Ionizable lipids containing biodegradable ester bonds and lipid nanoparticles containing the same

Abstract

The present invention relates to a novel ionizable lipid containing a biodegradable ester bond. The ionizable lipid containing an ester bond of the present invention stably delivers anionic drugs when preparing lipid nanoparticles, and is particularly effective in nucleic acid delivery, and can be useful in related technical fields such as lipid nanoparticle-mediated gene therapy.

Description

[Technical Field]

[0001] The present invention relates to a novel ionizable lipid containing a biodegradable ester bond. Specifically, the present invention relates to an ionizable lipid containing a biodegradable ester bond, lipid nanoparticles prepared using the same, and uses thereof. [Background technology]

[0002] Drug delivery systems (DDS) are technologies designed to efficiently deliver the required amount of drugs while minimizing side effects and maximizing efficacy and effectiveness. In particular, traditional viral vectors have been proven to be effective as drug delivery vehicles in gene therapy. However, their use as gene delivery systems has been limited due to several drawbacks, such as immunogenicity, limited size of injected DNA, and difficulty in mass production.

[0003] Therefore, as an alternative to viral systems, the method of transporting nucleic acids into cells has mainly been to transport them by mixing them with positively charged lipids or polymers (termed lipid-DNA conjugates (lipoplexes) and polymer-DNA conjugates (polyplexes), respectively) (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 conjugates are widely used at the cellular level because they bind to nucleic acids and effectively deliver nucleic acids into cells. However, when injected locally in vivo, they often cause inflammation in the body (Filonand and Phillips, Biochim. Biophys / Acta, 1329, 345-356, 1997), and when injected intravascularly, they accumulate mainly in tissues such as the lungs, liver, and spleen, which are first-pass organs (Renet et al., Gene Therapy. 7, 764-768, 2000).

[0004] Under these circumstances, the present inventors have made extensive efforts to develop a novel substance that has an excellent drug encapsulation rate and can efficiently deliver anionic drugs such as nucleic acids to target organs or cells. As a result, they have completed the present invention by confirming the excellent drug delivery effect of the novel ionizable lipid containing a biodegradable ester bond of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0005] One object of the present invention is to provide an ionizable lipid containing a biodegradable ester bond of a novel structure, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0006] Another object of the present invention is to provide lipid nanoparticles comprising said 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. [Means for solving the problem]

[0008] This will be explained in detail as follows. Note that each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. In other words, all combinations of all elements disclosed in the present invention belong to the scope of the present invention. Furthermore, the scope of the present invention should not be considered to be limited by the specific descriptions set forth below.

[0009] To achieve the above object, the present inventors have conducted extensive research and have found that when lipid nanoparticles are prepared using an ionizable lipid containing an ester bond represented by the following formula 1, the lipid nanoparticles can deliver drugs stably and effectively with minimal side effects such as hepatotoxicity, thereby completing the present invention.

[0010] Ionizable lipids containing ester bonds To achieve the above object, one embodiment of the present invention is an ionizable lipid represented by the following formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0011] [ka] where A is [ka] or [ka] and R1 and R2 are each independently -H, -C 1-6 Alkyl, -C 1-6 Alkyl-NR3R4, or -C 2-12 Alkyl-(C=O)-Y-(CH2) xCH=CH-C 4-12 alkyl, R3 and R4 are each independently -H, -C 1-6 Alkyl, or -C 2-12 Alkyl-(C=O)-Y-(CH2) x CH=CH-C 4-12 alkyl, R5 is -C 1-6 Alkyl-NR3R4 or -C 2-12 Alkyl-(C=O)-Y-(CH2) x CH=CH-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 5, n is an integer from 1 to 11, x is an integer from 1 to 5, l is an integer from 2 to 10, and p is an integer of 0 to 2.

[0012] Although not limited thereto, the compound represented by the formula 1 specifically includes the following compounds in the formula 1: A is, [ka] and R1 and R2 are each independently -H, -C 1-3 Alkyl, -C 1-4 Alkyl-NR3R4, or -C 4-8 Alkyl-(C=O)-Y-(CH2) x CH=CH-C 4-8 alkyl, R3 and R4 are each independently -H, -C 1-3 Alkyl, or -C 4-8 Alkyl-(C=O)-Y-(CH2) x CH=CH-C 4-8 alkyl, Y is —O—; m is an integer from 0 to 3, n is an integer from 3 to 7, x is an integer from 1 to 3, l is an integer from 2 to 6, and p may be 1.

