Cationic lipid compound, production method thereof, and applications

Abstract

This application relates to the field of drug carriers, and specifically discloses a cationic lipid compound, a method for producing the same, and applications thereof. The cationic lipid compound includes a compound represented by Formula I. In Formula I, n = 1, 2, 3, or 4, and R1 and R2 are each independently selected from secondary long-chain alkyl esters represented by Formula II. In Formula II, n1 = 2, 4, or 6, n2 = 5, 7, 9, or 11, and n3 = 5, 7, 9, or 11. The above cationic lipid compound can be combined with phospholipids, cholesterol, and polyethylene glycol lipids to produce an LNP carrier, and further encapsulate nucleic acid molecules to produce corresponding drugs. The production raw materials of the cationic lipid compound described in this application are easy to obtain, the synthesis steps are simple, the constructed LNP carrier has a good drug delivery effect, the corresponding drug can effectively enter target cells or activate the body's immune response, and it has practical application value.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of Chinese Patent Application No. 2022106678978, filed on June 14, 2022. The disclosure content of this application is hereby incorporated by reference in its entirety into this specification.

[0002] This application relates to the technical field of drug carriers, and more specifically, to cationic lipid compounds, their manufacturing methods, and applications.

Background Art

[0003] Gene therapy is to treat diseases by introducing a target gene into a patient's body to correct or compensate for diseases caused by defective and abnormal genes. It is a new technology born from the combination of modern medicine and molecular biology. As a new means of disease treatment, gene therapy has achieved success in several applications, and the scientific breakthroughs achieved have promoted the continuous development of gene therapy into mainstream medicine. Common gene drugs include plasmid DNA (pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA), small hairpin RNA (shRNA), and messenger RNA (mRNA). mRNA is a class of single - stranded ribonucleic acid containing specific genetic information, which can deliver the carried genetic information to ribosomes in cells and instruct them to synthesize specific target proteins in the body as a template. Compared with conventional protein drugs that are difficult to cross the cell membrane, due to the action mechanism of mRNA, it can deliver transmembrane or intracellular proteins to the body, so that more diseases can be treated or prevented. mRNA has become a trend in future drug development due to the above - mentioned advantages.

[0004] The key to gene therapy is to deliver and function gene drugs to target cells in the body. However, when exogenous genes are directly introduced into the body, they are decomposed by nucleases in the body and decomposed into low-molecular nucleotides before entering the target cells, resulting in the loss of therapeutic effects. Therefore, the key to realizing gene therapy is to construct an efficient and safe gene delivery system.

[0005] When delivering genes, gene carriers need to go through multiple complex processes such as reaching target cells through blood circulation, cell uptake, endosomal escape, intracellular movement, and release of gene substances from the carrier. The main obstacles are extracellular obstacles caused by the complex blood environment and intracellular obstacles caused by lysosomal enzyme degradation. Therefore, how to find a good gene carrier so that the target gene can reach the target site and function is an urgent problem to be solved by researchers of gene carriers.

[0006] Currently, gene delivery carrier systems are mainly divided into two categories. One is the viral carrier system, and the other is the non-viral carrier system. Viral carriers are natural carrier resources. The viral genome has a simple structure, high transfection efficiency, and strong target cell specificity, but its directivity is low, carrying capacity is low, and it has immunogenicity and potential tumorigenicity, so it is difficult to meet the requirements of clinical applications. Therefore, the non-viral carrier system with diversity, no immunogenicity, and easy production control has attracted attention in recent years and has been applied in many therapeutic fields.

[0007] Generally used non-viral carrier systems are mainly lipid carriers. Lipid carriers usually contain the positive charge of cationic lipids and bind to negatively charged gene drugs through electrostatic action, thereby concentrating and packaging the gene material into particles with relatively small particle sizes to form lipid nanoparticles (LNPs). LNPs show advantages that cannot be matched by other types of liposomes in the manufacture of gene delivery carriers and cell transfection. The relatively small particle size of the complex reduces the possibility of being recognized, phagocytosed, and removed by macrophages in the body, and improves the bioavailability of the drug in vivo. At the same time, in the case of tumor tissue, the relatively small particle size of the complex makes it easier to enter the tumor parenchyma from the gaps between vascular endothelial cells due to the enhanced permeability and retention effect, increasing the accumulation of the drug in the tumor tissue. In transfection, since the cell surface is slightly negatively charged, positively charged liposomes are easily adsorbed to the cell surface and enter the cell through mechanisms such as endocytosis, greatly increasing the transfection ability of the liposomes.

[0008] Currently, due to characteristics such as its simple structure, convenient operation, and high biological safety, LNP has become the most widely applied non-viral carrier. However, the manufacturing processes of most LNPs are complex and difficult for large-scale production. Therefore, how to manufacture a non-viral carrier system with a simple manufacturing method and good therapeutic effect has become an urgent problem to be solved.

Summary of the Invention

[0009] In order to improve the drug delivery effect of gene carriers and simplify their synthesis steps, the present application provides a cationic lipid compound, its manufacturing method, and applications. Such a cationic lipid compound is positively charged and easier to enter cells, so it can deliver drugs better, exhibit better therapeutic effects, and has simple synthesis, easy manufacture, and practical application value.

[0010] In a first aspect, the present application provides a cationic lipid compound that employs the following technical solutions.

[0011] A cationic lipid compound, the cationic lipid compound comprising a compound shown in formula I: [ka] In Formula I, n=1, 2, 3 or 4; R1 and R2 are each independently selected from secondary long chain alkyl esters shown in Formula II: [ka] In Formula II, n1=2, 4, or 6, n2=5, 7, 9, or 11, n3=5, 7, 9, or 11, and the wavy line represents the point of attachment of Formula II to the rest of Formula I.

[0012] In this application, an amphiphilic derivative obtained by combining 3-((2-(dimethylamino)ethane)(methyl)amino)propionic acid with a secondary long-chain alkyl ester is used as the cationic lipid component in the gene drug carrier system. Because it has a positive charge, it can better deliver gene drugs and achieve better gene therapy effects. Such cationic lipid compounds have simple synthesis steps, are readily available from raw materials, and can be produced on a large scale.

[0013] In this application, the cationic lipid compound contains a total of three ester groups, and the spatial positions of these three ester groups directly affect the function of the cationic lipid compound, while the number of carbon atoms on the alkyl chain affects the spatial structure of the ester groups. Tests have shown that when n=1, 2, 3, or 4, n1=2, 4, or 6, n2=5, 7, 9, or 11, and n3=5, 7, 9, or 11, the cationic compound can achieve optimal loading and delivery of gene drugs, thereby achieving optimal therapeutic effects.

[0014] As a preferred technical solution, the cationic lipid compounds described in the present application include compounds shown in formula III, formula IV, formula V, formula VI, formula VII, formula VIII or formula IX. [Chemistry] [Chemistry] [Chemistry]

[0015] In a second aspect, the present application provides a method for producing a cationic lipid compound that employs the following technical solution.

[0016] A method for producing a cationic lipid compound, wherein the method for producing the cationic lipid compound comprises: a step of performing an alkylation reaction and a reduction reaction in sequence using a bromoester compound as a raw material; and a step of further subjecting the compound to a condensation reaction with an acid chloride compound, performing two steps of substitution reaction and a primary hydrolysis reaction in sequence, and finally subjecting the compound to an esterification reaction with a monohydric secondary alcohol compound to produce the cationic lipid compound.

[0017] As a preferred technical solution, the method for producing the cationic lipid compound comprises: (1) a step of subjecting a bromoester compound to an alkylation reaction with TosMIC (p-toluenesulfonyl isonitrile) and NaH to produce an intermediate product 2; (2) a step of reacting the intermediate product 2 under acidic conditions to produce an intermediate product 3; (3) a step of subjecting the intermediate product 3 to a reduction reaction with NaBH4 to produce an intermediate product 4; (4) a step of subjecting the intermediate product 4 to a condensation reaction with an acid chloride compound to produce an intermediate product 5; (5) a step of subjecting the intermediate product 5 to a substitution reaction with NaI to produce an intermediate product 6; (6) a step of subjecting the intermediate product 6 to a substitution reaction with N,N,N'-trimethylethylenediamine to produce an intermediate product 7; (7) a step of subjecting the intermediate product 7 to a hydrolysis reaction with LiOH to produce an intermediate product 8. (8) Reacting the intermediate product 8 with a monohydric secondary alcohol-based compound by an esterification reaction to obtain the cationic lipid compound.

