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 produced using the same, and uses thereof. [Background technology]

[0002] Drug delivery systems (DDS) are technologies designed to reduce side effects of drugs, maximize efficacy and effectiveness, and efficiently deliver the required amount of drugs. In particular, traditional viral carriers have been proven to be effective as drug carriers in gene therapy, but the use of viruses as gene delivery systems has been limited due to several shortcomings, such as immunogenicity, limited size of injected DNA, and difficulty in mass production.

[0003] As an alternative to the viral system, the main method for transporting nucleic acids into cells has been to transport them in a mixture 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 the 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 have the disadvantage of accumulating in tissues such as the lungs, liver, and spleen, which are primary passage organs (Renet et al., Gene Therapy. 7, 764-768, 2000).

[0004] Under these circumstances, the present inventors 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, the present inventors 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 pharma- ceutically acceptable salt thereof.

[0006] Another object of the present invention is to provide lipid nanoparticles comprising said ionizable lipid, its stereoisomer or a pharma- ceutically 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 specifically described as follows. Each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. That is, all combinations of all elements disclosed in the present invention belong to the scope of the present invention. In addition, the scope of the present invention cannot be considered to be limited by the specific descriptions described below.

[0009] In order to achieve the above object, the present inventors have conducted extensive research and have found that when an ionizable lipid containing an ester bond represented by the following formula 1 is used to prepare lipid nanoparticles, it can stably and effectively deliver drugs and has few 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 pharma- ceutically acceptable salt thereof.

[0011] [ka] Here, 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 from 0 to 2.

[0012] Although not limited thereto, the compound represented by the formula 1 specifically includes the following 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] In addition, 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 the compounds listed in Table 1 below.

[0015] [Table 1] TIFF2025504405000008.tif184161

[0016] In the present invention, the term "alkyl" means, unless otherwise specified, a straight or branched chain non-cyclic saturated hydrocarbon. For example, "C 1-6 "Alkyl" can mean 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 in an equivalent range as long as it has the same effect as 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 lipid analogue. The ionizable lipid can change its charge state depending on the surrounding pH, and thus plays a role in encapsulating an anionic drug in the lipid nanoparticle 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 an ester bond and a double bond is bound to an amine head containing at least one primary amine, and has the advantages of excellent efficacy such as tissue specificity and gene expression rate in vivo when delivering nucleic acid, being easily decomposed in vivo after drug delivery, and having no significant side effects such as hepatotoxicity, compared to other known ionizable lipids.

[0018] In the present invention, the term "stereoisomer" refers to a compound of the present invention having the same chemical or molecular formula, but different in stereochemistry. Each such stereoisomer and mixtures thereof are included within the scope of the present invention as well. Unless otherwise indicated, 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 be in the form of a "pharmaceutical acceptable salt". As the salt, an acid addition salt formed with a pharmaceutical acceptable free acid can be useful, but is not limited thereto. The term "pharmaceutical 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 non-harmful effective concentration in patients, and the side effects caused by the salt do not reduce the beneficial efficacy of the compound represented by formula 1.

[0020] The acid addition salts can be prepared in the usual manner, for example by dissolving the compound in an excess of aqueous acid and precipitating the salt with a water-miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. Equal molar amounts of the compound and the acid or alcohol in water can be heated and then the mixture can be 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 the compound in a solution of excess alkali metal hydroxide or alkaline earth metal hydroxide, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. In this case, the metal salt can be, for example, sodium, potassium, or calcium salt, but is not limited thereto. Corresponding silver salts can also be obtained by reacting the alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0023] Lipid nanoparticles comprising ionizable lipids containing ester bonds - Patent Application 20070229633 Another aspect of the present invention is a lipid nanoparticle comprising an ionizable lipid represented by the above formula 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof. The lipid nanoparticle of the present invention may comprise one type of ionizable lipid represented by formula 1, or may comprise two or more types. In addition, it may further comprise 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, structured lipids, and PEG-lipids, but are not limited thereto.

[0025] The phospholipids surround and protect the core formed by the interaction of the ionizable lipid and the drug in the lipid nanoparticles, and bind to the phospholipid bilayer of the target cell to facilitate cell membrane passage and endosomal escape during intracellular delivery of the drug. The phospholipids can be any phospholipid that can promote the fusion of lipid nanoparticles, and examples of such phospholipids include dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EGG), and phospholipids that can promote the fusion of lipid nanoparticles. phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (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-phosphoethanolamine phoethanolamine, 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-diundecanoyl-sn-glycero-phosphocholine, DUPC, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine ...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, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine 3-phosphoethanolamine), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, or a mixture thereof.

