Ionizable lipids containing sulfides, and lipid nanoparticles containing the same.

Abstract

The present invention relates to a novel ionizable lipid containing sulfide. The ionizable lipid of the present invention can be useful in related technical fields such as lipid nanoparticle-mediated gene therapy by electrostatically interacting with an anionic drug during the preparation of lipid nanoparticles, thereby enabling the drug to be encapsulated in the lipid nanoparticles with high efficiency and the drug to be stably delivered to the body.

Description

[Technical Field]

[0001] The present invention relates to ionizable lipids containing sulfides, and more specifically, to ionizable lipids containing sulfides, lipid nanoparticles produced using the same, and their uses. [Background technology]

[0002] A drug delivery system (DDS) is a technology designed to efficiently deliver the required amount of drug by mitigating drug side effects and maximizing its efficacy and effects. In particular, while conventional viral carriers have proven effective as drug carriers in gene therapy, the use of viruses as gene delivery systems is limited by several drawbacks, including immunogenicity, limitations on the size of injected DNA, and the difficulty of mass production.

[0003] Therefore, as an alternative to viral systems, methods for transporting nucleic acids into cells by mixing them with positively charged lipids or polymers (named lipoplexes and polyplexes, respectively) are currently mainly used (Hirko et al., Curr, Med, Chem., 10, 1185-1193, 2003; Merdan et al., Adv.Drug.Deliv.Rev., 54, 715-758, 2002; Spagnou et al., Biochemistry, 43, 13348-13386, 2004). In particular, lipid-DNA complexes are widely used at the cellular level because they bind to nucleic acids and deliver them effectively into cells. However, in vivo, local injection often causes inflammation (Filonand and Phillips, Biochim.Biophys / Acta, 1329, 345-356, 1997), and when injected intravenously, they have the disadvantage of accumulating mainly in tissues such as the lungs, liver, and spleen, which are the first-pass organs (Ren et al., Gene Therapy.7, 764-768, 2000). [Overview of the project] [Problems that the invention aims to solve]

[0004] The inventors of this invention have made diligent efforts to develop a novel substance that has excellent drug encapsulation rates and can efficiently deliver anionic drugs such as nucleic acids to desired organs or cells. As a result, they have completed this invention by confirming the excellent drug delivery effect of a novel ionizable lipid containing the sulfide of this invention. [Means for solving the problem]

[0005] One object of the present invention is to provide ionizable lipids containing a novel sulfide structure, stereoisomers thereof, or pharmaceutically acceptable salts thereof.

[0006] Another object of the present invention is to provide lipid nanoparticles comprising the ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0007] Another object of the present invention is to provide a drug delivery composition comprising the lipid nanoparticles and an anionic drug, which delivers the drug specifically to the liver or lungs. [Effects of the Invention]

[0008] The sulfide-containing ionizable lipids of the present invention interact electrostatically with anionic drugs during the production of lipid nanoparticles, thereby enabling the drugs to be encapsulated in the lipid nanoparticles with high efficiency, and thus enabling the drugs to be delivered specifically and stably to the liver or lungs. [Brief explanation of the drawing]

[0009] [Figure 1] The results of measuring the luminescence intensity of mRNA-encapsulated lipid nanoparticles containing the ionizable lipids of the present invention in in vitro efficacy studies using HeLa, HepG2, HEK 239, and MRC-5 cells are shown. [Figure 2]This image shows bioluminescence after intravenous injection into mice to confirm in vivo delivery of mRNA-encapsulated HHES lipid nanoparticles depending on the type of ionizable lipid in an in vivo efficacy study. [Figure 3] This graph compares the bioluminescence observed in the liver of mice after intravenous injection to confirm the in vivo delivery efficacy of mRNA-encapsulated HHES lipid nanoparticles based on the type of ionizable lipid. [Figure 4] This image shows bioluminescence after intramuscular injection into mice to confirm the in vivo delivery of mRNA-encapsulated HHES lipid nanoparticles depending on the type of ionizable lipid in an in vivo efficacy study. [Figure 5] This graph compares the bioluminescence observed after intramuscular injection into mice to confirm the in vivo delivery efficacy of mRNA-encapsulated HHES lipid nanoparticles based on the type of ionizable lipid. [Figure 6] This graph shows the blood EPO protein levels and MCP-1 values ​​after intravenous injection of hEPO mRNA-encapsulated lipid nanoparticles into mice to confirm their in vivo delivery efficacy and initial immunogenicity. [Figure 7] This photograph shows bioluminescence observed after intravenous injection into mice to confirm in vivo delivery of mRNA-encapsulated lipid nanoparticles in proportion to the DOTAP content, as observed in an in vivo efficacy study. [Figure 8] This graph compares the bioluminescence of the liver, spleen, and lungs of mice after intravenous injection to confirm the in vivo delivery efficacy of mRNA-encapsulated lipid nanoparticles in relation to the proportion of DOTAP they contain. [Figure 9] This graph compares the luminescence distribution in various organs after intravenous injection into mice, in order to confirm the in vivo delivery efficacy of mRNA-encapsulated lipid nanoparticles in relation to the proportion of DOTAP they contain. [Modes for carrying out the invention]