[0018] In the present application, the raw material in step (1) is a bromoester-based compound, and in the actual production process, it can be specifically selected according to the structure of the synthesized specific cationic lipid compound.

[0019] Preferably, the bromoester-based compound includes ethyl 8-bromooctanoate, ethyl 6-bromohexanoate, or ethyl 4-bromobutyrate.

[0020] Preferably, in step (1), TBAI (tetrabutylammonium iodide) is used as the catalyst for the reaction.

[0021] Preferably, in step (1), the reaction solvent includes a DMSO (dimethyl sulfoxide) solution.

[0022] Preferably, in step (1), the reaction conditions are to be carried out at room temperature.

[0023] Preferably, in step (2), the reaction solvent includes a mixed solution of DCM (dichloromethane) and concentrated sulfuric acid, and the volume ratio of the two is DCM: concentrated sulfuric acid = (3.5 to 8): 1.

[0024] In the present application, concentrated sulfuric acid provides an acidic environment for carrying out the reaction in step (2).

[0025] Preferably, in step (2), the reaction conditions are to be carried out at room temperature.

[0026] In the present application, the intermediate product 3 is a symmetric ketone-based compound.

[0027] Preferably, in step (3), the reaction solvent includes a mixed solution of THF (tetrahydrofuran) and ethanol, and the volume ratio of the two is THF: ethanol = (2 to 4): 1.

[0028] Preferably, in step (3), the reaction conditions are to be carried out at room temperature.

[0029] In the present application, intermediate product 4 is an alcohol-based compound.

[0030] In the present application, the acid chloride-based compound in step (4) is a general term for a class of compounds with different numbers of carbon atoms and unsubstituted terminals by halogen, and in the actual production process, it can be specifically selected according to the structure of the synthesized specific cationic lipid compound.

[0031] Preferably, the acid chloride-based compound includes 3-chloropropionyl chloride, 2-chloroacetyl chloride, 4-chlorobutyryl chloride or 5-chlorovaleryl chloride.

[0032] Preferably, in step (4), the solvent for the reaction includes a DCM solution containing pyridine, where the concentration of pyridine is 73 - 134 mM.

[0033] Preferably, in step (4), the reaction conditions are to be carried out at room temperature.

[0034] Preferably, in step (5), the solvent for the reaction includes an acetone solution.

[0035] Preferably, in step (5), the reaction conditions are to be carried out at 68 - 72 °C, and the reaction temperature may be, for example, 68 °C, 70 °C, 72 °C, 68 - 70 °C, 68 - 72 °C or 70 - 72 °C, etc.

[0036] Preferably, in step (6), the solvent for the reaction includes a DCM solution.

[0037] Preferably, in step (6), the reaction conditions are to be carried out at room temperature.

[0038] Preferably, in step (7), the solvent for the reaction comprises a mixed solution of ethanol and water, and the volume ratio of the two is ethanol: water = 1: (1.5 to 3).

[0039] Preferably, in step (7), the reaction conditions are to be carried out at room temperature.

[0040] Preferably, in step (8), DMAP (4-dimethylaminopyridine) and DCC (dicyclohexylcarbodiimide) are used as the catalyst for the reaction.

[0041] Preferably, in step (8), the solvent for the reaction comprises a DCM solution.

[0042] Preferably, in step (8), the reaction conditions are to be carried out at room temperature.

[0043] In the present application, in step (8), the monovalent secondary alcohol compound is a monohydric alcohol containing different numbers of carbon atoms, and the hydroxy group is not located at the 1-position, and in the actual production process, it can be specifically selected according to the structure of the synthesized specific cationic lipid compound.

[0044] Preferably, the monovalent secondary alcohol compound comprises 13-pentacosanol, 11-heneicosanol, 9-heptadecanol, 7-pentadecanol or 9-heneicosanol.

[0045] In a third aspect, the present application provides an LNP carrier employing the following technical solution.

[0046] An LNP carrier, wherein the LNP carrier comprises any one or a combination of at least two of the cationic lipid compounds described in the first aspect.

[0047] In the present application, the cationic lipid compound in the LNP carrier may be a single cationic lipid compound or a combination of a plurality of cationic lipid compounds.

[0048] Preferably, in terms of molar fraction, the molar fraction of the cationic lipid compound in the LNP carrier is 15% to 70%, for example, 15%, 30%, 45%, 60%, 70%, 15% to 30%, 15% to 45%, 15% to 60%, 15% to 70%, 30% to 45%, 30% to 60%, 30% to 70%, 45% to 60%, 45% to 70% or 60% to 70%, etc. This molar fraction is the molar ratio of the lipid materials in the LNP carrier and does not include the drug (e.g., nucleic acid drug) contained.

[0049] In the present application, when there is one type of cationic lipid compound in the LNP carrier, its molar fraction in the LNP carrier is 15% to 70% (for example, it may be 15%, 30%, 45%, 60%, 70%, 15% to 30%, 15% to 45%, 15% to 60%, 15% to 70%, 30% to 45%, 30% to 60%, 30% to 70%, 45% to 60%, 45% to 70% or 60% to 70%, etc.). When there are multiple types of cationic lipid compounds in combination in the LNP carrier, the molar fraction of the total amount of the combination of multiple types of cationic lipid compounds in the LNP carrier is 15% to 70% (for example, it may be 15%, 30%, 45%, 60%, 70%, 15% to 30%, 15% to 45%, 15% to 60%, 15% to 70%, 30% to 45%, 30% to 60%, 30% to 70%, 45% to 60%, 45% to 70% or 60% to 70%, etc.). In some embodiments, the above molar fraction is the molar ratio of the lipid materials in the LNP carrier and does not include the drug (e.g., nucleic acid drug) contained.

[0050] In some embodiments, in terms of molar fraction, the LNP carrier further contains 5% to 30% phospholipid (for example, it may be 5% to 20% or 8% to 30%) and 15% to 65% cholesterol. This molar fraction is the molar ratio of the lipid materials in the LNP carrier and does not include the drug (e.g., nucleic acid drug) contained.

[0051] In some embodiments, in terms of molar fraction, the LNP carrier further comprises 5% - 30% (which may be, for example, 5% - 20% or 8% - 30%) of phospholipid, 15% - 65% of cholesterol, and 1.5% - 3% (which may be, for example, 1% - 3% or 0.5% - 2%) of polyethylene glycol lipid. The molar fraction is the molar ratio of lipid materials in the LNP carrier and does not include the drug (e.g., nucleic acid drug) contained.

[0052] Preferably, the molar fraction of the phospholipid in the LNP carrier is 5% - 40%, such as 8%, 10%, 15%, 20%, 25%, 30%, 5% - 10%, 8% - 10%, 5% - 15%, 8% - 15%, 5% - 20%, 8% - 20%, 5% - 25%, 8% - 25%, 5% - 30%, 8% - 30%, 10% - 15%, 10% - 20%, 10% - 25%, 10% - 30%, 15% - 20%, 15% - 25%, 15% - 30%, 20% - 25%, 20% - 30% or 25% - 30%, etc. The molar fraction is the molar ratio of lipid materials in the LNP carrier and does not include the drug (e.g., nucleic acid drug) contained.

[0053] Preferably, the molar fraction of the cholesterol in the LNP carrier is 10% - 65%, such as 10%, 15%, 30%, 45%, 65%, 15% - 30%, 15% - 45%, 15% - 65%, 30% - 45%, 30% - 65% or 45% - 65%, etc. The molar fraction is the molar ratio of lipid materials in the LNP carrier and does not include the drug (e.g., nucleic acid drug) contained.

