen or -(C=O)-R 1 And,

[0077] The above R c is -NH2 or And,

[0078] The above R 1 It is a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms, and

[0079] The above X is a polymer represented by [EEZ]5, and

[0080] The above E is And,

[0081] The above Z is

[0082] and It is one of the selected ones.

[0083] (where R n In the case of this hydrogen, R c Is and R n This -(C=O)-R 1 In the case of R c is -NH2)

[0084] The term 'saturated hydrocarbon' used in the present invention refers to a hydrocarbon consisting of single bonds, and 'unsaturated hydrocarbon' refers to a hydrocarbon having one or more double or triple bonds. The saturated or unsaturated hydrocarbons having 6 to 22 carbon atoms used in the present invention may include straight-chain, branched, or a combination thereof, and preferably form lipid chains to impart hydrophobic interactions within lipid nanoparticles.

[0085] The term “alkyl group” as used in the present invention refers to an alkane from which one hydrogen atom has been removed, and preferably can be expressed as -(CH2)a-CH3. According to one aspect, R1 may be an alkyl group having 6 to 17 carbon atoms.

[0086] The term 'polymer' as used in the present invention may refer to a structure formed by the repetition of a single monomer or by the repeated arrangement of one or more different monomers. In the present invention, since X is a polymer represented as [EEZ]5, it may have a structure in which the repeating unit of EEZ is repeated 5 times, and thus may be an oligomeric structure containing a total of 15 residue units. Such a repeating structure provides a balance between the lipid portion and the hydrophilic portion, thereby providing properties favorable for particle formation and stability.

[0087] In the present invention, E may be a glutamate residue that forms a main chain through a γ-carboxyl group. That is, by being connected through the γ-carboxyl group of the glutamate side chain rather than through a general α-carboxyl group, the arrangement of the repeating unit and the stereochemical properties of the chain can be controlled. Such a γ-bonded glutamate repeating structure can provide hydrophilic and negative charge properties and can contribute to colloidal stability on the surface or interface of lipid nanoparticles.

[0088] In the present invention, Z may be any one selected from seven types of structures, and preferably may be a residue derived from γ-aminobutyric acid, a residue derived from serine, a residue derived from O-methylserine, a residue derived from O-ethylserine, a residue derived from threonine, a residue derived from tyrosine, or a residue derived from tryptophan. Depending on the selection of Z as above, hydrophilicity, hydrophobicity, hydrogen bonding ability, and steric volume may vary, and as a result, the formation ability, encapsulation efficiency, particle stability, and in vivo delivery characteristics of lipid nanoparticles can be controlled.

[0089] In the present invention, the above R c may be a terminal structure comprising an amide group or an amino acid-derived residue, and preferably may provide a structure capable of binding to a terminal acyl group by including a lysine-derived residue. Accordingly, the compound of the present invention may include both a form in which a lipid hydrocarbon chain is introduced at the N-terminal side and a form in which a lipid hydrocarbon chain is introduced at the C-terminal side via a lysine-derived residue. That is, according to the above proviso, R n In the case of this hydrogen, R c go and R n This -(C=O)-R 1 In the case of R c It is limited to a structure where it is -NH2.

[0090] As used in the present invention, the term 'stereoisomer' refers to a compound having the same molecular formula and the same bonding order but differing spatial arrangements of atoms, and 'racemic mixture' refers to a form in which optical isomers are mixed. Additionally, 'pharmaceuticalally acceptable salt' refers to a salt that is not harmful to the body and is acceptable for pharmaceutical use, and may include, for example, addition salts with inorganic or organic acids, or salts with bases.

[0091] According to one aspect, the compound may be any one or more selected from the group consisting of the following chemical formulas 1-1 to 1-15:

[0092] [Chemical Formula 1-1]

[0093]

[0094] [Chemical Formula 1-2]

[0095]

[0096] [Chemical Formula 1-3]

[0097]

[0098] [Chemical Formula 1-4]

[0099]

[0100] [Chemical Formula 1-5]

[0101]

[0102] [Chemical Formula 1-6]

[0103]

[0104] [Chemical Formula 1-7]

[0105]

[0106] [Chemical Formula 1-8]

[0107]

[0108] [Chemical Formula 1-9]

[0109]

[0110] [Chemical Formula 1-10]

[0111]

[0112] [Chemical Formula 1-11]

[0113]

[0114] [Chemical Formula 1-12]

[0115]

[0116] [Chemical Formula 1-13]

[0117]

[0118] [Chemical Formula 1-14]

[0119]

[0120] [Chemical Formula 1-15]

[0121]

[0122] According to another embodiment of the present invention, a lipid nanoparticle composition comprising the above compound is provided. The lipid nanoparticle may mean nano-sized spherical or substantially spherical particles composed of lipids.