[0010] Specifically, it is as follows. On the other hand, each of the descriptions and embodiments disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Also, the scope of the present invention should not be limited by the following specific description.

[0011] As a result of the research efforts of the inventors to achieve the above object, an ionizable lipid containing a sulfide represented by the following Chemical Formula 1 and lipid nanoparticles containing the same were completed. Further, it was confirmed that the lipid nanoparticles can stably and effectively deliver a drug, such as a nucleic acid, to the liver or lung.

[0012] Ionizable lipids containing sulfides One aspect of the present invention for achieving the above object relates to an ionizable lipid represented by the following Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. [Chemical Formula 1]

[0013] [Chemical Structure]

[0014] In the above Chemical Formula 1, A is

[0015] [Chemical Structure]

[0016] and R1 and R2 are each independently -H, -C , 2-8 , 4-12 , alkyl, -C 1-6 alkyl-NR3R4, or -C 2-12 alkyl-(C=O)-Y-(CH2) x S-C 4-12 alkyl, and are each any one selected therefrom, R3 and R4 are each independently -C 2-8 alkyl, R5 is -C2-12 Alkyl-(C=O)-Y-(CH2) x S-C 4-12 is alkyl, Y is -O- or NR6, R6 is -H or -C 1-3 is alkyl, m is an integer from 0 to 3, n is independently an integer from 2 to 7, x is an integer from 1 to 3, p is an integer from 0 to 2.

[0017] Without being limited thereto, the compound represented by the above Chemical Formula 1 is specifically in the above Chemical Formula 1, A is,

[0018]

Chemical formula

[0019] and, R5 is -C 2-12 Alkyl-(C=O)-Y-(CH2) x S-C 4-12 is alkyl, Y is -O-, m is an integer from 0 to 3, n is independently an integer from 2 to 7, x is an integer from 1 to 3, p may be 1.

[0020] Also, according to a specific example of the present invention, the compound represented by the above Chemical Formula 1 may contain any one or more selected from the group consisting of the compounds described in Table 1 below.

[0021]

Table 1

[0022] In this invention, the term "alkyl" means a straight-chain or branched-chain acyclic saturated hydrocarbon unless otherwise specified. For example, "C 1-6 "Alkyl" can mean an alkyl group containing 1 to 6 carbon atoms. Even if a simple substituent is added to the alkyl structure of the present invention, all such additions are included within the scope of the present invention, as long as they have the same effect as the ionizable lipids of the present invention.

[0023] In the present invention, "ionizable lipid" refers to an amine-containing lipid that can be easily protonated, and is also named a lipid analog. For example, it may be a lipid whose charge state changes depending on the surrounding pH. It plays a role in enabling the anionic drug to be encapsulated in lipid nanoparticles with high efficiency through electrostatic interaction with the drug, and contributes to the formation of the lipid nanoparticle structure. The ionizable lipid of the present invention is characterized in that an alkyl chain containing a sulfide is bonded to the amine-containing head portion. Specifically, the ionizable lipid may be a compound having similar properties to lipids produced by the reaction of an amine containing a piperazine structure with an alkyl sulfide.

[0024] The ionizable lipid is an ionizable compound having properties similar to those of a lipid, and can play a role in enabling the drug (e.g., anionic drugs and / or nucleic acids) to be encapsulated with high efficiency within the lipid nanoparticles through electrostatic interactions with the drug.