[0026] The structural lipids provide morphological rigidity to lipid packing within lipid nanoparticles and are distributed in the core and surface of nanoparticles to improve nanoparticle stability. The structural lipids may be, for example, cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or mixtures thereof, but are not limited thereto.

[0027] The PEG-lipid in the present invention refers to a form in which a lipid and PEG are conjugated, and means a lipid having a hydrophilic polymer, polyethylene glycol polymer, bound to one end. The PEG-lipid contributes to the particle stability of nanoparticles in serum in lipid nanoparticles and prevents aggregation between nanoparticles. In addition, the PEG-lipid protects nucleic acids from decomposition enzymes during in vivo delivery of nucleic acids, strengthens the stability of nucleic acids in the body, and increases the half-life of drugs encapsulated in nanoparticles. The PEG-lipid may be, for example, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, or a mixture thereof, but is not limited thereto.

[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 the ionizable lipid:phospholipid:structural lipid:PEG-lipid may be 10-40:10-30:40-70:1-5. The molar ratio may also be, but is not limited to, 20-30:15-25:45-55:1-5.

[0029] The lipid nanoparticles of the present invention have a pKa of 6.0 to 7.0, specifically 6.0 to 6.5, and therefore are positively charged under acidic pH conditions, and can easily form complexes with therapeutic agents such as negatively charged nucleic acids and anionic drugs through electrostatic interactions, and can encapsulate anionic drugs with high efficiency, and can be used as a drug delivery composition for intracellular or in vivo drugs. Therefore, the lipid nanoparticles of the present invention can be usefully used to deliver not only nucleic acids but also any form of drug that is anionic. That is, the lipid nanoparticles of the present invention can be produced in a form that finally contains an anionic drug (encapsulated form).

[0030] In the present invention, the term "encapsulation" refers to encapsulating a delivery substance to surround it for efficient in vivo uptake, and drug encapsulation efficiency refers to the content of drug encapsulated in lipid nanoparticles relative to the total content of 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 / 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 in 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 the anionic drug-containing lipid nanoparticles according to the present invention as an active ingredient.

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

[0038] The lipid nanoparticles of the present invention form a stable complex with anionic drugs such as nucleic acids, and exhibit low cytotoxicity and effective cellular absorption, 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 the type of nucleic acid used, and have unlimited potential for use as a drug delivery composition.

[0039] The term "treatment" in the present invention refers to intervention to modify 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, relieving symptoms, reducing all direct or indirect pathological consequences associated with the disease, preventing metastasis, reducing the rate of disease progression, alleviating or palliating 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, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof, and an anionic drug as an active ingredient. In addition, the term "prevention" refers to any action that inhibits 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 prophylactic purposes, they are administered to an individual in a therapeutically effective amount.

[0040] The term "therapeutically effective amount" used in the present invention refers to an effective amount of anionic drug-containing lipid nanoparticles. Specifically, "therapeutically 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 according to factors including the type and severity of the individual, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as a single 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 once or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain 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 the present invention can be determined by an expert depending on various factors such as the patient's condition, age, sex, and complications. The active ingredient of the composition of the present invention is excellent in safety, so it can be used even at or above the determined dosage.

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

[0042] The composition of the present invention may further include one or more additional pharma- ceutically acceptable carriers for administration. As the pharma-ceutically acceptable salt, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary. In addition, the composition of the present invention may be formulated in the form of an injection such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets by additionally adding diluents, dispersants, surfactants, binders, and lubricants. Thus, the composition of the present invention may be a patch, liquid, pill, capsule, granule, tablet, suppository, etc. These preparations may be prepared by conventional methods used in the art for formulation or methods disclosed in the literature [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and may be formulated into various preparations according to each disease or component.