[0054] In some embodiments, in terms of molar fraction, the LNP carrier further comprises a polyethylene glycol lipid, wherein the molar fraction of the polyethylene glycol lipid in the LNP carrier is 0.5% to 3%, for example, 1.5%, 2%, 2.5%, 3%, 0.5% to 2%, 1% to 2%, 1.5% to 2%, 0.5% to 2.5%, 1% to 2%, 1.5% to 2.5%, 0.5% to 3%, 1% to 2%, 1.5% to 3%, 2% to 2.5%, 1% to 3%, 2% to 3%, or 2.5% to 3%, etc. This molar fraction is the molar ratio of the lipid materials in the LNP and does not include the drug (e.g., nucleic acid drug) contained therein.

[0055] In some embodiments of the present application, in terms of molar fraction, the LNP carrier comprises 15% to 70% (e.g., 15%, 30%, 45%, 60%, 70%, 30% to 70%, 15% to 30%, 15% to 45%, 15% to 60%, 15% to 70%, 30% to 45%, 30% to 60%, 30% to 70%, 45% to 60%, 45% to 70% or 60% to 70% etc.) of a cationic lipid compound, 5% to 30% (e.g., 8%, 10%, 15%, 20%, 25%, 30%, 5% to 10%, 8% to 10%, 5% to 15%, 8% to 15%, 5% to 20%, 8% to 20%, 5% to 25%, 8% to 25%, 5% to 30%, 8% to 30%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 15% to 20%, 15% to 25%, 15% to 30%, 20% to 25%, 20% to 30% or 25% to 30% etc.) of a phospholipid and 10% to 65% (e.g., 15%, 30%, 45%, 65%, 15% to 30%, 15% to 45%, 15% to 65%, 30% to 45%, 30% to 65% or 45% to 65% etc.) of cholesterol.In some embodiments, in terms of molar fraction, the LNP carrier comprises 15% to 70% (e.g., 15%, 30%, 45%, 60%, 70%, 30% - 70%, 15% - 30%, 15% - 45%, 15% - 60%, 15% - 70%, 30% - 45%, 30% - 60%, 30% - 70%, 45% - 60%, 45% - 70% or 60% - 70%, etc.) of a cationic lipid compound, 5% to 30% (e.g., 8%, 10%, 15%, 20%, 25%, 30%, 5% - 10%, 8% - 10%, 5% - 15%, 8% - 15%, 5% - 20%, 8% - 20%, 5% - 25%, 8% - 25%, 5% - 30%, 8% - 30%, 10% - 15%, 10% - 20%, 10% - 25%, 10% - 30%, 15% - 20%, 15% - 25%, 15% - 30%, 20% - 25%, 20% - 30% or 25% - 30%, etc.) of a phospholipid, 10% to 65% (e.g., 15%, 30%, 45%, 65%, 15% - 30%, 15% - 45%, 15% - 65%, 30% - 45%, 30% - 65% or 45% - 65%, etc.) of cholesterol, and 0.5% to 2% (e.g., 1.5%, 2%, 2.5%, 3%, 0.5% - 2%, 1% - 2%, 1.5% - 2%, 0.5% - 2.5%, 1% - 2%, 1.5% - 2.5%, 0.5% - 3%, 1% - 2%, 1.5% - 3%, 2% - 2.5%, 2% - 3% or 2.5% - 3%, etc.) of a polyethylene glycol lipid. The molar fraction is the molar ratio of lipid materials in the LNP and does not include the drug (e.g., nucleic acid drug) contained.

[0056] In the present application, by optimizing the components and their respective addition amounts of the LNP carrier, the technical effect of synergistic gain is obtained, the loading amount of the nucleic acid drug is higher, the particle size distribution of the LNP is more controllable, the transfection effect on target cells is stronger, thereby improving the action effect of the drug, reducing toxic side effects, and having a more excellent therapeutic effect.

[0057] In the fourth aspect, the present application provides a drug adopting the following technical solution.

[0058] A drug, wherein the drug comprises the LNP carrier described in the third aspect.

[0059] Preferably, the drug further comprises a nucleic acid drug. In some embodiments, the drug is a gene drug. In some embodiments, the gene drug includes, but is not limited to, plasmid DNA (pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA), small hairpin RNA (shRNA), and messenger RNA (mRNA). In some embodiments, the drug belonging thereto is mRNA. In some embodiments, the drug belonging thereto is mRNA encoding the SARS-CoV-2 virus spike glycoprotein (S protein).

[0060] Preferably, in the drug, the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug (i.e., the N / P molar ratio) is (2 to 15):1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, (2 to 3):1, (2 to 4):1, (2 to 5):1, (2 to 6):1, (3 to 4):1, (3 to 5):1, (3 to 6):1, (4 to 5):1, (4 to 6):1, or (5 to 6):1, etc.

[0061] In some embodiments, in the drug, the number of parts by mass of the nucleic acid drug (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein) occupying the drug-loaded LNP (drug loading amount) is 1% to 10%, for example, 1% to 7%, 1% to 6%, 2% to 6%, 2% to 5%, or 3% to 5%, etc.

[0062] In the present application, the ratio of the cationic lipid compound to the nucleic acid drug directly affects the drug encapsulation rate and subsequent drug release effect. In some other aspects of the present application, when the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2-15):1, the drug-carrying structure has better stability and the best release efficiency in cells. In some aspects of the present application, according to the verification of tests, when the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2-6):1, the drug encapsulation rate is the highest, the therapeutic effect is the best, and the utilization rate of raw materials can be improved and the production cost can be saved. In some aspects of the present application, when the mass fraction (drug loading amount) of the nucleic acid drug in the drug-carrying LNP is 3%-10%, the drug encapsulation rate is relatively good, and the utilization rate of raw materials can be improved and the production cost can be saved.

[0063] In the present application, the particle size and particle size distribution of the drug affect the drug-carrying capacity and subsequent practical applications. In some aspects of the present application, the average particle size of the drug is 30 nm to 300 nm, and may be, for example, 30 nm to 270 nm, 30 nm to 250 nm, 30 nm to 200 nm, 50 nm to 200 nm, 50 nm to 150 nm, or 60 nm to 120 nm. In some aspects of the present application, the polydispersity (PDI) of the drug is less than 0.15, and may be, for example, less than 0.13, less than 0.12, less than 0.11, less than 0.1, or less than 0.09. In some aspects of the present application, the polydispersity (PDI) of the drug is 0.02 to 0.25, and may be, for example, 0.02 to 0.20, 0.05 to 0.20, 0.05 to 0.25, 0.05 to 0.1, or 0.05 to 0.25. In some aspects of the present application, the PDI of the drug belonging thereto is less than 0.1.

[0064] In some embodiments of the present application, the drug comprises an LNP carrier and mRNA (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein), wherein the mRNA drug loading amount is 2-6% (wt.), and the LNP carrier here comprises 60 mol% - 70 mol% of a TM3 lipid compound, 7 mol% - 12 mol% of a phospholipid (e.g., HSPC), 20 mol% - 25 mol% of cholesterol, and 1 mol% - 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 150 nm and 200 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 85%. In some embodiments, the N / P value of the above drug is 5.5 - 6.5.

[0065] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 5% - 7%, and the LNP carrier here comprises 60 mol% - 70 mol% of a TM3 lipid compound, 10 mol% - 15 mol% of a phospholipid (e.g., HSPC), 15 mol% - 25 mol% of cholesterol, and 1 mol% - 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 50 nm and 100 nm. In some embodiments, the PDI of the above drug is less than 0.1 or less than 0.05. In some embodiments, the encapsulation efficiency of the above drug is greater than 80% or greater than 90%.

[0066] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 4% - 5%. Here, the LNP carrier comprises 60 mol% - 70 mol% of a TM3 lipid compound, 5 mol% - 8 mol% of a phospholipid (such as HSPC), 25 mol% - 35 mol% of cholesterol, and 1 mol% - 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 80% or greater than 90%.