[0025] In this invention, the term "stereoisomer" means a compound of the present invention having the same chemical or molecular formula but being stereoisomerically different. Such stereoisomers and mixtures thereof are also included within the scope of this invention. Unless otherwise specified, solid line bonds (-) linked to chiral carbon atoms represent solid wedge-shaped bonds indicating an absolute arrangement of stereocenters.

[0026] [ka]

[0027] or dotted wedge-shaped connection

[0028] [ka]

[0029] It may include.

[0030] The compound of Chemical Formula 1 of the present invention may exist in the form of a "pharmaceutically acceptable salt." While acid addition salts formed with pharmaceutically acceptable free acids are useful as salts, they are not limited to these. In the present invention, the term "pharmaceutically acceptable salt" means any organic or inorganic acid addition salt or base addition salt of the compound having a concentration that is relatively non-toxic and harmless to the patient, and whose side effects do not diminish the beneficial efficacy of the compound represented by Chemical Formula 1.

[0031] Acid addition salts can be produced by conventional methods, for example, by dissolving a compound in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone, or acetonitrile. Alternatively, equimolar amounts of the compound and an acid or alcohol in water may be heated, and the mixture may be evaporated or the precipitated salt may be filtered by suction.

[0032] In this case, organic acids and inorganic acids can be used as free acids. As inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid may be used, and as organic acids, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, or hydroiodic acid may be used, but are not limited to these.

[0033] Furthermore, pharmaceutically acceptable metal salts can also be produced using bases. Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the insoluble compound salt, and then evaporating and drying the filtrate. In this case, the metal salt can be, but is not limited to, sodium salts, potassium salts, or calcium salts. Corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0034] Lipid nanoparticles containing ionizable lipids including sulfides Another aspect of the present invention is lipid nanoparticles comprising an ionizable lipid represented by the above chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The lipid nanoparticles of the present invention may contain one ionizable lipid represented by the chemical formula 1, or two or more thereof. Furthermore, depending on the purpose, they may further contain ionizable lipids other than those of the present invention.

[0035] The lipid nanoparticles may further contain, but are not limited to, one or more selected from the group consisting of helper lipids, structural lipids, and PEG-lipids.

[0036] The aforementioned auxiliary lipids can be any auxiliary lipid capable of promoting the fusion of lipid nanoparticles, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), and egg phosphatidylcholine (egg 1,2-dioleoyl-3-trimethylammonium-propane (1,2-dioleoyl-3-trimethylammonium-propane) opane (DOTAP), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate (18-PA), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0) PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-diun 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 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-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (1,It may be 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, or a mixture thereof.

[0037] In the present invention, PEG-lipid refers to a form in which lipids and PEG are conjugated, and means a lipid to which a hydrophilic polymer, polyethylene glycol polymer, is bound. Within lipid nanoparticles, the PEG-lipid contributes to the particle stability of the nanoparticles in serum and plays a role in preventing aggregation between nanoparticles. Furthermore, the PEG-lipid can protect nucleic acids from degrading enzymes, enhance the in vivo stability of nucleic acids, and extend the half-life of drugs encapsulated within nanoparticles. The PEG-lipid may be, but is not limited to, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, or mixtures thereof.

[0038] The structural lipids maintain the particle shape within the lipid nanoparticles and play a role in improving the stability of the nanoparticles by being dispersed in the core and on the surface of the nanoparticles. The structural lipids may be, but are not limited to, cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, or mixtures thereof.

[0039] As a specific example, when producing lipid nanoparticles by mixing the ionizable lipids, auxiliary lipids, structural lipids, and PEG lipids of the present invention, the molar ratio of ionizable lipids:auxiliary lipids:structural lipids:PEG lipids may be 10-40:10-70:40-70:1-5. Furthermore, the molar ratio may also be 15-35:10-30:45-65:1-4, or 20-30:15-25:50-60:1-3, but is not limited to these.

[0040] The lipid nanoparticles of the present invention exhibit a positive charge under acidic pH conditions, and therefore readily form complexes with negatively charged nucleic acids and therapeutic agents such as anionic drugs through electrostatic interactions, enabling highly efficient encapsulation of anionic drugs. This allows them to be used as intracellular or in vivo drug delivery compositions. Therefore, the lipid nanoparticles of the present invention can be usefully used for the delivery of not only nucleic acids but also all types of anionic drugs. In other words, the lipid nanoparticles of the present invention can ultimately be manufactured in a form further containing anionic drugs (encapsulated form).