[0013] Furthermore, the compound represented by the formula 1 may be, but is not limited to, the compound represented by the formula 1, A is, [ka] and R3 and R4 are each independently -H, -C 1-3 Alkyl, or -C 4-8 Alkyl-(C=O)-Y-(CH2) x CH=CH-C 4-8 alkyl, R5 is -C 1-4 Alkyl-NR3R4 or -C 4-8 Alkyl-(C=O)-Y-(CH2) x CH=CH-C 4-8 is alkyl, Y is —O—; m is an integer from 0 to 3, n is an integer from 3 to 7, x is an integer from 1 to 3, l is an integer from 2 to 6, and p may be 1.

[0014] According to an embodiment of the present invention, the compound represented by Formula 1 may be selected from the group consisting of compounds set forth in Table 1 below.

[0015] [Table 1] TIFF0007828458000007.tif184161

[0016] In the present invention, the term "alkyl" means, unless otherwise specified, a straight or branched chain acyclic saturated hydrocarbon. For example, "C 1-6 The term "alkyl" can refer to an alkyl having 1 to 6 carbon atoms. In the alkyl structure of the present invention, even if a simple substituent is added, it is included in the scope of the present invention within an equivalent range as long as it has an effect equivalent to that of the ionizable lipid of the present invention.

[0017] In the present invention, the term "ionizable lipid" refers to an amine-containing lipid that can be easily protonated and is also called a lipidoid. The charge state of the ionizable lipid changes depending on the ambient pH, allowing the drug to be efficiently encapsulated in lipid nanoparticles through electrostatic interactions with anionic drugs, thereby contributing to the formation of the lipid nanoparticle structure. The ionizable lipid of the present invention is characterized by a structure in which an alkyl chain containing an ester bond and a double bond is attached to an amine head containing at least one primary amine. Compared to other known ionizable lipids, the ionizable lipid has excellent efficacy in terms of tissue specificity and gene expression rate in vivo during nucleic acid delivery, is easily degraded in vivo after drug delivery, and has minimal side effects such as hepatotoxicity.

[0018] As used herein, the term "stereoisomer" refers to compounds of the present invention that have the same chemical or molecular formula but are stereochemically distinct. Each such stereoisomer and mixtures thereof are likewise included within the scope of the present invention. Unless otherwise specified, a solid bond (-) connected to an asymmetric carbon atom is replaced by a solid wedge bond indicating the absolute configuration of the stereocenter. [ka] or dashed wedge bond [ka] may include:

[0019] The compound of Formula 1 in the present invention can exist in the form of a "pharmaceutically acceptable salt." Examples of useful salts include, but are not limited to, acid addition salts formed with pharmaceutically acceptable free acids. The term "pharmaceutically acceptable salt" in the present invention refers to any and all organic or inorganic acid addition salts or base addition salts of the compound represented by Formula 1, which have a relatively non-toxic and harmless effective concentration in patients, and whose side effects do not reduce the beneficial effects of the compound.

[0020] Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Equal molar amounts of the compound and the acid or alcohol in water are heated, and the mixture is then evaporated to dryness, or the precipitated salt can be filtered off with suction.

[0021] In this case, the free acid may be an organic acid or an inorganic acid. The inorganic acid may be hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid. The organic acid may be 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, carbonic acid, vanillic acid, or hydroiodic acid, but is not limited thereto.

[0022] Pharmaceutically acceptable metal salts can also be prepared using bases. Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving a compound in a solution of excess alkali metal hydroxide or alkaline earth metal hydroxide, filtering the undissolved compound salt, and then evaporating and drying the filtrate. Examples of metal salts that can be prepared include, but are not limited to, sodium, potassium, or calcium salts. Corresponding silver salts can also be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

[0023] Lipid nanoparticles containing ionizable lipids containing ester bonds - Patent Application 20070122999 Another aspect of the present invention is lipid nanoparticles comprising an ionizable lipid represented by the above-mentioned formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The lipid nanoparticles of the present invention may contain one type of ionizable lipid represented by formula 1, or two or more types. Furthermore, they may further contain an ionizable lipid other than that of the present invention, depending on the purpose.

[0024] The lipid nanoparticles may further include at least one selected from the group consisting of phospholipids, structural lipids, and PEG-lipids, but are not limited thereto.