[0043] The embodiments of the present invention can be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Moreover, 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, "including" a certain component does not exclude other components, and means that other components can be further included, unless otherwise specified to the contrary. Effect 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 description of the drawings]

[0045] [Figure 1]This shows the results of pKa measurement of 244-9-cis lipid nanoparticles by TNS experiments. [Diagram 2] In order to examine the hepatocyte targeting potential of siRNA-encapsulated lipid nanoparticles containing the ionizable lipid of the present invention through in vivo efficacy experiments, the results of confirming the siFVII (Factor VII) knockout effect are shown. [Diagram 3] To confirm the in vivo delivery of hEPO mRNA-encapsulated 244-9-cis lipid nanoparticles, mice were intravenously injected with the nanoparticles, after which blood was collected and the hEPO levels were analyzed. [Figure 4] To confirm the in vivo delivery of mFLuc-encapsulated 244-9-cis lipid nanoparticles, a photograph was taken confirming bioluminescence after intramuscular injection into mice. [Diagram 5] To confirm the cytotoxicity of 244-9-cis lipid nanoparticles, lipid nanoparticles without encapsulating nucleic acid drugs were prepared, and the lipid nanoparticles were treated at different concentrations in four types of 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, lipid nanoparticles encapsulating mRNA were prepared and administered to mice, after which the AST and ALT levels were examined and compared with those of ALC-0315 lipid. [Figure 7] To confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, lipid nanoparticles encapsulating mRNA were prepared and administered, after which the levels of ALP and ALB were examined 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. After 24 hours, liver tissue images were taken to compare the hepatotoxicity with that of ALC-0315. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0048] Specifically, compounds 1 to 4 were prepared by adding 1.5 equivalents of 9-bromo nonanoic acid, DIC (N,N-diisopropyl carbodiimide), and 0.2 equivalents of DMAP (4-dimethylaminopyridine) per cis-2-nonen-1-ol to DCM (dichloromethane) solvent, reacting overnight at 25°C, and purifying with hexane / ethyl acetate (5:1 v / v ratio) using a CombiFlash column. After removing all the solvent, the product was dissolved in ethanol, and in the case of a substance having a primary amine (amine head), compound 1 was added at 0.3 equivalents, compound 2 and compound 4 were added at 0.2 equivalents, and compound 3 was added at 0.15 equivalents, and the reaction was carried out for 3 days. Ionizable lipids were synthesized using a CombiFlash column and purification was performed with DCM / MeOH (9:1 v / v ratio).

[0049] Specifically, compounds 5 to 9 were prepared by adding 1.5 equivalents of DIC (N,N-diisopropylcarbodiimide) and 0.2 equivalents of DMAP (4-dimethylaminopyridine) to a DCM (dichloromethane) solvent per cis-2-nonen-1-ol, 9-bromononanoic acid (9-bromo nonanoic acid), 5-bromopentanoic acid (5-bromo pentanoic acid) (compound 6), 6-bromohexanoic acid (6-bromo hexanoic acid) (compound 7), 7-bromoheptanoic acid (7-bromo heptanoic acid) (compound 8), and 8-bromooctanoic acid (8-bromo octanoic acid) (compound 9), and reacting overnight at 25°C. The mixture was purified with hexane / ethyl acetate (5:1 v / v ratio) using a CombiFlash column. After removing all the solvent, the product was dissolved in ethanol, and 1 equivalent of DIPEA (N,N-diisopropylethylamine) and 0.3 equivalent of a substance with a primary amine (amine head) were added and reacted for 3 days. The ionizable lipid was synthesized by purifying with DCM / MeOH (9:1 v / v ratio) using a CombiFlash column.

[0050] As an example, a 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] TIFF2025504405000013.tif169161

[0054] Example 1-2. Confirmation of synthesis of ester bond-containing ionizable lipids In order to confirm the synthesis of the ionizable lipid produced 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 MS analysis was performed. The equipment used for the analysis was 6230 LC / MS manufactured by Agilent Technologies (Palo Alto, USA), and the separation column was Zorbax SB-C18 (100 mm x 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 lipid nanoparticles encapsulating siRNA 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 in which ionizable lipids, cholesterol, phospholipids, and lipid-PEG were dissolved were mixed at a volume ratio of 1:3, respectively, at a flow rate of 12 ml / min using a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) to prepare lipid nanoparticles.