[0067] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 4% - 5%. Here, the LNP carrier comprises 60 mol% - 70 mol% of a TM3 lipid compound, 10 mol% - 15 mol% of a phospholipid (such as HSPC), 15 mol% - 25 mol% of cholesterol, and 2 mol% - 3 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 80% or greater than 90%.

[0068] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 2% - 3%. Here, the LNP carrier comprises 25 mol% - 35 mol% of a TM3 lipid compound, 5 mol% - 12 mol% of a phospholipid (such as HSPC), 50 mol% - 60 mol% of cholesterol, and 1 mol% - 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 50 nm and 100 nm. In some embodiments, the PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 80% or greater than 90%.

[0069] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 5% to 6%. Here, the LNP carrier comprises 35 mol% to 45 mol% of a TM3 lipid compound, 12 mol% to 18 mol% of a phospholipid (such as HSPC), 40 mol% to 50 mol% of cholesterol, and 0.5 mol% to 1 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 80% or greater than 90%.

[0070] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug (such as mRNA encoding the SARS-CoV-2 virus spike glycoprotein), wherein the LNP carrier comprises 60 mol% to 70 mol% of a TM6 lipid compound, 10 mol% to 15 mol% of a phospholipid (such as HSPC), 15 mol% to 25 mol% of cholesterol, and 2 mol% to 3 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 90%. In some embodiments, the N / P value in the above drug is 5.5 to 6.2.

[0071] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein), where the N / P value is 5.5 to 6.2, and the LNP carrier herein comprises 25 mol% to 35 mol% of a TM6 lipid compound, 5 mol% to 15 mol% of a phospholipid (e.g., HSPC), 55 mol% to 65 mol% of cholesterol, and 1 mol% to 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 150 nm and 200 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 85%.

[0072] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein), where the N / P value is 3.8 to 4.2, and the LNP carrier herein comprises 45 mol% to 55 mol% of a TM6 lipid compound, 5 mol% to 15 mol% of a phospholipid (e.g., HSPC), 35 mol% to 45 mol% of cholesterol, and 1 mol% to 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 50 nm and 100 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 85%.

[0073] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein), wherein the N / P value is 5.5 to 6.2, and the LNP carrier herein comprises 60 mol% to 70 mol% of a TM7 lipid compound, 10 mol% to 15 mol% of a phospholipid (e.g., HSPC), 15 mol% to 25 mol% of cholesterol, and 2 mol% to 3 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.15. In some embodiments, the encapsulation efficiency of the above drug is greater than 70%.

[0074] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein), wherein the N / P value is 5.5 to 6.2, and the LNP carrier herein comprises 25 mol% to 35 mol% of a TM7 lipid compound, 5 mol% to 15 mol% of a phospholipid (e.g., HSPC), 55 mol% to 65 mol% of cholesterol, and 1 mol% to 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 50 nm and 1100 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 95%.

[0075] In some embodiments of the present application, the drug comprises an LNP carrier and a nucleic acid drug (e.g., mRNA encoding the SARS-CoV-2 virus spike glycoprotein), where the N / P value is between 3.8 and 4.2, and the LNP carrier here comprises 45 mol% - 55 mol% of a TM7 lipid compound, 5 mol% - 15 mol% of a phospholipid (e.g., HSPC), 35 mol% - 45 mol% of cholesterol, and 1 mol% - 2 mol% of DSPE-PEG2000. In some embodiments, the average particle size of the above drug is between 100 nm and 150 nm. In some embodiments, the particle size distribution PDI of the above drug is less than 0.1. In some embodiments, the encapsulation efficiency of the above drug is greater than 90%.

[0076] In some embodiments, the drug in the present application can be used to treat or prevent several diseases such as COVID infection, viral and bacterial infections, tumors, metabolic diseases, autoimmune diseases, cardiovascular diseases, and genetic diseases.

[0077] In the fifth embodiment, the present application provides a method for manufacturing the above drug adopting the following technical solutions.

[0078] A method for manufacturing a drug, wherein the method for manufacturing the drug comprises weighing and dissolving other components except the cationic lipid compound to produce a stock solution; adding the cationic lipid compound to the stock solution and mixing to obtain a lipid-alcohol phase; diluting the nucleic acid drug to produce a nucleic acid aqueous phase; mixing the lipid-alcohol phase and the nucleic acid aqueous phase to produce an intermediate product of the drug, followed by dialysis to obtain the drug.

[0079] Preferably, when dissolving the phospholipid, cholesterol, and polyethylene glycol lipid, the solvent used contains ethanol (e.g., anhydrous ethanol).

[0080] Preferably, when diluting the nucleic acid drug, it is diluted using an acidic buffer salt solution, for example, diluted using an acidic buffer salt solution with pH = 4.0.

[0081] In the present application, in order to remove the solvent, dialysis is performed using a dialysis device. In some embodiments, after the dialysis step, the production method of the present application further includes dispersing the LNP in a biocompatible injection buffer.

[0082] In the sixth aspect, the present application provides a vaccine adopting the following technical solutions.

[0083] A vaccine, wherein the vaccine contains the LNP carrier described in the third aspect.

[0084] In the seventh aspect, the present application provides the application of the cationic lipid compound described in the first aspect in the production of an LNP carrier, a drug or a vaccine, or the application of the LNP carrier described in the third aspect in the production of a drug and / or a vaccine.

[0085] In summary, the present application has the following beneficial effects. 1. The present application combines 3 - ((2 - (dimethylamino)ethane)(methyl)amino)propionic acid and a secondary long - chain alkyl ester to obtain an amphiphilic derivative as the cationic lipid component in the gene drug carrier system. Because it has a positive charge, it is easier to adsorb to the cell surface and enter the cell, the drug delivery efficiency is higher, in some embodiments the gene transfection efficiency is higher, the therapeutic effect is better, the production raw materials are easy to obtain, the production method is simple, the synthesis efficiency is high, and it provides conditions for large - scale production.

[0086] 2. The LNP carrier prepared from the above cationic lipid compound has good drug loading and delivery efficiency. In some embodiments, the encapsulation efficiency can reach 88% or more. After preparing the drug together with the nucleic acid molecule, the related protein can be efficiently expressed in vivo. Therefore, it can exert the effect of treating diseases as a therapeutic drug or activate immunity as a vaccine, and has a broad application prospect. In some embodiments, the LNP carrier prepared from the above cationic lipid compound can accurately and effectively enter the cell, express the related protein, and exert the therapeutic effect.

Brief Description of Drawings

[0087]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Mode for Carrying Out the Invention

[0088] The present application provides a cationic lipid compound containing a compound represented by Formula I,

Chemical formula

Chemical formula

[0089] In some aspects of the present application, n = 1, 2 or 3.

[0090] In some aspects of the present application, n = 2.

[0091] In some aspects of the present application, n1 in R1 is the same as the value of n1 in R2.

[0092] Specifically, the cationic lipid compound contains TM1 represented by Formula III, TM2 represented by Formula IV, TM3 represented by Formula V, TM4 represented by Formula VI, or TM5 represented by Formula VII.

[0093] Specifically, taking TM3 as an example, the present application describes a method for producing the cationic lipid compound. A schematic diagram of its synthetic route is shown in Figure 1, and the specific process is as follows.

[0094] (1) Ethyl 8-bromooctanoate is subjected to an alkylation reaction with TosMIC and NaH at room temperature in a DMSO solution using TBAI as a catalyst to produce intermediate product 2. 18 - 22 mmol of ethyl 8-bromooctanoate is dissolved in 55 - 65 mL of anhydrous DMSO, stirred at 8 - 15 °C for 5 - 10 min, 8 - 12 mmol of TosMIC is added, stirred for 5 - 10 min, 23 - 28 mmol of NaH is added in several portions, and finally 1.8 - 2.2 mmol of TBAI is further added. The temperature is gradually raised to room temperature and stirred for 1 - 3 h.