[0025] The phospholipid surrounds and protects the core formed by the interaction of the ionizable lipid and the drug within the lipid nanoparticle, and binds to the phospholipid bilayer of the target cell, facilitating cell membrane passage and endosomal escape during intracellular delivery of the drug. The phospholipid may be any phospholipid that can promote fusion of lipid nanoparticles, and examples thereof include dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EGG), and the like. phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine amine (DSPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine (dipalmitoylphosphatidylethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphate (18-PA), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16:0PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (1-palmitoyl-2-oleoyl-sn-glycero-3-phos phoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (1,2-diundecanoyl-sn-glycero-phosphocholine, DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (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 (1-hexadecyl-sn-glycero-3-phosphocholine, C16 Lyso PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine The glycerol may be 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, or a mixture thereof.

[0026] The structural lipid provides morphological rigidity to the lipid packing within the lipid nanoparticle and is dispersed in the core and surface of the nanoparticle, improving the stability of the nanoparticle. The structural lipid may be, for example, but is not limited to, cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or a mixture thereof.

[0027] In the present invention, PEG-lipid refers to a conjugated lipid and PEG, i.e., a lipid having a hydrophilic polymer, polyethylene glycol, attached to one end. The PEG-lipid contributes to the particle stability of lipid nanoparticles in serum and prevents aggregation of nanoparticles. Furthermore, the PEG-lipid protects nucleic acids from degradative enzymes during in vivo delivery, thereby enhancing the stability of nucleic acids in vivo and increasing the half-life of drugs encapsulated in nanoparticles. The PEG-lipid may be, for example, but is not limited to, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, or a mixture thereof.

[0028] As a specific example, when the ionizable lipid of the present invention is mixed with a phospholipid, a structural lipid, and a PEG lipid to produce lipid nanoparticles, the molar ratio of ionizable lipid:phospholipid:structural lipid:PEG-lipid may be 10-40:10-30:40-70:1-5. Alternatively, the molar ratio may be 20-30:15-25:45-55:1-5, but is not limited thereto.

[0029] The lipid nanoparticles of the present invention exhibit a pKa of 6.0 to 7.0, specifically 6.0 to 6.5, and are therefore positively charged under acidic pH conditions. They readily form complexes with negatively charged therapeutic agents, such as nucleic acids and anionic drugs, through electrostatic interactions, and can encapsulate anionic drugs with high efficiency, making them suitable for use as intracellular or in vivo drug delivery compositions. Therefore, the lipid nanoparticles of the present invention can be useful for delivering not only nucleic acids but also any form of anionic drug. In other words, the lipid nanoparticles of the present invention can be ultimately produced in a form that additionally contains anionic drugs (encapsulated form).

[0030] In the present invention, the term "encapsulation" refers to the process of surrounding a delivery substance and encapsulating it for efficient in vivo uptake, and the drug encapsulation efficiency refers to the content of the drug encapsulated in the lipid nanoparticle relative to the total content of the drug used in the preparation.

[0031] 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 conjugate, but is not limited thereto as long as it can form lipid nanoparticles together with the ionizable lipid of the present invention and can be delivered stably and efficiently.

[0032] The nucleic acid in the present invention may be, but is not limited to, siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme, or a mixture thereof.

[0033] In the lipid nanoparticles, the weight ratio of ionizable lipid to nucleic acid may be 1 to 20, for example, 5 to 15, more specifically 8 to 12, but is not limited thereto.

[0034] The lipid nanoparticles of the present invention may have a diameter of, for example, 50 to 90 nm, but are not limited thereto.

[0035] Drug delivery and pharmaceutical compositions comprising lipid nanoparticles Another aspect of the present invention is a drug delivery composition comprising anionic drug-containing lipid nanoparticles according to the present invention.

[0036] Another aspect of the present invention is a pharmaceutical composition comprising, as an active ingredient, the anionic drug-containing lipid nanoparticles according to the present invention.

[0037] The lipid nanoparticles and anionic drugs are as described above.

[0038] The lipid nanoparticles of the present invention form stable complexes with anionic drugs such as nucleic acids, exhibiting low cytotoxicity and effective cellular uptake, making them effective for delivering anionic drugs. Therefore, the lipid nanoparticles have preventive or therapeutic effects on related diseases depending on the type of anionic drug and nucleic acid used, and have unlimited potential for use as drug delivery compositions.