[0041] In this invention, the term "encapsulation" means surrounding a delivery substance and encapsulating it for efficient absorption into the body, and the drug encapsulation rate (encapsulation efficiency) refers to the content of the drug encapsulated within the lipid nanoparticles relative to the total drug content used in the manufacture.

[0042] The anionic drug may be a nucleic acid, a small molecule compound, a peptide, a protein, a protein-nucleic acid structure, or an anionic biopolymer-drug complex, but is not limited to these, as long as it can form lipid nanoparticles with the ionizable lipids of the present invention and be delivered stably and efficiently.

[0043] In the present invention, the nucleic acid may be, but is not limited to, small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), sgRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme, or mixtures thereof.

[0044] The weight ratio of total lipids to nucleic acids in the lipid nanoparticles may be 1 to 20, more specifically 5 to 15, and more specifically 7 to 12, but is not limited thereto.

[0045] In the present invention, the lipid nanoparticles may have a diameter of, for example, 40 to 150 nm, more specifically 50 to 140 nm, and more specifically 60 to 130 nm, but are not limited thereto.

[0046] In yet another embodiment of the present invention, the lipid nanoparticles may further contain 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as an auxiliary lipid.

[0047] As a specific example, when producing lipid nanoparticles by mixing the ionizable lipid of the present invention with DOTAP, structural lipids, and PEG lipids, the DOTAP may be present in an amount of 10 to 70 mol% relative to the total lipids of the lipid nanoparticles. Specifically, the molar ratio of ionizable lipid:DOTAP:structural lipid:PEG lipid may be 10 to 40:10 to 70:1 to 70:1 to 5. Furthermore, the molar ratio may also be 15 to 35:15 to 65:5 to 65:1 to 4, or 20 to 30:20 to 60:10 to 60:1 to 3, but is not limited to these.

[0048] The lipid nanoparticles containing DOTAP may further contain an anionic drug, and the lipid nanoparticles may have a diameter of, for example, 30 to 100 nm, more specifically 40 to 100 nm, and more specifically 50 to 90 nm, but are not limited thereto.

[0049] Drug delivery and pharmaceutical compositions containing lipid nanoparticles Another aspect of the present invention is a drug delivery composition comprising anionic drug-containing lipid nanoparticles according to the present invention.

[0050] Another aspect of the present invention is a pharmaceutical composition comprising an anionic drug-containing lipid nanoparticles according to the present invention as an active ingredient.

[0051] Lipid nanoparticles and anionic drugs are as described above.

[0052] The lipid nanoparticles of the present invention form stable complexes with anionic drugs such as nucleic acids and possess low cytotoxicity and effective cell water absorption, making them effective for the delivery of anionic drugs. Therefore, depending on the type of anionic drug and nucleic acid used, the lipid nanoparticles may have preventive or therapeutic effects against related diseases and can be used without limitation as a drug delivery composition. Furthermore, since the lipid nanoparticles can be delivered specifically to the liver or lungs at the time of administration, desired tissues can be targeted as needed.

[0053] In the present invention, the term "treatment" refers to intervention to alter the natural processes of an individual or cells with a disease, which is performed during the progression of a pathological condition or to prevent it. Desired therapeutic effects include preventing the onset or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, reducing or temporarily alleviating a disease state, improving recovery, or improving prognosis. In particular, the present invention includes all actions that improve the course of a disease by administering lipid nanoparticles containing sulfide-containing ionizable lipids, their stereoisomers or pharmaceutically acceptable salts thereof, and anionic drugs as active ingredients. The term "prevention" also refers to all actions that suppress or delay the onset of a disease by administering the lipid nanoparticles. When the lipid nanoparticles of the present invention are used for therapeutic or preventive purposes, they are administered to the individual in a therapeutically effective amount.

[0054] As used in this invention, the term "therapeutably effective amount" refers to an effective amount of anionic drug-containing lipid nanoparticles. Specifically, "therapeutably effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the individual's species and severity, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, route of administration and elimination rate, duration of treatment, concomitant drugs, and other well-known factors in the medical field. The pharmaceutical composition of this invention may be administered as a single therapeutic agent, in combination with other therapeutic agents, or sequentially or simultaneously with commercially available therapeutic agents. It may also be administered as a single dose or multiple doses. Considering all of the above factors, it is important to administer the amount that provides the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of this invention can be determined by a professional based on various factors such as the patient's condition, age, sex, and comorbidities. Because the active ingredient of the composition of this invention is highly safe, it can be used even in doses exceeding the prescribed dosage.