[0123] According to one aspect, the compound may replace PEGylated lipids to increase colloidal stability and prevent aggregation between particles. That is, the compound of the present invention can be used as a substitute lipid that performs at least some of the surface stabilization function performed by conventional PEGylated lipids while reducing the possibility of PEG-derived side effects.

[0124] According to one aspect, the composition may further include one or more selected from the group consisting of ionized lipids, helper lipids, and structure-maintaining lipids, and preferably may include all of the ionized lipids, helper lipids, and structure-maintaining lipids.

[0125] The ionized lipid is an ionizable compound having properties similar to lipids and can serve to encapsulate drugs, such as anionic drugs and / or nucleic acids, with high efficiency through electrostatic interaction with said drugs. The ionizable lipid may be protonated at a pH below the pKa of the ionized lipid and may be substantially neutral at a pH above the pKa. In one example, the lipid nanoparticle may comprise a protonated ionizable lipid and / or an ionizable lipid exhibiting neutrality. The ionized lipid may be a cationic lipid, and a non-limiting example is SM-102.

[0126] The above helper lipids can play a role in promoting the fusion of lipid nanoparticles or assisting in particle formation, and include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoreyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), It may be one or more selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE), but preferably may be DSPC.

[0127] The above-mentioned structure-maintaining lipid may refer to a lipid that imparts structural rigidity within lipid nanoparticles and is dispersed in the core and surface of the nanoparticles to enhance the stability of the nanoparticles. The above-mentioned structure-maintaining lipid may be one or more selected from the group consisting of cholesterol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene, but preferably may be cholesterol.

[0128] According to one aspect, the composition may comprise 8 to 12 mol% of the compound, 45 to 55 mol% of ionized lipids, 8 to 12 mol% of helper lipids, and 28 to 32 mol% of structure-maintaining lipids. According to one aspect, the composition may comprise 10 mol% of the compound, 50 mol% of ionized lipids, 10 mol% of helper lipids, and 30 mol% of structure-maintaining lipids. Such compositional ranges may be advantageous for ensuring the formation, stability, and formulation suitability of lipid nanoparticles while allowing the compound of the present invention to function as a substitute for PEGylated lipids.

[0129] According to one aspect, the lipid nanoparticle composition described above may contain a therapeutic or prophylactic agent within it, and the therapeutic or prophylactic agent may preferably be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (DsiRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof, and most preferably may be messenger RNA (mRNA). When the therapeutic or prophylactic agent is messenger RNA, the lipid nanoparticle may preferably have a diameter of 110 to 250 nm and a zeta potential of -45 to -30 mV.

[0130] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided. The vaccine composition may preferably be an mRNA vaccine and may further comprise a pharmaceutically acceptable carrier or adjuvant.

[0131] The vaccine composition of the present invention may be administered via, for example, an oral or parenteral route. Here, parenteral refers to a broad route of administration and includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intranasal, sublingual, intravertebral, inhalation, ocular, rectal, vaginal, and ventricular administration.

[0132] When formulating the above composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration may include tablets, pills, powders, granules, capsules, troches, etc., and such solid formulations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, or gelatin, with one or more compounds or compositions according to the present invention. In addition, lubricants such as magnesium stearate or talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, or syrups, and may include wetting agents, sweeteners, flavoring agents, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents or suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used.

[0133] According to another embodiment of the present invention, a method for manufacturing lipid nanoparticles is provided. The manufacturing method may include the steps of: preparing an organic phase comprising an ionized lipid, a helper lipid, a structure-maintaining lipid, and a compound according to any one of claims 1 to 4; contacting the organic phase with an aqueous phase to form lipid nanoparticles; and recovering the lipid nanoparticles. The aqueous phase may include a therapeutic or prophylactic agent, and the therapeutic or prophylactic agent may be one or more selected from messenger RNA (mRNA), interfering RNA (siRNA), microRNA (miRNA), self-amplifying RNA (saRNA), single guide RNA (sgRNA), guide RNA, plasmid DNA (pDNA), or antisense oligonucleotide. The contact between the organic phase and the aqueous phase can be performed by known methods such as stirring, mixing, emulsification, or microfluidic mixing, and the formed lipid nanoparticles can be recovered through processes such as solvent exchange, filtration, concentration, or buffer replacement.