[0095] The progress of the reaction is monitored by TLC. When the reaction is complete, the system is cooled in an ice-water bath, 145 - 155 mL of ice water is added to quench it, and extraction is carried out 3 times with 90 - 110 mL of DCM each time. All organic phases are collected, washed with 90 - 110 mL of water, and further washed 2 times with 145 - 155 mL of saturated sodium bicarbonate each time. After drying and concentration, purification is carried out by a silica gel column to obtain intermediate product 2.

[0096] (2) Intermediate product 2 is reacted at room temperature in an acidic mixed solution of DCM and concentrated sulfuric acid (volume ratio (3.5 - 8):1) to produce intermediate product 3. 3.9 - 4.2 g of intermediate product 2 is weighed, dissolved in 45 - 55 mL of DCM solution, and stirred for 3 - 7 min. 7 - 12 mL of concentrated sulfuric acid is added, and stirred at room temperature for 2 - 5 h.

[0097] The progress of the reaction was monitored by TLC. After the reaction was completed, 45 - 55 mL of water was added to the system, and after uniform mixing, it was allowed to stand for layering. The aqueous layer was extracted with 45 - 55 mL of DCM. All the organic phases were combined, and the organic phase was eluted with 45 - 55 mL of saturated sodium bicarbonate. After drying and concentration, it was purified by silica gel column to obtain intermediate product 3.

[0098] (3) Intermediate product 3 was subjected to a reduction reaction with NaBH₄ at room temperature in a mixed solution of THF and ethanol (volume ratio (2 - 4):1) to produce intermediate product 4. 3 - 5 mmol of intermediate product 3 was weighed and dissolved in 45 - 55 mL of a mixed solution of THF and ethanol (volume ratio (2 - 4):1), and stirred at 0 - 4 °C for 3 - 7 min. 3 - 5 mmol of NaBH₄ was gradually added in several portions and reacted at room temperature for 3 - 5 h.

[0099] The progress of the reaction was detected by TLC. After the reaction was completed, 90 - 110 mL of ice water was added to the system for quenching, and extracted three times with 90 - 110 mL each time using DCM. All the organic phases were combined, and the organic phase was eluted with 45 - 55 mL of saturated sodium bicarbonate. After drying and concentration, it was purified by silica gel column to obtain intermediate product 4.

[0100] (4) Intermediate product 4 was subjected to a condensation reaction with 3 - chloropropionyl chloride at room temperature in a DCM solution containing pyridine (pyridine concentration 73 - 134 mM) to produce intermediate product 5. 4 - 6 mmol of intermediate product 4 was weighed and dissolved in 45 - 55 mL of DCM solution, and 4 - 6 mmol of pyridine was further added. It was stirred at 0 - 4 °C for 3 - 7 min, and 7 - 9 mmol of 3 - chloropropionyl chloride was gradually added and reacted at room temperature for 0.5 - 2 h.

[0101] The progress of the reaction was detected by TLC. After the reaction was completed, 90 - 110 mL of ice water was added to the system for quenching, washed with 90 - 110 mL of water, and further washed twice with 140 - 160 mL each time using saturated sodium bicarbonate solution. After drying and concentration, it was purified by silica gel column to obtain intermediate product 5.

[0102] (5) React the intermediate product 5 in an acetone solution with NaI under the conditions of 68 - 72 °C to carry out a substitution reaction to produce intermediate product 6. Weigh 3 - 5 mmol of intermediate product 5, dissolve it in 45 - 55 mL of acetone solution, gradually add 3 - 6 mmol of NaI, raise the temperature to 68 - 72 °C, and react for 12 - 18 h.

[0103] Detect the progress of the reaction by TLC. After the reaction is completed, elute the organic phase with 45 - 55 mL of saturated sodium thiosulfate, and after drying and concentration, purify it by silica gel column to obtain intermediate product 6.

[0104] (6) React the intermediate product 6 in a DCM solution with N,N,N'-trimethylethylenediamine under room temperature conditions to carry out a substitution reaction to produce intermediate product 7. Weigh 3 - 5 mmol of intermediate product 6, dissolve it in 45 - 55 mL of DCM solution, add 5 - 7 mmol of N,N,N'-trimethylethylenediamine, and stir at room temperature for 1 - 3 h for reaction.

[0105] Detect the progress of the reaction by TLC. After the reaction is completed, elute the organic phase with 45 - 55 mL of saturated sodium bicarbonate, and after drying and concentration, purify it by silica gel column to obtain intermediate product 7.

[0106] (7) React the intermediate product 7 in a mixed solution of ethanol and water (volume ratio 1:(1.5 - 3)) with LiOH under room temperature conditions to carry out a hydrolysis reaction to produce intermediate product 8. Weigh 4 - 6 mmol of intermediate product 7 and 18 - 22 mmol of LiOH, dissolve them in 90 - 110 mL of a mixed solution of ethanol and water (volume ratio 1:(1.5 - 3)), and stir at room temperature for 4 - 7 h.

[0107] The progress of the reaction was detected by TLC. After the reaction was completed, ethanol was rotary dried and then acidified with 1.5 - 2.5 M hydrochloric acid until the pH reached about 2, and extracted three times with 90 - 110 mL of DCM each time. All the organic phases were combined, eluted with 45 - 55 mL of saturated sodium bicarbonate, dried and concentrated, and then purified by a silica gel column to obtain the intermediate product 8.

[0108] (8) The intermediate product 8 was subjected to an esterification reaction with 9 - heptadecanol at room temperature under the action of DMAP and DCC in a DCM solution to produce the cationic lipid compound TM3. 4 - 6 mmol of the intermediate product 8, 14 - 16 mmol of DMAP and 16 - 20 mmol of 9 - heptadecanol were weighed, dissolved in 55 - 65 mL of DCM, stirred for 3 - 7 min, 13 - 17 mmol of DCC was added, and stirred overnight at room temperature.

[0109] The progress of the reaction was detected by TLC. After the reaction was completed, it was washed three times with 55 - 65 mL each time with water. Further, it was washed twice with 45 - 55 mL each time using saturated sodium bicarbonate. After drying and concentration, it was purified by a silica gel column to obtain the cationic lipid compound TM3.

[0110] The present application further provides an LNP carrier. In terms of molar fraction, the LNP carrier contains 15% - 70% of a cationic lipid compound, 8% - 30% of a phospholipid, 15% - 65% of cholesterol, and 1.5% - 3% of a polyethylene glycol lipid.

[0111] The present application further provides an LNP carrier. In terms of molar fraction, the LNP carrier contains 15% - 70% of a cationic lipid compound, 8% - 40% of a phospholipid, and 10% - 65% of cholesterol.

[0112] The present application further provides a drug, which comprises an LNP carrier and a nucleic acid drug. Here, in the drug, the drug loading amount of the nucleic acid drug is between 1.0% and 10.0%. In some embodiments, the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is 2:1 to 15:1, 2:1 to 10:1, 2:1 to 6:1, or 4:1 to 6:1.

[0113] In some embodiments, the drug can be prepared by the following method.

[0114] Accurately weigh the lipids according to the formulation, add ethanol, and stir and dissolve in a magnetic stirrer. Add the mRNA stock solution to a calculated amount of citrate buffer with pH = 4 and mix uniformly.

[0115] Absorb the above lipid ethanol solution and mRNA-citrate buffer aqueous solution with a syringe, use an LNP mixing pump, and collect a stable solution in the mixing stage after startup. Add the above mixed mRNA-LNP intermediate product to a dialysis bag, add pre-cooled dialysis buffer at 4°C, place it in a refrigerator at 2 - 8°C, and stir and dialyze overnight at 200 rpm.

[0116] Filter and sterilize with a 0.22 μm sterile needle filter (Millipore), and place it in a sterile enzyme-free centrifuge tube. Use Ribogreen detection reagent (thermo) to detect the mRNA loading amount and encapsulation rate of the filtered mRNA-LNP. Use a PCS detector to detect the zeta average particle size of the filtered mRNA-LNP.

[0117] In some embodiments, the drug can be prepared by the following method.