[0039] The term "treatment" in the present invention refers to intervention to alter the natural processes of an individual or cell with a disease, which can be performed during the progression of a pathological condition or to prevent it. The intended therapeutic effects include preventing the onset or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences associated with the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily alleviating the disease state, and improving the prognosis. In particular, the present invention includes any action that improves the course of a disease by administering lipid nanoparticles containing an ester-bond-containing ionizable lipid, its stereoisomer, or a pharmaceutically acceptable salt thereof, and an anionic drug as active ingredients. The term "prevention" also refers to any action that suppresses or delays 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 an individual in a therapeutically effective amount.

[0040] The term "therapeutically effective amount" used herein refers to an effective amount of anionic drug-containing lipid nanoparticles. Specifically, "therapeutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors well known in the medical field, including the type and severity of the individual, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors. The pharmaceutical composition of the present invention can be administered as a sole therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. It can also be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy with the minimum amount without side effects, and this can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention can be determined by a specialist depending on various factors, such as the patient's condition, age, sex, and comorbidities. The active ingredient of the composition of the present invention is highly safe, so it can be used at a dosage higher than the determined dosage.

[0041] The composition containing the lipid nanoparticles as an active ingredient can be administered by oral, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, pulmonary, and nasal injection, but is not limited thereto.

[0042] The compositions of the present invention may further comprise one or more pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable salts may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to the compositions to formulate them into injectable forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Therefore, the compositions of the present invention may be in the form of patches, liquids, pills, capsules, granules, tablets, suppositories, and the like. These formulations may be prepared by conventional methods used in the art or by methods disclosed in the literature [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and may be formulated into various formulations depending on the disease or ingredient.

[0043] The embodiments of the present invention can be modified into various different forms, and the scope of the present invention is not limited to the embodiments described below. Furthermore, the embodiments of the present invention are provided to more completely explain the present invention to those having average knowledge in the art. Furthermore, throughout the specification, the term "comprise" a certain element does not exclude other elements, and means that other elements may also be included, unless otherwise specified to the contrary. [Effects of the Invention]

[0044] The ionizable lipid containing an ester bond of the present invention stably delivers anionic drugs when preparing lipid nanoparticles, and is particularly effective in nucleic acid delivery, and can be useful in related technical fields such as lipid nanoparticle-mediated gene therapy. [Brief explanation of the drawings]

[0045] [Figure 1]This shows the results of pKa measurement by TNS experiments of 244-9-cis lipid nanoparticles. [Figure 2] In vivo efficacy experiments were conducted to examine the hepatocyte targeting potential of siRNA-encapsulated lipid nanoparticles containing the ionizable lipid of the present invention, and the results of confirming the siFVII (Factor VII) knockout effect are shown below. [Figure 3] To confirm the in vivo delivery of hEPO mRNA-encapsulated 244-9-cis lipid nanoparticles, mice were intravenously injected, and blood samples were collected and analyzed for hEPO levels. [Figure 4] This is a photograph showing bioluminescence after intramuscular injection into mice to confirm in vivo delivery of mFLuc-encapsulated 244-9-cis lipid nanoparticles. [Figure 5] To confirm the cytotoxicity of 244-9-cis lipid nanoparticles, lipid nanoparticles without nucleic acid drugs were prepared and treated with the lipid nanoparticles at different concentrations in four cell lines: HepG2, MEF, Hela, and KB-GFP, and the cytotoxicity was confirmed. [Figure 6] To confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, mRNA-encapsulated lipid nanoparticles were prepared and administered to mice, after which the AST and ALT levels were measured and compared with those of ALC-0315 lipid. [Figure 7] To confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, mRNA-encapsulated lipid nanoparticles were prepared and administered, after which the ALP and ALB levels were measured and compared with those of ALC-0315 lipid. [Figure 8] To confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, lipid nanoparticles encapsulating mFLuc were prepared and administered to mice. Photographs of liver tissue were taken 24 hours later, comparing the hepatotoxicity with that of ALC-0315. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention will be described in detail below through Preparation Examples, Examples, and Experimental Examples, however, the following Preparation Examples, Examples, and Experimental Examples are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these Examples.

[0047] Example 1. Preparation of ester bond-containing ionizable lipids Example 1-1. Synthesis of ester-linked ionizable lipids Ester linkages and alkyl chains containing double bonds have been attached to a variety of amine head groups to prepare the ester linkage-containing ionizable lipids of the present invention.