[0055] The composition containing the aforementioned lipid nanoparticles as an active ingredient can be administered by oral, intramuscular, intravenous, arterial, subcutaneous, peritoneal, pulmonary, and nasal injection, but is not limited to these methods.

[0056] The composition of the present invention may further contain one or more pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable carriers include physiological saline, sterile water, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. Other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Therefore, the composition of the present invention may be in the form of patches, pills, capsules, granules, tablets, suppositories, etc. These formulations can be manufactured by conventional methods used in the art for formulation or by methods disclosed in the literature [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and can be formulated into various formulations depending on the disease or component.

[0057] Embodiments of the present invention can be modified into various other forms, and the scope of the invention is not limited to the embodiments described below. Furthermore, embodiments of the present invention are provided to more fully explain the invention to a person with average skill in the art. Moreover, throughout the specification, "including" a component means that, unless otherwise stated, other components may be included rather than being excluded. [Examples]

[0058] Modes for explaining the invention The configuration and effects of the present invention will be described in detail below through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[0059] Manufacturing example: Production of ionizable lipids containing sulfides.

[0060] [Reaction Equation 1]

[0061] [ka]

[0062] First, using the synthesis method of reaction formula 1 above, bromate and 2-((hexylthio)ethane-1-ol) were reacted in a 1:1 molar ratio to produce tail groups containing sulfides of various alkyl chain lengths.

[0063] [Reaction Equation 2]

[0064] [ka]

[0065] Next, using the synthesis method of reaction equation 2 described above, the tail group and N-(2-aminoethyl)piperazine, which will become the head group, were reacted in a molar ratio of 2.36:0.7 to synthesize an ionizable lipid having a linear structure containing sulfides, which was named 244nHHES (where n is the number of carbon atoms from the head group to the ester group).

[0066] Example 1-1: Preparation of Compounds 1-4 Specific examples of the ionizable lipids containing sulfides according to the present invention are shown in Table 2 below.

[0067] [Table 2]

[0068] As an example, the specific manufacturing method for compound 3 (2448HHES) is as follows: 3 mmol of 2-((hexylthio)ethane-1-ol and 3 mmol of 8-bromooctanoic acid were added to 1 ml of DCM (dichloromethane) solvent. Then, 4.6 mmol of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride containing DIC (N,N-Diisopropylcarbodiimide) and 0.6 mmol of DMAP (4-Dimethylaminopyridine) were added, and the mixture was stirred at room temperature for 18 hours. The reaction product was then filtered and evaporated under vacuum. The residue was purified by column chromatography using a RediSep Gold Resolution silica column (Teledyne Isco) with a CombiFlash RF system, by gradient elution from 100% hexane to a hexane:ethyl acetate volume ratio of 95:5. As a result, 2.36 mmol of 2-((hexylthio)ethyl 8-bromooctanoate was synthesized in 76.6% yield and named 8HHES.

[0069] Next, 2.36 mmol of 2-((hexylthio)ethyl 8-bromooctanoate), 0.7 mmol of N-(2-aminoethyl)piperazine, and 0.7 mmol of N,N-diisopropylethylamine were added to 1 ml of DMF (N,N-Dimethylmethanamide) solvent, stirred at 80°C for 3 days, and evaporated under vacuum. The residue was purified by column chromatography using a RediSep Gold Resolution silica column (Teledyne Isco) with a CombiFlash RF system, by gradient elution from 100% DCM to a DCM:MeOH:NH4OH volume ratio of 95:4.5:0.5. As a result, ionizable lipids were produced in a yield of 36.6%.

[0070] The compounds in the remaining examples were also prepared in the same manner as in reaction formulas 1 and 2 described above.

[0071] Examples 1-2. Confirmation of the synthesis of ionizable lipids containing sulfides. MS analysis was performed to confirm the synthesis of compounds 1-4 prepared in Example 1-1 above. The results are shown in Table 3 below.