[0118] Weigh 1.5 - 1.8 mg of phospholipid, 1.7 - 1.9 mg of cholesterol, and 0.8 - 1 mg of polyethylene glycol lipid, dissolve them in 0.8 - 1.5 mL of absolute ethanol under the condition of 55 - 65°C to produce a stock solution. Add 13 - 15 mg of a cationic lipid compound to the stock solution at room temperature, mix to obtain a lipid - alcohol phase, Dilute the nucleic acid drug with an acidic buffer salt solution until the final concentration reaches 0.5 - 0.7 mg / mL, and mix uniformly to produce a nucleic acid aqueous phase, Mix the lipid - alcohol phase and the nucleic acid aqueous phase at a volume ratio of 1:(2 - 5) at room temperature using a mixer to produce an intermediate product of the drug, dialyze using a dialysis device to remove the solvent, and replace it with a Tris solution having a concentration of 18 - 22 mM containing 0.2% - 0.5% sodium chloride and 4% - 6% sucrose to obtain the drug.

[0119] In some embodiments, the drug can be produced by the following method.

[0120] Weigh 1.5 - 1.8 mg of phospholipid, 1.7 - 1.9 mg of cholesterol, and 0.8 - 1 mg of polyethylene glycol lipid, dissolve them in 0.8 - 1.5 mL of absolute ethanol under the condition of 55 - 65 °C to produce a stock solution, Add 13 - 15 mg of a cationic lipid compound to the stock solution at room temperature, mix to obtain a lipid - alcohol phase, Dilute the nucleic acid drug with an acidic buffer salt solution until the final concentration reaches 0.05 - 0.3 mg / mL, and mix uniformly to produce a nucleic acid aqueous phase, Mix the lipid - alcohol phase and the nucleic acid aqueous phase at a volume ratio of 1:(2 - 5) at room temperature using a mixer to produce an intermediate product of the drug, dialyze using a dialysis device to remove the solvent, and replace it with a Tris solution having a concentration of 18 - 22 mM containing 0.2% - 0.5% sodium chloride and 4% - 6% sucrose to obtain the drug.

[0121] The listed examples The following listed examples illustrate some aspects of the present invention.

[0122] Example 1. A cationic lipid compound, including the compound shown in Formula I,

Chemical formula

Chemical formula

[0123] Example 2. The cationic lipid compound includes a compound represented by formula III, formula IV, formula V, formula VI or formula VII,

Chemical formula

Chemical formula

[0124] Example 3. A method for producing the cationic lipid compound according to Example 1 or 2, A step of performing an alkylation reaction and a reduction reaction in sequence using a bromoester compound as a raw material, Furthermore, it is condensed with an acid chloride compound, and two steps of substitution reaction and primary hydrolysis reaction are performed in sequence, and finally it is esterified with a monohydric secondary alcohol compound to produce the cationic lipid compound, a method characterized by including this.

[0125] Example 4. An LNP carrier, characterized by including any one or a combination of at least two of the cationic lipid compounds according to Example 1 or 2.

[0126] Example 5. In molar fraction, the LNP carrier contains 15% to 70% of the cationic lipid compound, Preferably, in terms of molar fraction, the LNP carrier further comprises 8% - 30% phospholipid, 5% - 65% cholesterol, and 1.5% - 3% polyethylene glycol lipid, and the LNP carrier according to Example 4 is characterized in this regard.

[0127] Example 6. A drug characterized by comprising the LNP carrier according to Example 4 or 5.

[0128] Example 7. The drug further comprises a nucleic acid drug. Preferably, in the drug, the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2 - 6):1, and the drug according to Example 6 is characterized in this regard.

[0129] Example 8. The method for manufacturing the drug is as follows. Weighing and dissolving other components except the cationic lipid compound to produce a stock solution. Adding the cationic lipid compound to the stock solution and mixing to obtain a lipid alcohol phase. Diluting the nucleic acid drug to produce a nucleic acid aqueous phase. Mixing the lipid alcohol phase and the nucleic acid aqueous phase to produce an intermediate product of the drug, followed by dialysis to obtain the drug, and the method for manufacturing the drug according to Example 6 or 7 is characterized in this regard.

[0130] Example 9. A vaccine characterized by comprising the LNP carrier according to Example 4 or 5.

[0131] Example 10. Application of the cationic lipid compound according to Example 1 or 2 in the manufacture of the LNP carrier, drug or vaccine, or application of the LNP carrier according to Example 4 or 5 in the manufacture of the drug and / or vaccine.

[0132] Hereinafter, in combination with Drawings 1 - 9, Production Examples 1 - 2 and Examples 1 - 2, the present application will be described in more detail.

[0133] Production Example Production Example 1 This production example provided a cationic lipid compound TM3 whose structural formula is shown in Formula V.

[0134] [Chemical formula]

[0135] The cationic lipid compound TM3 was produced by the following method, and the synthetic route diagram is as shown in Figure 1.

[0136] (1) Ethyl 8-bromooctanoate was subjected to an alkylation reaction with TosMIC and NaH at room temperature in a DMSO solution using TBAI as a catalyst to produce intermediate 2. In a 500 mL one-necked flask, 5 g of ethyl 8-bromooctanoate (20 mmol) was weighed, dissolved in 60 mL of anhydrous DMSO, stirred at 10 °C for 7 min, 1.9 g of TosMIC (10 mmol) was added, stirred for 7 min, 1 g of NaH (25 mmol) was added in portions, and finally 0.7 g of TBAI (2 mmol) was further added. The temperature was gradually raised from 10 °C to room temperature and stirred for 2 h.

[0137] The progress of the reaction was monitored by TLC. When the reaction was completed, the system was cooled in an ice-water bath, 150 mL of ice water was added to quench it, and extraction was carried out 3 times with 100 mL each time using DCM. All the organic phases were collected, washed with 100 mL of water, and further washed 2 times with 150 mL each time using saturated sodium bicarbonate. After drying and concentration, it was purified by a silica gel column to obtain intermediate 2. The detection result of the hydrogen spectrum of intermediate 2 is shown in Figure 2.

[0138] (2) Intermediate 2 was reacted in an acidic mixed solution of DCM and concentrated sulfuric acid (volume ratio 5:1) at room temperature to produce intermediate 3. In a 250 mL one-necked flask, 4.1 g of intermediate 2 was weighed, dissolved in 50 mL of a DCM solution, and stirred for 5 min. 10 mL of concentrated sulfuric acid was added, and it was stirred at room temperature for 3 h.

[0139] The progress of the reaction was monitored by TLC. After the reaction was completed, 50 mL of water was added to the system, and after uniform mixing, the mixture was allowed to stand for phase separation. The aqueous layer was extracted with 50 mL of DCM. All the organic phases were combined, and the organic phase was eluted with 50 mL of saturated sodium bicarbonate. After drying and concentration, it was purified by silica gel column to obtain intermediate product 3. The detection results of the hydrogen spectrum of intermediate product 3 are shown in Figure 3.

[0140] (3) Intermediate product 3 was subjected to a reduction reaction with NaBH4 at room temperature in a mixed solution of THF and ethanol (volume ratio 3:1) to produce intermediate product 4. In a 250 mL one-necked flask, 1.5 g of intermediate product 3 (4 mmol) was weighed and dissolved in 50 mL of a mixed solution of THF and ethanol (volume ratio 3:1). The solution was stirred at 0 °C for 5 min. 0.15 g of NaBH4 (4 mmol) was gradually added in several portions, and the reaction was carried out at room temperature for 4 h.

[0141] The progress of the reaction was detected by TLC. After the reaction was completed, 100 mL of ice water was added to the system to quench it, and extraction was carried out three times with 100 mL of DCM each time. All the organic phases were combined, and the organic phase was eluted with 50 mL of saturated sodium bicarbonate. After drying and concentration, it was purified by silica gel column to obtain intermediate product 4. The detection results of the hydrogen spectrum of intermediate product 4 are shown in Figure 4.