[0134] The composition according to the present invention may be administered in a pharmaceutically effective amount. In the present invention, a “pharmaceutically effective amount” means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses.

[0135] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0136] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0137] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0138]

[0139] Preparation Example 1. Design and Synthesis of Modified Peptide Lipid Compounds

[0140] The modified peptide lipid compound of the present invention was designed as a novel lipid compound to replace PEG-lipids among the components of lipid nanoparticles. In the present invention, all compounds were prepared using commercially available amino acid reagents.

[0141] Specifically, the modified peptide lipid compound of the present invention basically comprises a myristoyl group (Mys) or a lysine residue into which a myristoyl group is introduced [K(Mys)], a glutamic acid residue (Glu, E) forming a main chain through a γ-carboxyl group, and γ-aminobutyric acid (GABA), serine (Ser, S), and O-methylserine [Ser(Me), S Me ], O-ethylserine[Ser(Et), S Et It was designed to consist of a repeating structure including residues derived from ], threonine (Thr, T), tyrosine (Tyr, Y), and tryptophan (Trp, W).

[0142] All reagents used in the present preparation examples were commercially available. For example, glutamic acid derivatives, γ-aminobutyric acid (GABA) derivatives, serine derivatives, O-methylserine derivatives, O-ethylserine derivatives, threonine derivatives, tyrosine derivatives, tryptophan derivatives, lysine derivatives, and myristic acid or equivalent reagents for introducing myristoyl were used. The peptide backbone was synthesized by commissioning Peptron or Dandicure to obtain it according to the conventional solid phase peptide synthesis (SPPS) method.

[0143] The following modified peptide lipid compounds were prepared in this manner.

[0144] Among the compounds prepared, those having a myristoyl group at the N-terminus were as follows.

[0145] [Chemical Formula 1-1]

[0146]

[0147] [Chemical Formula 1-2]

[0148]

[0149] [Chemical Formula 1-3]

[0150]

[0151] [Chemical Formula 1-4]

[0152]

[0153] [Chemical Formula 1-5]

[0154]

[0155] [Chemical Formula 1-6]

[0156]

[0157] [Chemical Formula 1-7]

[0158]

[0159] All of the above compounds were prepared using commercially available amino acid derivatives and synthesized to have a structure that repeats the repeating unit [γ(EE)-Z] five times.

[0160]

[0161] Among the synthesized compounds, those having a lysine-myristoyl structure [K(Mys)] at the C-terminus were as follows.

[0162] [Chemical Formula 1-8]

[0163]

[0164] [Chemical Formula 1-9]

[0165]

[0166] [Chemical Formula 1-10]

[0167]

[0168] [Chemical Formula 1-11]

[0169]

[0170] [Chemical Formula 1-12]

[0171]

[0172] [Chemical Formula 1-13]

[0173]

[0174] [Chemical Formula 1-14]

[0175]

[0176] [Chemical Formula 1-15]

[0177]

[0178] The above C-terminal type compound can be prepared by first forming a C-terminal backbone containing a lysine residue, and then introducing a myristoyl group to the side chain amino group of lysine, or by a corresponding synthesis route.

[0179]

[0180] Comparative Manufacturing Example 1. Preparation of a Precursor

[0181] Mys-{γ(EEEMe)}5 and {γ(EEEMe)}5-K(Mys), which were used as prior art materials, were also prepared or obtained using the same solid-phase peptide synthesis method as described above or as described in Korean Patent Publication No. 10-2025-0144708. These compounds were used as comparative materials for comparison with the novel structure of the present invention.

[0182]

[0183] Experimental Example 1. Preparation and Physicochemical Characteristics Evaluation of Lipid Nanoparticles for mRNA Delivery Containing Novelly Modified Peptide Lipids

[0184] In order to determine whether the above-prepared modified peptide lipid compounds could replace PEGylated lipids to form lipid nanoparticles for mRNA delivery, lipid nanoparticles containing each compound were prepared and their physicochemical properties were evaluated.