[0048] Compounds 1 to 4 were prepared by adding 1.5 equivalents each of 9-bromononanoic acid, N,N-diisopropylcarbodiimide (DIC), and 0.2 equivalents of 4-dimethylaminopyridine (DMAP) to cis-2-nonen-1-ol in dichloromethane (DCM) overnight at 25 °C. The mixture was then purified using a CombiFlash column with hexane / ethyl acetate (5:1 v / v ratio). After evaporating the solvent, the product was dissolved in ethanol and added with 1 equivalent of N,N-diisopropylethylamine (DIPEA). For substances with primary amines (amine head groups), 0.3 equivalents of Compound 1, 0.2 equivalents of Compounds 2 and 4, and 0.15 equivalents of Compound 3 were added. The mixture was then reacted for 3 days. Purification was performed using a CombiFlash column with DCM / MeOH (9:1 v / v ratio) to synthesize ionizable lipids.

[0049] Compounds 5 to 9 were prepared by adding 1.5 equivalents of N,N-diisopropylcarbodiimide (DIC) and 0.2 equivalents of 4-dimethylaminopyridine (DMAP) to a DCM (dichloromethane) solvent containing cis-2-nonen-1-ol per 100 ml of 9-bromononanoic acid (5), 5-bromopentanoic acid (6), 6-bromohexanoic acid (7), 7-bromoheptanoic acid (8), and 8-bromooctanoic acid (9). The resulting mixture was then reacted overnight at 25°C and purified using a CombiFlash column with hexane / ethyl acetate (5:1 v / v ratio). After evaporating the solvent, the product was dissolved in ethanol, and 1 equivalent of DIPEA (N,N-diisopropylethylamine) and 0.3 equivalents of a primary amine-containing substance (amine head group) were added. The mixture was allowed to react for 3 days. Purification was performed using a CombiFlash column with DCM / MeOH (9:1 v / v ratio) to synthesize the ionizable lipid.

[0050] As an example, the specific reaction scheme for 244-9-cis is shown in the following reaction scheme 1.

[0051]

number

[0052] Specific example compounds of the ester bond-containing ionizable lipids of the present invention are shown in Table 2 below.

[0053] [Table 2] TIFF0007828458000012.tif169161

[0054] Example 1-2. Confirmation of synthesis of ester bond-containing ionizable lipids To confirm the synthesis of the ionizable lipid prepared in Example 1-1, MS analysis was performed. Specifically, the ionizable lipid was diluted in ethanol to a concentration of 0.5 ppm or less and subjected to MS analysis. The instrument used for the analysis was a 6230 LC / MS manufactured by Agilent Technologies (Palo Alto, USA), and the separation column was a Zorbax SB-C18 (100 mm × 2.1 mm id, 3.5 μm) manufactured by Agilent Technologies. The details of the MS analysis are shown in Table 3.

[0055] [Table 3]

[0056] From the above results, it was confirmed that an ionizable lipid containing an ester bond was successfully prepared in Example 1-1.

[0057] Example 2. Preparation of lipid nanoparticles Example 2-1. Formulation parameters Lipid nanoparticles containing ionizable lipids of the present invention were prepared in the weight ratios shown in Tables 4 and 5 below.

[0058] [Table 4]

[0059] [Table 5]

[0060] Example 2-2. Preparation of siRNA-encapsulated lipid nanoparticles The ionizable lipid prepared in Example 1-1, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≥99%, Sigma, Korea), phospholipid (DSPC) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol at a molar ratio of 26.5:20:52:1.5.

[0061] Then, siRNA was dissolved in 50 mM sodium acetate (Sigma, Korea) buffer, and the ethanol and acetate buffer containing dissolved ionizable lipids, cholesterol, phospholipids, and lipid-PEG were mixed at a volume ratio of 1:3 with a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) at a flow rate of 12 ml / min to produce lipid nanoparticles.

[0062] Example 2-3. Preparation of mRNA-encapsulated lipid nanoparticles The ionizable lipid prepared in Example 1-1, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≥99%, Sigma, Korea), phospholipid (DOPE) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol at a molar ratio of 26.5:20:52:1.5.

[0063] The mRNA was then dissolved in 10 mM sodium citrate (Sigma, Korea) buffer, and the ethanol and citrate buffer containing dissolved ionizable lipids, cholesterol, phospholipids, and lipid-PEG were mixed at a volume ratio of 1:3 with a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) at a flow rate of 12 ml / min to produce lipid nanoparticles.