[0072] [Table 3]

[0073] Example 2. Production of lipid nanoparticles containing nucleic acids Example 2-1. Production of lipid nanoparticles containing auxiliary lipids (DOPE) The ionizable lipids (2446HHES~2449HHES) prepared as described above, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≥99%, Sigma, Korea), DOPE (18:1(△9-Cis)PE(DOPE), Avanti, USA), and C16-PEG2000 ceramide (C16 PEG2000 Ceramid, Avanti, USA) were dissolved in ethanol in a molar ratio of 26.5:52:20:1.5. In addition, 30 μg of mRNA was diluted in 0.75 ml of sodium citrate as an RNA therapeutic agent.

[0074] Lipid nanoparticles containing nucleic acids were produced by mixing an organic phase (ethanol) containing ionizable lipids, cholesterol, accessory lipids, and lipid-PEG, and an aqueous phase (sodium acetate or sodium citrate) containing mRNA, at a flow rate of 12 ml / min using a microfluidic mixing apparatus (Benchtop Nanoassemblr; PNI, Canada). To produce lipid nanoparticles containing luciferase mRNA (SEQ ID NO: 1), the organic and aqueous phases were mixed in a weight ratio of mRNA:ionizable lipid of 1:10 to produce lipid nanoparticles. Subsequently, the ethanol was removed, and the molecules were dialyzed with PBS (Phosphate-buffered saline) for 16 hours using a 3500 MWCO dialysis cassette to match the pH of the lipid nanoparticles with the pH of the body.

[0075] Example 2-2. Production of lipid nanoparticles containing auxiliary lipids (DOTAP) Ionizable lipids (2448HHES):DOTAP (1,2-Dioleoyl-3-trimethylammonium, Avanti, USA):cholesterol:lipid-PEG (C16-PEG2000 ceramide) were dissolved in ethanol in molar ratios of 26.5:20 to 60:12 to 52:1.5. Lipid nanoparticles were then produced by mixing the organic and aqueous phases in a weight ratio of mRNA:ionizable lipids of 1:10 (Table 4). The produced lipid nanoparticles were dialyzed with PBS for 16 hours using a 3500 MWCO dialysis cassette to remove ethanol and match the pH of the lipid nanoparticles to the pH of the body.

[0076] [Table 4]

[0077] Experimental Example 1. Confirmation of the physicochemical properties of lipid nanoparticles Experimental Example 1-1. Measurement of Drug Encapsulation Rate Ribogreen analysis (Quant-iT TM Ribo Green (R) The drug encapsulation rate (encapsulation efficiency, %) of each lipid nanoparticle containing mRNA as a nucleic acid drug was measured using RNA (Invitrogen). The lipid nanoparticles were diluted in 50 μl of 1xTE buffer in a 96-well plate to a final mRNA concentration of 4-7 μg / ml. 50 μl of 1xTE buffer was added to the Triton-X untreated group (Triton-X LNP(-)), and 50 μl of 2% Triton-X buffer was added to the Triton-X treated group (Triton-X LNP(+)). By incubation at 37°C for 10 minutes, the lipid nanoparticles were degraded by Triton-X, releasing the encapsulated nucleic acid. Subsequently, 100 μl of Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNP(-) and Triton LNP(+) was measured using Infinite (R)The drug encapsulation rate (capsulation efficiency, %) was calculated using the following formula 1, measured with a 200 PRO NanoQuant (Tecan) at a wavelength bandwidth (exitation: 485 nm, emission: 528 nm).

[0078] [Mathematical formula 1] Drug encapsulation rate (%) = (Fluorescence of Triton LNP(+) - Fluorescence of Triton LNP(-)) / (Fluorescence of Triton LNP(+)) × 100

[0079] [Table 5]

[0080] As can be seen from Table 5 above, the lipid nanoparticles according to the present invention have been confirmed to be able to encapsulate drugs with high efficiency.

[0081] Experimental Example 1-2. Measurement of Particle Size We attempted to measure the size of lipid nanoparticles containing the manufactured nucleic acids. The RNA (luciferase mRNA) contained in each lipid nanoparticle manufactured in Example 2 above was diluted with PBS to a concentration of 1 μg / ml, and the diameter and polydispersity index (PDI) of the LNPs were measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano (Malvern Instruments, UK). The respective result values ​​are as follows (Table 6).