[0185] The lipid nanoparticles were composed to include SM-102 as an ionized lipid, DSPC as a helper lipid, cholesterol as a structure-maintaining lipid, and the modified peptide lipid compound of the present invention. The control group used Con_LNP containing PEG-DMG as a PEG-lipid.

[0186] The aqueous phase containing mRNA can be prepared under conditions of 50 mM sodium citrate buffer, 110 mM sodium chloride, and pH 4.0, and the lipid mixture can be dissolved in an organic phase containing ethanol or dimethyl sulfoxide (DMSO). Subsequently, lipid nanoparticles were formed by rapid mixing at 25°C using a thermoshaker. In the example, the N / P ratio was set to 6. After preparation, the solvent was replaced with physiological saline or phosphate buffered saline (PBS) using a centrifugation filter tube as needed.

[0187] The size (diameter), polydispersity index (PDI), and zeta potential of the synthesized lipid nanoparticles were measured using a dynamic light scattering particle size analyzer (Zetasizer, Malvern Panalytical Ltd.). The results are shown in Table 1.

[0188]

[0189]

[0190] The 'e' in (EE)-e used above represents the D-amino acid monomer of E (glutamic acid). The (EEE) used above Me )'s E Me It is a modified form of the -C(=O)-OH group of the glutamic acid monomer, which is -C(=O)-O-CH3.

[0191] As shown in Table 1 above, some compositions of lipid nanoparticles containing the modified peptide lipid compounds of the present invention exhibited an appropriate particle size of 120 to 220 nm and a low PDI of less than 0.2, enabling the formation of stable lipid nanoparticles for mRNA delivery. In particular, OGA085, OGA101, OGA105, OGA109, OGA117, OGA129, and OGA137 exhibited relatively uniform size and low PDI. Some C-terminal compositions exhibited relatively large particle sizes or high PDIs, but showed high encapsulation efficiency.

[0192] The above results show that the novel modified peptide lipid compound of the present invention can form stable lipid nanoparticles for mRNA delivery while replacing PEG-DMG.

[0193]

[0194] Experimental Example 2. Evaluation of in vitro transfection efficiency of mRNA delivery lipid nanoparticles containing novel modified peptide lipids

[0195] To verify the intracellular delivery and expression efficiency of the above-prepared mRNA delivery lipid nanoparticles, an in vitro transfection experiment using Huh7 cells was performed.

[0196] Specifically, Huh7 cells were seeded at a density of 80,000 cells per well in 24-well cell culture plates the previous day and cultured. The following day, 0.25 μg of each lipid nanoparticle composition containing mRNA for Enhanced Green Fluorescence Protein (EGFP) expression was applied to the cells based on the mRNA amount per well. The negative control (NC), other comparison group (RO), Con_LNP, and prior peptide lipid comparison group (Con_OGA) were compared together. After culturing for 24 hours following treatment, the cells were harvested and measured using a flow cytometer. Fluorescence signals were measured in the FITC channel, and the FITC mean value was used as an indicator of mRNA transfection efficiency.

[0197] The results are shown in Figure 1. As shown in Figure 1, lipid nanoparticles including OGA093, OGA105, OGA117, OGA085, OGA109, and OGA101 exhibited significantly higher average FITC values ​​compared to NC and RO. In particular, OGA093 and OGA105 showed high fluorescence values ​​among the experimental groups, and OGA117, OGA085, OGA109, and OGA101 also showed significant fluorescence expression. In addition, Con_LNP showed a very high average FITC value, and lipid nanoparticles OGA129 and OGA137 containing novel modified peptide lipid compounds showed relatively high fluorescence values.

[0198] The above results show that lipid nanoparticles containing the novel modified peptide lipid compound of the present invention exhibit excellent mRNA delivery and expression efficiency in Huh7 cells, and are particularly advantageous for improving intracellular transfection efficiency.

[0199]

[0200] Experimental Example 3. Comparison of Relative Transfection Efficiency of Novel Modified Peptide Lipids Compared to Precursor

[0201] In order to confirm whether the novel modified peptide lipid structure of the present invention exhibits improved transfection efficiency compared to a prior material, the relative fluorescence ratio to OGA050 was compared with the reference material OGA050, and the prior peptide lipid comparison group Con_OGA was also compared. The method of preparation and composition of the reference material OGA050 are as described in Korean Published Patent No. 10-2025-0144708.