[0064] Experimental Example 1: Confirmation of physicochemical properties of lipid nanoparticles Experimental Example 1-1. Measurement of particle size, polydispersity index (PDI), zeta potential, and drug encapsulation rate In this experimental example, we attempted to confirm the physicochemical properties of lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) or FVII siRNA (SEQ ID NO: 2 and SEQ ID NO: 3) prepared in Examples 2-3. Specifically, after preparing 244-9-cis lipid nanoparticles encapsulating mRNA or siRNA, they were diluted with PBS to a concentration of 1 μg / ml. The diameter, polydispersity index (PDI), and zeta potential of the lipid nanoparticles were then measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0065] Next, the encapsulation efficiency (drug encapsulation rate, %) of the mRNA- or siRNA-encapsulated lipid nanoparticles was measured using Ribogreen analysis (Quant-iT™ RiboGreen® RNA, Invitrogen). The mRNA-encapsulated lipid nanoparticles were diluted with 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(+)). After incubation at 37°C for 10 minutes, the lipid nanoparticles were degraded by Triton-X, releasing the encapsulated nucleic acid. Then, 100 μl of Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNP(-) and Triton LNP(+) was measured using an Infinite® 200 PRO NanoQuant (Tecan) at a wavelength bandwidth (excitation: 485 nm, emission: 528 nm), and the drug encapsulation rate (encapsulation efficiency, %) was calculated using the following formula 1.

[0066]

number

[0067] The respective result values ​​are as follows (Table 6).

[0068] [Table 6]

[0069] Experimental Example 1-2. pKa measurement In this experimental example, the pKa of the firefly luciferase mRNA (SEQ ID NO: 1) lipid nanoparticles formulated in Example 2-3 was calculated by in vitro TNS (2-(p-toluidino)naphthalene-6-sulfonic acid) analysis.

[0070] Specifically, the pH of a solution containing 20 mM sodium phosphate, 25 mM citric acid, 20 mM ammonium acetate, and 150 mM sodium chloride was adjusted from pH 3.5 to pH 11 in 0.5 increments using 0.1 N sodium hydroxide and / or 0.1 N hydrochloric acid to prepare solutions with various pH units. 100 μl of each solution was added to a black 96-well plate, and a 300 μM TNS stock solution was added to each solution to a final concentration of 6 μM. Formulated lipid nanoparticles were added to the mixed solution to a final concentration of 20 μM, and fluorescence intensity was measured (excitation: 325 nm, emission: 435 nm) using a Tecan instrument. The pKa for the lipid nanoparticles was calculated as the pH value at which half of the maximum fluorescence was reached.

[0071] As a result, it was confirmed that the lipid nanoparticles of the present invention have a pKa value of approximately 6.2 and exhibit an s-shaped curve on the graph (Figure 1). Anionic TNS becomes lipophilic by interacting with positively charged ionizable lipids. As the pH approaches the pKa value of each LNP, the lipophilicity of TNS decreases, and more water molecules quench the TNS fluorescence. Therefore, lipid nanoparticles with a pKa of 6.0 to 7.0 have excellent in vivo drug delivery efficiency. Furthermore, lipid nanoparticles that exhibit an "s-shaped curve" on the graph showing fluorescence as a function of pH indicate that they easily interact with endosomal membranes and can easily escape from the endosome upon acidification.

[0072] Experimental Example 1-3. Measurement by Cryo-TEM The internal structure of the lipid nanoparticles was measured by cryo-TEM.

[0073] Specifically, lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) were concentrated to a final total lipid concentration of 15-25 mg / ml, and 2-4 μl of the LNP solution was placed on a copper grid and blotted. Then, the internal images of the lipid nanoparticles were measured using a cryo-TEM (FEI Tecnai F20 G2) at the Characterization Center of the Korea Advanced Institute of Science and Technology (KIST).

[0074] Experimental Example 2: Confirmation of in vitro efficacy of mRNA-encapsulated lipid nanoparticles To confirm the efficacy of mRNA-encapsulating lipid nanoparticles in vitro, screening was performed using lipid nanoparticles synthesized with various amine head groups.

[0075] The test was carried out using lipid nanoparticles (Examples 1, 2, 4 and 5) synthesized with various amine head groups, and after treating HeLa cells with 20 ng of lipid nanoparticles encapsulating mFLuc (SEQ ID NO: 1), the level of luminescence was measured 24 hours later.