[0062] Example 2-3. Preparation of lipid nanoparticles encapsulating mRNA 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] Then, the mRNA was dissolved in 10 mM sodium citrate (Sigma, Korea) buffer, and the ethanol and citrate buffer in which the ionizable lipids, cholesterol, phospholipids, and lipid-PEG were dissolved were mixed at a volume ratio of 1:3, respectively, at a flow rate of 12 ml / min using a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) 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, 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 Example 2-3 were examined. Specifically, after preparing 244-9-cis lipid nanoparticles encapsulating mRNA or siRNA, the lipid nanoparticles were diluted with PBS so that the concentration of RNA contained in each lipid nanoparticle was 1 μg / ml. Then, the diameter, polydispersity index (PDI), and zeta potential of the lipid nanoparticles were measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0065] Next, the encapsulation efficiency (drug encapsulation rate, %) of the lipid nanoparticles encapsulating the mRNA or siRNA was measured by Ribogreen analysis (Quant-iT™ RiboGreen™ RNA, Invitrogen). The lipid nanoparticles encapsulating the mRNA were diluted with 50 μl of 1xTE buffer so that the final concentration of mRNA was 4 to 7 μg / ml in a 96-well plate. 50 μl of 1xTE buffer was added to the Triton-X non-treated group (Triton-x LNP(-)), and 50 μl of 2% Triton-X buffer was added to the Triton-X treated group (Triton-X LNP(+)). The lipid nanoparticles were decomposed with Triton-X by incubation at 37°C for 10 minutes, and the encapsulated nucleic acid was released. 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 (registered trademark) 200 PRO NanoQuant (Tecan) with a wavelength bandwidth (excitation: 485 nm, fluorescence: 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 intervals 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 with each pH was added to a black 96-well plate, and 300 μM TNS stock solution was added to each of the solutions to a final concentration of 6 μM. Formulated lipid nanoparticles were added to the mixed solution to a final concentration of 20 μM, and the fluorescence intensity was measured using a Tecan instrument (excitation: 325 nm, emission: 435 nm). Here, 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 about 6.2 and show an s-shaped curve on the graph (Figure 1). Anionic TNS becomes lipophilic by interacting with positively charged ionizable lipids, and as the pH value approaches the pKa value of each LNP, the lipophilicity of TNS decreases and more water molecules quench the TNS fluorescence, so lipid nanoparticles with a pKa of 6.0 to 7.0 have excellent in vivo drug delivery efficiency. In addition, lipid nanoparticles that show an "s-shaped curve" on the graph showing fluorescence by pH are easy to 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 total lipid final concentration of 15-25mg / ml, and 2-4μl of LNP solution was placed on a copper grid and blotted. Then, the internal image of the lipid nanoparticles was 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 lipid nanoparticles encapsulating mRNA in vitro, a screening was performed using lipid nanoparticles synthesized with various amine head groups.

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

[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, it was found that the compound 5 (244-9-cis) lipid nanoparticles showed the highest expression effect.

[0077] [Table 7]

[0078] Experimental Example 3: Confirmation of the in vivo effect 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 by the method of Example 2-2. Based on the concentration of siRNA contained in the lipid nanoparticles, 244-9-cis lipid nanoparticles encapsulating siRNA prepared at a concentration of 0.03, 0.1, or 0.3 mg / kg were intravenously injected into C57BL / 6 female mice aged 7 weeks. After 3 days, blood was collected and analyzed according to the protocol of the COASET FVII assay kit. A standard curve was drawn using blood from mice administered PBS to measure the amount of FVII expression.

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

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

[0082] Specifically, 244-9-cis lipid nanoparticles encapsulating mFluc (SEQ ID NO: 1) were prepared by the method of Example 2-3. The prepared lipid nanoparticles (2 μg based on mRNA) were then intravenously injected into 7-week-old C57BL / 6 mice, and 3 hours later, 0.25 mg / kg luciferin was intraperitoneally administered, and bioluminescence was observed using an IVIS (PerkinElmer, USA) device. As a result, it was 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 in the same manner as above. Lipid nanoparticles prepared at a dose of 0.1 mg / kg based on mRNA were intravenously injected into 7-week-old C57BL / 6 mice, and blood was collected 3, 6, 9, 24, and 48 hours later, and hEPO quantitative analysis was performed using an hEPO ELISA kit. As a result, the lipid nanoparticles containing the ionizable lipid of the present invention effectively delivered nucleic acid in vivo, and hEPO was detected at a considerable level in the blood, and this effect was confirmed by the area under the curve being about 1.7 times higher than that of the lipid nanoparticles prepared with the control group 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 by intramuscular injection into mice, after which bioluminescence was confirmed, confirming gene expression.