[0142] (4) Intermediate product 4 was subjected to a condensation reaction with 3-chloropropionyl chloride at room temperature in a DCM solution containing pyridine (pyridine concentration 100 mM) to produce intermediate product 5. In a 250 mL one-necked flask, 1.8 g of intermediate product 4 (5 mmol) was weighed and dissolved in 50 mL of DCM solution. Then 0.4 g of pyridine (5 mmol) was added, and the solution was stirred at 0 °C for 5 min. 1 g of 3-chloropropionyl chloride (8 mmol) was gradually added, and the reaction was carried out at room temperature for 1 h.

[0143] The progress of the reaction was detected by TLC. After the reaction was completed, 100 mL of ice water was added to the system to quench it, and it was washed with 100 mL of water. Further, it was washed twice with 150 mL each time using a saturated sodium bicarbonate solution, and after drying and concentration, it was purified by a silica gel column to obtain intermediate product 5. The detection results of the hydrogen spectrum of intermediate product 5 are shown in Figure 5.

[0144] (5) Intermediate product 5 was subjected to a substitution reaction with NaI in an acetone solution under the condition of 70 °C to produce intermediate product 6. In a 250 mL one-necked flask, 1.8 g of intermediate product 5 (4 mmol) was weighed, dissolved in 50 mL of an acetone solution, 0.8 g of NaI (5 mmol) was gradually added, the temperature was raised to 70 °C, and the reaction was carried out for 16 h.

[0145] The progress of the reaction was detected by TLC. After the reaction was completed, the organic phase was eluted with 50 mL of saturated sodium thiosulfate, and after drying and concentration, it was purified by a silica gel column to obtain intermediate product 6.

[0146] (6) Intermediate product 6 was subjected to a substitution reaction with N,N,N'-trimethylethylenediamine in a DCM solution under room temperature conditions to produce intermediate product 7. In a 250 mL one-necked flask, 2.2 g of intermediate product 6 (4 mmol) was weighed, dissolved in 50 mL of a DCM solution, 0.6 g of N,N,N'-trimethylethylenediamine (6 mmol) was added, and the mixture was stirred at room temperature for 2 h for reaction.

[0147] The progress of the reaction was detected by TLC. After the reaction was completed, the organic phase was eluted with 50 mL of saturated sodium bicarbonate, and after drying and concentration, it was purified by a silica gel column to obtain intermediate product 7. The detection results of the hydrogen spectrum of intermediate product 7 are shown in Figure 6.

[0148] (7) Intermediate product 7 was subjected to a hydrolysis reaction with LiOH in a mixed solution of ethanol and water (volume ratio 1:2) under room temperature conditions to produce intermediate product 8. In a 250 mL single-neck flask, 3.8 g of intermediate product 7 (5 mmol) and 0.5 g of LiOH (20 mmol) were weighed, dissolved in a 100 mL mixed solution of ethanol and water (volume ratio 1:2), and stirred at room temperature for 5 h.

[0149] The progress of the reaction was detected by TLC. After the reaction was completed, ethanol was rotary dried and then acidified with 2 M hydrochloric acid until the pH reached about 2, and extracted three times with 100 mL of DCM each time. All the organic phases were combined, eluted with 50 mL of saturated sodium bicarbonate, dried and concentrated, and then purified by silica gel column to obtain intermediate product 8.

[0150] (8) Intermediate product 8 was esterified with 9-heptadecanol at room temperature in a DCM solution under the action of DMAP and DCC to produce the cationic lipid compound TM3. In a 250 mL single-neck flask, 3.6 g of intermediate product 8 (5 mmol), 1.8 g of DMAP (15 mmol) and 4.6 g of 9-heptadecanol (18 mmol) were weighed, dissolved in 60 mL of DCM, stirred for 5 min, 3.1 g of DCC (15 mmol) was added, and stirred overnight at room temperature.

[0151] The progress of the reaction was detected by TLC. After the reaction was completed, it was washed three times with 60 mL of water each time. It was further washed twice with 50 mL of saturated sodium bicarbonate each time. After drying and concentration, it was purified by silica gel column to obtain the cationic lipid compound TM3. The detection result of the hydrogen spectrum of TM3 is shown in Figure 7.

[0152] In the above reaction, the statistical results of the mass and yield of the products obtained in each step are shown in Table 1.

[0153]

Table 1

[0154] Production Example 2 This manufacturing example provides a drug TP1, where the nucleic acid drug is an mRNA encoding the SARS-CoV-2 virus spike glycoprotein (S protein) and is manufactured by the following method.

[0155] Weighed 1.64 mg of HSPC, 1.83 mg of cholesterol and 0.89 mg of DSPE-PEG2000, dissolved them in 1 mL of absolute ethanol under the condition of 60 °C, and prepared a stock solution.

[0156] Added 13.41 mg of TM3 to the stock solution at room temperature, mixed them to obtain a lipid-alcohol phase.

[0157] Dissolved 1.6 mL of 1 mg / mL mRNA in 1.4 mL of D-citric acid solution with pH 4.0, mixed them uniformly to prepare a nucleic acid aqueous phase.

[0158] Mixed 1 mL of the lipid-alcohol phase and 3 mL of the nucleic acid aqueous phase at room temperature by a mixer to prepare an intermediate product of the drug.

[0159] Removed ethanol and citric acid from the intermediate product by a dialysis device, and replaced them with a Tris solution with a concentration of 20 mM containing 0.4% sodium chloride and 5% sucrose to manufacture the drug TP1.

[0160] Performance test For the drug TP1 manufactured in Manufacturing Example 2, a performance test including specifically the detection of particle size, dispersity and encapsulation efficiency was carried out.

[0161] Detection of particle size and dispersity Using a laser scattering particle size analyzer (PCS, Dandong Bettersize BeNano 180 Zeta pro laser particle size analyzer), the particle size and dispersity (PDI) of nanoparticles were measured. A 671 nm solid laser was used as the incident light, the dynamic light scattering test was carried out at 25 °C, and the scattering optical path was detected by 173° backscattering. The average value detected continuously three times was used as the detection data.

[0162] Detection of encapsulation efficiency The detection of the encapsulation rate adopted the fluorescence quantitative detection method, and Quant-it TM RiboGreen RNA Assay Kit was used as the detection reagent kit. After adding Triton for demulsification, the nucleic acid in the formulation was released. Then, the specific nucleic acid dye ribogreen was added, and the ALLSHENG Feyond-A300 microplate reader was set with an excitation light wavelength of 470 nm and an emission light wavelength of 525 nm to detect the absorbance value of the sample. The total nucleic acid content was calculated according to the standard curve. Furthermore, for the LNP sample without Triton treatment, the specific nucleic acid dye ribogreen was added, and the ALLSHENG Feyond-A300 microplate reader was set with an excitation light wavelength of 470 nm and an emission light wavelength of 525 nm to detect the absorbance value of the sample. The free nucleic acid content was calculated according to the standard curve. The formula is as follows.

[0163] Encapsulation rate = (total nucleic acid content - free nucleic acid content) / total nucleic acid content × 100%.

[0164] The detection results of particle size, dispersion and encapsulation rate are shown in Table 2.

[0165]

Table 2

[0166] As can be seen from Table 2, the drug TP1 obtained by manufacturing according to the formulation and method of Production Example 2 has an average particle size of 169 nm and a PDI of 0.07. It has an appropriate particle size and a uniform particle size distribution, and at the same time has a relatively high encapsulation rate and good drug loading capacity, providing conditions for subsequent practical applications.

[0167] Production Example 3 This production example provides the production of 7 drugs, where the nucleic acid drug is mRNA encoding luciferase. The specific lipid formulation information is shown in Table 3.

[0168]

Table 3

[0169] Manufacturing method: Based on the above formulation, the lipid was accurately weighed, anhydrous ethanol was added, and it was stirred and dissolved in a magnetic stirrer. The mRNA stock solution was added to a calculated amount of citrate buffer with pH = 4 and uniformly mixed. The above lipid ethanol solution and the mRNA-citrate buffer aqueous solution were aspirated with a syringe, and a LNP mixing pump was used to collect a stable solution at the mixing stage after startup. The mRNA-LNP intermediate product after the above mixing was added to a dialysis bag, pre-cooled dialysis buffer at 4 °C was added, and it was placed in a refrigerator at 2 - 8 °C and dialyzed with stirring at 200 rpm overnight. It was filtered through a 0.22 μm sterile needle filter (Millipore) for sterilization and placed in a sterile enzyme-free centrifuge tube.