[0082] [Table 6]

[0083] Experimental Example 2. Confirmation of the efficacy of nucleic acid-encapsulated lipid nanoparticles in a test tube. To confirm the efficacy of lipid nanoparticles encapsulating nucleic acids in a test tube, a screening was performed using lipid nanoparticles synthesized with multiple tail groups. The test involved delivering luciferase mRNA to HeLa, HepG2, HEK 293, and MRC-5 cells using lipid nanoparticles (compounds 1-4) synthesized with multiple tail groups, and then measuring the luminescence intensity to confirm the expression of the gene.

[0084] Specifically, one day before transforming the cells with lipid nanoparticles, HeLa, HepG2, HEK 293, and MRC-5 cells (Korea Cell Line Bank) are placed in a white plate (96-well) in a 0.01 × 10⁶ layer. 6 Cells were dispensed into individual cells / well and cultured in DMEM media (SH30022, Hyclone, USA) at 37°C and 0.5-3% CO2. Using the mRNA contained in the lipid nanoparticles as a reference, cells were treated with lipid nanoparticles containing luciferase mRNA at a dose of 25 ng / well. After 24 hours, Bright-Glo TM Luciferase assay solution (promega, USA) was applied to 100 μl / well, and after being left at room temperature for 10 minutes, the luminescence intensity of the lysed cells was measured using an Infinite M200 emission analyzer (Tecan, USA).

[0085] As can be seen in Figure 1, the luminescence intensity was significantly increased in all lipid nanoparticles compared to the positive control group, SM-102.

[0086] Experimental Example 3. Confirmation of the in vivo efficacy of lipid nanoparticles. Experimental Example 3-1. Effects of Luciferase mRNA-Inclusion Lipid Nanoparticles Lipid nanoparticles encapsulating luciferase mRNA were prepared, and each nanoparticle was dialyzed with PBS for 16 hours to remove ethanol. Seven-week-old female C57BL / 6 mice (Orient bio) were intravenously or intramuscularly injected with lipid nanoparticles containing mRNA at a dose of 0.1 mg / kg, based on the mRNA contained in the lipid nanoparticles. Six hours later, 0.25 mg / kg of luciferin was administered intraperitoneally, and bioluminescence was confirmed using an IVIS (PerkinElmer, USA) instrument. Mice administered with luciferase mRNA-encapsulated lipid nanoparticles were sacrificed, organs were removed, and the biodistribution of lipid particles in each organ was confirmed using the IVIS instrument.

[0087] As a result of intravenous injection, as shown in Figures 2 and 3, luciferase mRNA was encapsulated by whole-body imaging and ex vivo organ imaging. It was confirmed that in the case of lipid nanoparticles containing HHES-ionizable lipids, a liver-specific high luminescence intensity was observed.

[0088] Furthermore, intramuscular injection confirmed that lipid nanoparticles containing luciferase mRNA and HHES-ionizable lipids emitted light at the injection site (Figures 4 and 5).

[0089] Experimental Example 3-2. Effects of hEPO mRNA-encapsulated lipid nanoparticles Lipid nanoparticles containing hEPO mRNA (SEQ ID NO: 2) were administered to mice intravenously at an mRNA dose of 0.5 mg / kg. Blood was collected 6 hours after injection, and serum was separated from the blood. hEPO and MCP-1 levels were confirmed using the hEPO ELISA kit (DEP00; R&D systems, Minneapolis, MN, USA) and the MCP-1 ELISA kit (BMS281; Thermo Fisher, Waltham, MA, USA), respectively.

[0090] As a result, as shown in Figure 6, the lipid nanoparticles containing the ionizable lipids of the present invention showed significantly higher blood EPO protein levels compared to MC-3, and the MCP-1 value was significantly lower compared to SM-102. MCP-1 is an indicator of the initial immunogenicity of lipid nanoparticles, and it was confirmed that the lipid nanoparticles containing the ionizable lipids of the present invention have significantly lower initial immunogenicity compared to SM-102.

[0091] Experimental Example 4. Confirmation of the properties and effects of lipid nanoparticles containing auxiliary lipids (DOTAP). Experimental Example 4-1. Confirmation of the properties of lipid nanoparticles based on the DOTAP content ratio. The physicochemical properties of the lipid nanoparticles produced in Example 2-2 were confirmed and are shown in Table 7 below. Excellent drug encapsulation rates were also confirmed when the auxiliary lipid was used as DOTAP.