[0202] Specifically, Huh7 cells were treated with each mRNA delivery lipid nanoparticle in the same manner as in Experimental Example 2, and FITC fluorescence was measured using a flow cytometer. Subsequently, the fluorescence value of OGA050 was set to a reference value of 1, and the relative fluorescence ratio of each sample was calculated. The results are shown in Figure 2.

[0203] As shown in Figure 2, OGA085 and OGA093 showed relative fluorescence ratios of approximately 3.4 and approximately 9.3, respectively, OGA105 showed approximately 6.5, OGA129 showed approximately 4.7, OGA117 and OGA137 showed approximately 4.1 and approximately 3.8, respectively, OGA109 showed approximately 2.6, and OGA101 showed approximately 1.6 relative fluorescence ratios.

[0204] From the above results, it was confirmed that transfection efficiency increased in the C-terminal structure, and also γ-aminobutyric acid (GABA), serine (Ser, S), O-methylserine [Ser(Me), S Me ], O-ethylserine[Ser(Et), S Et It was confirmed that transfection efficiency increased in structures containing ], tyrosine (Tyr, T).

[0205] In other words, it was confirmed that the novel modified peptide lipid compound of the present invention can go beyond simply replacing PEG-lipids and significantly improve intracellular mRNA delivery and expression efficiency depending on structural modification.

[0206]

[0207] Experimental Example 4. Evaluation of in vivo biodistribution of mRNA delivery lipid nanoparticles containing novel modified peptide lipids

[0208] In order to confirm the in vivo distribution characteristics of lipid nanoparticles for mRNA delivery containing the novel modified peptide lipid compound of the present invention, an in vivo bio-distribution experiment was performed to compare signals by organ after administration to animals.

[0209] Specifically, each lipid nanoparticle composition containing Firefly luciferase (Fluc) mRNA was administered to two 8-week-old female C57BL / 6J rats at a dose of 10 μg based on mRNA amount via tail vein injection. Six hours after administration, 150 mg / kg of luciferin dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS) was injected intraperitoneally per rat, and major organs were excised 20 minutes later. The excised organs were the brain (Br), heart (Ht), lung (Lu), liver (Li), spleen (Sp), kidney (Kd), and lymph nodes (LN). Images of each organ were acquired using a bioimaging device, and the average radiance for each organ was calculated. Subsequently, the spleen / liver radiance ratio was calculated to evaluate the signal distribution of the liver relative to the spleen. The results are shown in Figure 3.

[0210] As shown in Fig. 3, various lipid nanoparticles containing novel modified peptide lipid compounds exhibited improved spleen tropism compared to the prior art-based composition or Con-LNP. Fig. 3b shows imaging for each lipid compound, and Fig. 3c shows a graph quantifying the spleen-to-liver ratio. In particular, OGA085, OGA093, OGA105, OGA117, OGA089, OGA101, OGA109, OGA129, and OGA137 showed higher spleen-to-liver ratios compared to Con-LNP. This suggests that the novel modified peptide lipids of the present invention can significantly increase the spleen-to-liver ratio, thereby mitigating liver-dominant distribution and increasing delivery to the spleen.

[0211] As shown in Figure 3a, the average spleen / liver radiance increased from 0.02 to 0.36 compared to the prior art material. This demonstrates that the novel modified peptide lipid compound of the present invention can significantly alter the distribution characteristics of lipid nanoparticles within the body, and implies high potential for application in fields where spleen targeting is particularly advantageous.

[0212]

[0213] Although the embodiments have been described above with reference to the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, suitable results may be achieved even if the described techniques are performed in a different order than described, and / or if the components of the described system, structure, device, circuit, etc. are combined or assembled in a form different from described, or replaced or substituted by other components or equivalents.

[0214] Therefore, other embodiments, other manufacturing examples, and equivalents to the claims also fall within the scope of the claims set forth below.

[0215]

[0216] This invention was completed with support from the institution's proprietary projects, the Future Source Biomedical Engineering Technology Development Project (Development of disability overcoming technology based on disease state monitoring (2E33770:20250101~20251231:)), the Future Source Biomedical Convergence Technology Development Project (Development of immunity-based autism treatment (2E33790:20250101~20251231:)), and the National R&D Project (Establishment and advancement of a natural killer cell-friendly delivery platform for non-viral-mediated gene manipulation (2N78400:20250101~20251231:)).