[0076] As a result, as shown in Table 7 below, it was confirmed that the luminescence intensity increased significantly in most of the lipid nanoparticles tested, and in particular, compound 5 (244-9-cis) lipid nanoparticles were found to show the highest expression effect.

[0077] [Table 7]

[0078] Experimental Example 3: Confirmation of the in vivo effects of lipid nanoparticles Experimental Example 3-1. Delivery effect of siRNA-encapsulated lipid nanoparticles Since FVII is specifically expressed in hepatocytes, we attempted to confirm the hepatocyte targeting potential of lipid nanoparticles through the FVII (Factor VII) knockout effect using siFVII.

[0079] Specifically, 244-9-cis lipid nanoparticles encapsulating FVII-targeting siRNA (SEQ ID NO: 2 and SEQ ID NO: 3) were prepared using the method described in Example 2-2. 244-9-cis lipid nanoparticles encapsulating siRNA prepared at concentrations of 0.03, 0.1, or 0.3 mg / kg (based on the siRNA concentration contained in the lipid nanoparticles) were intravenously injected into 7-week-old female C57BL / 6 mice. Three days later, blood was collected and analyzed according to the protocol provided with the COASET FVII assay kit. A standard curve was generated using blood from mice administered PBS, and FVII expression levels were measured.

[0080] As a result, as shown in Figure 2, lipid nanoparticles prepared using the ionizable lipid of the present invention suppressed FVII expression in vivo in a manner dependent on the encapsulated siRNA concentration, confirming that the lipid nanoparticles of the present invention can effectively deliver nucleic acids to target hepatocytes. In particular, lipid nanoparticles containing the ionizable lipid of the present invention showed superior FVII expression suppression effects at all doses to lipid nanoparticles prepared using FDA-approved ALC-0315.

[0081] Experimental Example 3-2. Confirmation of the delivery efficacy of mRNA-encapsulated lipid nanoparticles via intravenous injection To investigate mRNA delivery by 244-9-cis lipid nanoparticles, luciferase mRNA was delivered to mice, and bioluminescence was confirmed to confirm gene expression.

[0082] Specifically, 244-9-cis lipid nanoparticles encapsulating mFluc (SEQ ID NO: 1) were prepared using the method described in Example 2-3. The prepared lipid nanoparticles (2 μg of mRNA) were then intravenously injected into 7-week-old C57BL / 6 mice. Three hours later, 0.25 mg / kg of luciferin was intraperitoneally administered, and bioluminescence was monitored using an IVIS (PerkinElmer, USA) device. The results confirmed that most of the lipid nanoparticles were delivered to the liver (Table 8).

[0083] [Table 8]

[0084] Next, lipid nanoparticles encapsulating hEPO mRNA (SEQ ID NO: 4) were prepared using the same method as above. Lipid nanoparticles prepared at a dose of 0.1 mg / kg mRNA were intravenously injected into 7-week-old C57BL / 6 mice, and blood samples were taken 3, 6, 9, 24, and 48 hours later. hEPO was quantitatively analyzed using an hEPO ELISA kit. As a result, lipid nanoparticles containing the ionizable lipid of the present invention effectively delivered nucleic acids in vivo, and significant levels of hEPO were detected in the blood. This effect was confirmed by an area under the curve that was approximately 1.7-fold higher than that of lipid nanoparticles prepared with the control ALC-0315 (Table 9 and Figure 3).

[0085] [Table 9]

[0086] Experimental Example 3-3. Delivery effect of mRNA-encapsulated lipid nanoparticles by intramuscular injection Luciferase mRNA (SEQ ID NO: 1) was delivered to mice by intramuscular injection, and bioluminescence was confirmed to confirm gene expression.

[0087] Specifically, mFluc-loaded lipid nanoparticles (244-9-cis) were prepared as described above, and 2 μg (mRNA basis) was injected intramuscularly into 7-week-old C57BL / 6 mice. Three hours later, 0.25 mg / kg of luciferin was intraperitoneally administered, and bioluminescence was monitored using an IVIS (PerkinElmer, USA).

[0088] As a result, it was confirmed that most of the lipid nanoparticles were successfully delivered to the injection site (Table 10 and Figure 4).

[0089] [Table 10]

[0090] Experimental Example 4: Confirmation of cytotoxicity of lipid nanoparticles CCK-8 (tetrazolium salt) forms orange formazan through reduction by mitochondrial dehydrogenase in living cells, making it possible to confirm cell viability through absorbance analysis.