[0092] [Table 7]

[0093] Experimental Example 4-2. Confirmation of in vivo efficacy based on DOTAP content ratio. Seven-week-old female C57BL / 6 mice (Orient bio) were intravenously injected with lipid nanoparticles containing luciferase mRNA at a dose of 0.1 mg / kg, based on the mRNA contained in the lipid nanoparticles as a reference. Six hours later, 0.25 mg / kg of luciferin was administered intraperitoneally, and bioluminescence was confirmed using an IVIS (PerkinElmer, USA) instrument. The mice administered with lipid nanoparticles containing luciferase mRNA were sacrificed, their organs were removed, and the biodistribution of lipid particles in each organ was confirmed using the IVIS instrument.

[0094] As a result, whole-body imaging and in vitro organ imaging confirmed that lipid nanoparticles containing luciferase mRNA exhibited high luminescence intensity in the lungs (Figure 7). Therefore, it was confirmed that the ionizable lipids and DOTAP-containing lipid nanoparticles of the present invention deliver nucleic acids specifically to the lungs.

[0095] From the above description, those skilled in the art in which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, the above embodiments should be understood to be illustrative and not limiting in all respects. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, which are described in more detail below.

Claims

1. An ionizable lipid represented by the following chemical formula 1, its stereoisomer, or a pharmaceutically acceptable salt thereof. [Chemical formula 1] 【Chemistry 1】 Here, A is 【Chemistry 2】 And, R ３ and R ４ Each of them is independently C ２－８ It is alkyl, R ５ is, -C ２－１２ Alkyl-(C=O)-Y-(CH ２ ) ｘ S-C ４－１２ It is alkyl, Y is -O-, m is an integer between 0 and 3. n is an independent integer between 2 and 7. x is an integer between 1 and 3. p is between 0 and 2.

2. The ionizable lipid according to Claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein p is 1.

3. The ionizable lipid described above is selected from the group consisting of compounds 1 to 4 listed in the table below, as described in claim 1, an ionizable lipid, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. Table 1

4. Lipid nanoparticles comprising an ionizable lipid according to any one of claims 1 to 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

5. The lipid nanoparticles according to claim 4, further comprising one or more selected from the group consisting of auxiliary lipids, structural lipids, and PEG-lipids.

6. The aforementioned auxiliary lipids are DOPE, DSPC, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, DOTAP, phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, POPE, DOPS, DLPC, DMPC, DUPC, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesterylhem Lipid nanoparticles according to claim 5, which are one or more selected from the group consisting of succinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin.

7. The lipid nanoparticle according to claim 5, wherein the PEG-lipid is one or more selected from the group consisting of PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

8. The lipid nanoparticle according to claim 5, wherein the structural lipid is one or more selected from the group consisting of cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and α-tocopherol.

9. The lipid nanoparticles according to claim 5, wherein the lipid nanoparticles contain ionizable lipids: auxiliary lipids: structural lipids: PEG-lipids in a molar ratio of 10-40:10-70:40-70:0.5-5.

10. The lipid nanoparticles according to claim 5, further comprising an anionic drug.

11. The lipid nanoparticle according to claim 10, wherein the anionic drug is one or more selected from the group consisting of nucleic acids, small molecule compounds, peptides, proteins, protein-nucleic acid structures, and anionic biopolymer-drug complexes.

12. The lipid nanoparticle according to claim 11, wherein the nucleic acid is one or more selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), sgRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.

13. The lipid nanoparticle according to claim 12, wherein the weight ratio of total lipids to nucleic acids in the lipid nanoparticle is 1 to 20.

14. The lipid nanoparticles according to claim 10, wherein the lipid nanoparticles have an average diameter of 40 nm to 150 nm.

15. A drug delivery composition comprising lipid nanoparticles as described in claim 10.

16. The drug delivery composition according to claim 15, wherein the drug delivery composition is delivered specifically to the liver.

17. The drug delivery composition according to claim 15, further comprising DOTAP as the lipid nanoparticles, which are delivered specifically to the lungs.

18. The drug delivery composition according to claim 17, wherein the DOTAP is contained in an amount of 10 to 70 mol% relative to the total lipids of the lipid nanoparticles.

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