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

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

[0089] [Table 10]

[0090] Experimental Example 4: Confirmation of cytotoxicity of lipid nanoparticles CCK-8 (tetrazolium salt) is reduced by mitochondrial dehydrogenase in living cells to form orange formazan, 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 5 Then, the lipid nanoparticle components excluding mRNA were mixed using a microfluidic mixer (Benchtop Nanoassemblyr; PNI, Canada) to produce 244-9-cis lipid nanoparticles without encapsulating mRNA. 24 hours after cell seeding, lipid nanoparticles (based on ionizable lipids) were treated in amounts of 0.5 μg, 5 μg, 50 μg, or 100 μg per well. 24 hours after lipid nanoparticle treatment, Cell Counting Kit-8 (Sigma-Aldrich, 96992) was added at 10 μl per well. After 4 hours of incubation, the absorbance at 450 nm was measured using 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, they are present in the blood at low concentrations, but when liver cells are damaged, they flow out and the concentration increases. ALP (Alkaline phosphatase) is an enzyme in the cells surrounding the bile ducts of the liver, and increases with diseases such as bile duct blockage and cessation of bile secretion in the liver. ALB (Albumin) is produced in the liver, and albumin values ​​are characterized by a decrease in acute hepatitis such as cirrhosis.

[0094] To confirm liver toxicity, 244-9-cis lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) were prepared and administered once to 7-week-old C57BL / 6 mice at a dose of 2 mg / kg of mRNA. ALC-0315 was used as a control group. 24 hours after administration, blood was collected to confirm 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 lipid profile was generally less hepatotoxic than that of the FDA-approved ALC-0315 lipid.

[0096] From the above description, a person skilled in the art to which the present invention pertains can understand that the present invention can be embodied in other specific forms without changing the technical idea or essential features 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, and their equivalent concepts, rather than the above detailed description.

Claims

1. An ionizable lipid represented by the following formula 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 Here, A is 【Chemistry 2】 or 【Chemistry 3】 and R １ and R ２ are each independently -H, -C １－６ Alkyl, -C １－６ Alkyl-NR ３ R ４ , or -C ２－１２ Alkyl-(C═O)-Y-(CH ２ ) ｘ CH=CH-C ４－１２ alkyl, R ３ and R ４ are each independently -H, -C １－６ Alkyl, or -C ２－１２ Alkyl-(C═O)-Y-(CH ２ ) ｘ CH=CH-C ４－１２ alkyl, R ５ is -C １－６ Alkyl-NR ３ R ４ Or -C ２－１２ Alkyl-(C═O)-Y-(CH ２ ) ｘ CH=CH-C ４－１２ is alkyl, Y is -O- or NR ６ and R ６ 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 from 0 to 2.

2. A is, 【Chemistry 4】 and R １ and R ２ are each independently -H, -C １－３ Alkyl, -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 -H, -C １－３ Alkyl, or -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 2. The ionizable lipid of claim 1, wherein p is 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

3. A is, 【Chemistry 5】 and R ３ and R ４ are each independently -H, -C １－３ Alkyl, or -C ４－８ Alkyl-(C═O)-Y-(CH ２ ) ｘ CH=CH-C ４－８ alkyl, 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 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 2. The ionizable lipid of claim 1, wherein p is 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

4. The ionizable lipid according to claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of compounds 1 to 11 in the following table: 【Table 1】

5. A lipid nanoparticle comprising the ionizable lipid according to any one of claims 1 to 4, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

6. The lipid nanoparticle of claim 5, further comprising at least one selected from the group consisting of phospholipids, structured lipids, and PEG-lipids.

7. 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 1,2-diphosphocholine, 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 6, which is any one or more selected from the group consisting of sphingomyelin.

8. The lipid nanoparticle according to claim 6, wherein the PEG-lipid is at least one selected from the group consisting of PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

9. The lipid nanoparticle of claim 6, wherein the structural lipid is any one or more selected from the group consisting of cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid and alpha-tocopherol.

10. The lipid nanoparticle of claim 6, comprising a molar ratio of ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate of 10-40:10-30:40-70:1-5.

11. The lipid nanoparticle of claim 6, further comprising an anionic drug.

12. The lipid nanoparticle of claim 11, wherein the anionic drug is at least one selected from the group consisting of a nucleic acid, a low molecular weight compound, a peptide, a protein, a protein-nucleic acid structure, and an anionic biopolymer-drug conjugate.

13. The lipid nanoparticle according to claim 12, 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.

14. The lipid nanoparticle of claim 13, wherein the weight ratio of ionizable lipid to nucleic acid in the lipid nanoparticle is 1 to 20.

15. A drug delivery composition comprising the lipid nanoparticles described in claim 11.

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