[0091] Specifically, various types of cells (HeLa, HepG2, MEF, KB-GFP) were seeded (0.4x10) in a clear 96-well plate (SPL, 30096). 5 The mRNA-free lipid nanoparticle components were then mixed using a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) to produce 244-9-cis lipid nanoparticles without mRNA encapsulation. 24 hours after cell seeding, 0.5 μg, 5 μg, 50 μg, or 100 μg of lipid nanoparticles (based on ionizable lipids) were added per well. 24 hours after lipid nanoparticle treatment, 10 μl of Cell Counting Kit-8 (Sigma-Aldrich, 96992) was added per well. After 4 hours of incubation, absorbance at 450 nm was measured using an Infinite® 200 PRO NanoQuant (Tecan).

[0092] As a result, it was confirmed that no cytotoxicity was observed up to 5 μg in each cancer cell line (FIG. 5).

[0093] Experimental Example 5: Confirmation of hepatotoxicity of lipid nanoparticles AST (Aspartate transaminase) and ALT (Alanine transaminase) are values ​​that can be used to measure the presence or absence of diseases such as hepatocellular disease and hepatitis. Normally present in the blood at low concentrations, they leak out when liver cells are damaged, resulting in increased concentrations. ALP (Alkaline phosphatase) is an enzyme in the cells surrounding the bile ducts of the liver, and increases with diseases such as bile duct obstruction and cholesteatosis in the liver. ALB (Albumin) is produced in the liver, and albumin levels tend to decrease in acute hepatitis such as cirrhosis.

[0094] To confirm hepatotoxicity, 244-9-cis lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) were prepared and administered to 7-week-old C57BL / 6 mice at a single dose of 2 mg / kg of mRNA. ALC-0315 was used as a control. 24 hours after administration, blood samples were collected to check the levels of AST, ALT, ALP, and ALB (Figures 6 and 7). Photographs of liver tissue analysis for each experimental group are shown in Figure 8.

[0095] The results showed that the drug generally showed less liver toxicity than the FDA-approved ALC-0315 lipid.

[0096] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts thereof.

Claims

1. An ionizable lipid represented by the following formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 Here, A is 【Transformation 3】 and R ５ is —C 1-4 alkyl-NR 3 R 4 or —C 4-8 alkyl-(C═O)—Y—(CH 2 ) x CH═CH—C 4-8 alkyl; R ３ and R ４ are each independently selected from —H, —C 1-3 alkyl, and —C 4-8 alkyl-(C═O)—Y—(CH 2 ) x CH═CH—C 4-8 alkyl; Y is —O—; m is an integer from 0 to 3; n is an integer from 3 to 7, x is an integer from 1 to 3, l is an integer from 2 to 6, and p is 1.

2. The ionizable lipid of claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the ionizable lipid is selected from the group consisting of Compounds 4 to 11 listed in the following table: Table 1 。

3. A lipid nanoparticle comprising the ionizable lipid according to claim 1 or 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

4. The lipid nanoparticles according to claim 3, further comprising any one or more selected from the group consisting of phospholipids, structural lipids, and PEG-lipids, The structural lipid is at least one selected from the group consisting of cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol. Lipid nanoparticles.

5. The phospholipids include 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-cholesterylhemisuccinoyl-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. The lipid nanoparticle according to claim 4, which is any one or more selected from the group consisting of sphingomyelin.

6. The PEG-lipid is any 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. The lipid nanoparticle of claim 4.

7. The lipid nanoparticle of claim 4, wherein the lipid nanoparticle comprises an ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate in a molar ratio of 10-40:10-30:40-70:1-5.

8. The lipid nanoparticle of claim 4, further comprising an anionic drug.

9. The lipid nanoparticle of claim 8, wherein the anionic drug is at least one selected from the group consisting of nucleic acids, small molecular weight compounds, peptides, proteins, protein-nucleic acid structures, and anionic biopolymer-drug conjugates.

10. The lipid nanoparticle of claim 9, wherein the nucleic acid is at least one selected from the group consisting of siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, and DNAzyme.

11. The lipid nanoparticle of claim 10, wherein the weight ratio of ionizable lipid to nucleic acid in the lipid nanoparticle is 1 to 20.

12. A drug delivery composition comprising the lipid nanoparticles of claim 8.

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