Claims

1. A compound represented by the following chemical formula 1, its stereoisomers, its racemic mixture, or its pharmaceutically acceptable salt: In the above chemical formula 1, The above R n It is hydrogen or -(C=O)-R 1 And, The above R c is -NH2 or And, The above R 1 It is a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms, and The above X is a polymer represented by [EEZ]5, and The above E is And, The above Z is and It is one of the selected ones. (where R n In the case of this hydrogen, R c Is and R n This -(C=O)-R 1 In the case of R c is -NH2) 2. In Paragraph 1, The above R 1 A compound having 6 to 17 carbon atoms as an alkyl group, a stereoisomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof.

3. In Paragraph 1, The above E is a compound that is a glutamic acid residue forming a main chain through a γ-carboxyl group, a stereoisomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof.

4. In Paragraph 1, The above compound is characterized as being one or more selected from the group consisting of the following chemical formulas 1-1 to 1-15, a stereoisomer thereof, a racemic mixture thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1-1] [Chemical Formula 1-2] [Chemical Formula 1-3] [Chemical Formula 1-4] [Chemical Formula 1-5] [Chemical Formula 1-6] [Chemical Formula 1-7] [Chemical Formula 1-8] [Chemical Formula 1-9] [Chemical Formula 1-10] [Chemical Formula 1-11] [Chemical Formula 1-12] [Chemical Formula 1-13] [Chemical Formula 1-14] [Chemical Formula 1-15] 5. A lipid nanoparticle composition comprising a compound of any one of claims 1 to 4, an ionized lipid, a helper lipid, and a structure-maintaining lipid.

6. A lipid nanoparticle composition according to claim 5, wherein the compound replaces PEGylated lipids to increase colloidal stability and prevent aggregation between particles.

7. In claim 6, the helper lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinoreyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dillinoreoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diol-leoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Palmitoyloleoylphosphatidylethanolamine (POPE), Distearoyl-phosphatidyl-ethanolamine (DSPE), A lipid nanoparticle composition characterized by being one or more selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoil-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

8. A lipid nanoparticle composition according to claim 6, wherein the structure-maintaining lipid is one or more selected from the group consisting of cholesterol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.

9. In Paragraph 6, A lipid nanoparticle composition characterized by comprising 8 to 12 mol% of the compound, 45 to 55 mol% of ionized lipid, 8 to 12 mol% of helper lipid, and 28 to 32 mol% of structure-maintaining lipid.

10. In Paragraph 9, A lipid nanoparticle composition characterized by comprising 10 mol% of the compound, 50 mol% of ionized lipid, 10 mol% of helper lipid, and 30 mol% of structure-maintaining lipid.

11. A lipid nanoparticle composition according to any one of claims 5 to 10, wherein the lipid nanoparticle composition comprises a therapeutic or prophylactic agent within.

12. A lipid nanoparticle composition according to claim 11, wherein the therapeutic or prophylactic agent is selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (DsiRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

13. In paragraph 12, the above therapeutic or prophylactic agent is messenger RNA, and A lipid nanoparticle composition characterized in that the lipid nanoparticles have a diameter of 110 to 250 nm and a zeta potential of -45 to -30 mV.

14. A vaccine composition comprising the lipid nanoparticle composition of claim 11.

15. A method for manufacturing lipid nanoparticles, A step of preparing an organic phase comprising an ionized lipid, a helper lipid, a structure-maintaining lipid, and a compound according to any one of claims 1 to 4; A step of forming lipid nanoparticles by contacting the above organic phase with an aqueous phase; and Step of recovering the above lipid nanoparticles A method for manufacturing lipid nanoparticles comprising 16. In Paragraph 15, The above-mentioned invention is a method for manufacturing lipid nanoparticles comprising a therapeutic or prophylactic agent.

17. In Paragraph 16, A method for preparing lipid nanoparticles, wherein the above-mentioned therapeutic or prophylactic agent is one or more selected from the group consisting of mRNA, siRNA, miRNA, saRNA, sgRNA, guide RNA, pDNA, or antisense oligonucleotides.

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