[0170] The mRNA loading amount and encapsulation rate of the filtered mRNA-LNP were detected using the Ribogreen detection reagent (Thermo). The zeta average particle size of the filtered mRNA-LNP was detected using a PCS detector. The detection results are shown in Table 4.

[0171]

Table 4

[0172] Cryo-electron microscopy: 2.5 μL of the LNP solution was taken, a grid (Quantifoil Cu R1.2 / 1.3, 300 mesh) was dropped, and a cryo-electron microscopy sample was prepared using a Vitrobot Mark IV (ThermoFisher Scientific). Imaging was performed using a Talos F200C equipped with a Ceta 4k×4k camera. The cryo-electron microscopy photograph of the drug HG5-06 is shown in Figure 10. The cryo-electron microscopy photograph of the drug HG5-023 is shown in Figure 11.

[0173] Production Example 4 A method similar to Production Example 3 was adopted, and this production example provides the production of a drug containing TM6 and TM7, where the nucleic acid drug is an mRNA encoding the SARS-CoV-2 virus spike glycoprotein (S protein). The nuclear magnetic resonance spectrum of TM7 is shown in Figure 12.

[0174] The formulation information of the obtained LNP, as well as the particle size and encapsulation efficiency information, are shown in Table 5.

Table 5

[0175] Example 1 In this example, the expression level of the drug TP1 produced in Production Example 2 in in vitro cultured cells was detected, and the steps are as follows.

[0176] (1) Cell culture and collection: The 293T cell line, which is a derivative of the human fetal kidney cell line 293, was obtained by subculture in the laboratory and cultured in RPMI-1640 medium containing 10% FBS, and subcultured 2-3 times a week. Cells with a cell density of about 80% were used for seeding. After counting the number of cells, the cell concentration was adjusted to 2×10 5 / mL using the medium, and in a 48-well plate, 0.5 mL of the above cell suspension was added to each well, that is, the number of cells in each well was about 1×10 5 . Three parallel wells were set for each group, and three parallel wells were also set for the blank control group.

[0177] (2) Co-incubation of cells and drug: Based on the total mRNA concentration for fluorescence detection of mRNA, a drug containing 1 μg of mRNA was added to each well, and incubated for 18, 24, 48, and 72 h respectively to observe the expression level of the protein within different incubation times.

[0178] (3) Detection of protein expression: The cell culture plates were centrifuged at 800 g for 5 min, the supernatants were taken, and diluted 10-fold, 50-fold, and 100-fold with ddH2O respectively to prepare the samples to be measured. Based on the expressed protein, a standard curve was plotted using the SARS-CoV-2 virus spike glycoprotein (S protein) as a standard, and detected by ELISA method.

[0179] (4) Result analysis: The OD value of the blank control was subtracted from the OD value of the measured sample to calibrate the absorbance value of the standard curve. A standard curve was plotted with the standard concentration on the x-axis, and the concentration of the corresponding S protein of the sample OD value was calculated based on the standard curve.

[0180] The detection results of the protein expression level of drug TP1 in 293T cells are shown in Figure 8. As can be seen from Figure 8, drug TP1 can be effectively introduced into 293T cells to express S protein, and with the increase of the incubation time, the expression level also continuously increased, and the protein expression level was also positively correlated with the dosage. From the above results, it was shown that the LNP carrier can introduce and release drugs into cells, so it can be used for the treatment of related diseases and has practical application value.

[0181] Example 2 This example detected the immunogenicity of drug TP1 prepared in Preparation Example 2 in vivo, and the steps were as follows.

[0182] Six 6- to 8-week-old female BALB / c mice were taken and drug TP1 was injected intramuscularly (i.m.) on the 1st and 14th days respectively. The titers of anti-S protein antibodies in mouse sera were detected by ELISA method on the 10th, 21st, 28th, and 35th days after the first administration.

[0183] The detection results of the titer of anti-S protein antibody in mouse serum were shown in Figure 9. As can be seen from Figure 9, antibodies were produced on the 10th day after the first administration. After an additional injection on the 14th day, the expression level of antibodies increased significantly on the 21st day, and continued to be expressed continuously over time, and still continued to be expressed on the 35th day. According to the above results, the drug can effectively activate the immune response of animals, and the antibody titer in the animal body is significantly improved after administration. Therefore, it can stimulate the living body to generate an immune response corresponding to the vaccine, indicating that the effect is significant.

[0184] Example 3 This example considered the drug effects of different formulations of TM6 drug and TM7 drug in Example 4 in cell and animal models.

[0185] Evaluation of in vitro cell transfection activity of the formulation: The cells cryopreserved at 37°C were thawed, transferred to 5 mL of culture medium, centrifuged at 1000 rpm for 5 min, then the supernatant was discarded, the cells were resuspended with the culture medium, 10 μL was taken, stained with trypan blue, and the number / survival rate was measured. The remaining cell suspension was transferred to a cell culture flask and placed in an incubator at 37°C for static culture. When the cells grew to an abundance of about 90%, they were digested, centrifuged, and the cells were collected. After resuspension, they were counted and the cell concentration was adjusted to 2×10^5 / ml. 0.5 ml of the cell suspension was added to each well of a 48-well plate (Corning). Groups were set based on specific experiments, and 3 to 4 parallel wells were set in each group. 1 μg of the corresponding mRNA-LNP of mRNA was added to each well. After administration, the cell culture plate was shaken and then returned to the cell incubator for continued culture. After culturing the cells for 24 hours, the cell culture supernatant was transferred to the corresponding EP tube, centrifuged at 1300 rpm for 5 min, and then used for detection.

[0186] Detection of reporter gene concentration: The above cell culture collected samples were diluted at a certain ratio, and the dilution ratio was determined by preliminary experiments. The diluted samples were detected by an Elisa kit.

[0187] Figure 14 compares the cell transfection effects of TM6 and TM7 drugs with various lipid formulations, and compares them with drugs with similar lipid formulations containing TM3. The transfection activities of drugs containing TM6 and TM7 were slightly higher than those of drugs containing TM3. However, the activity of the TM3 drug with HG-23 lipid formulation was also very high. In short, the cationic TM series of LNPs have certain differences in particle size under different lipid formulation conditions, but all have excellent cell transfection activities.

[0188] The inventors further evaluated the in vivo immune effects of these drugs. The animal model was 6-8-week-old female Balb / c mice housed in a SPF-grade animal breeding center. Administered i.m. according to the test protocol, with n = 6 in each group and the dosage per animal being 10 μg. Two weeks after immunization, blood was collected from the orbital cavity after anesthesia, about 50 μl of whole blood was collected into a 1.5 mL EP tube, left at room temperature for 0.5 h, then centrifuged at 7000 g for 10 min, collected, diluted at a certain ratio, and the OD value was detected by (SARS-Cov2~2 Spike RBD Antibody Titer Assay Kit (mouse) SinoBiological). The results are shown in Figure 15.

[0189] According to the result analysis of the mouse experiment, the three different lipid structures and their formulations have equivalent titers of antibodies that induce anti-COVID S protein. Here, both the HG-06 formulation of TM6 and the HG-23 formulation of TM7 are excellent and have clinical development value.

[0190] Example 4: TMEA is a cationic lipid structure disclosed in CN105085292B, the same as the hydrophilic and ionizable end groups of TM3, both being 3-((2-(dimethylamino)ethane)(methyl)amino)propionic acid, but with different hydrophobic structures. Therefore, the inventors compared the constructed mRNA-loaded LNPs. Table 6 compares three different lipid formulations and found that the in vivo protein expression activities of LNPs obtained from TM3 were all much superior to those of TMEA LNPs.

[0191] In the in vivo protein expression experiment, Balb / c mice aged 6 - 8 weeks were selected, 10 μg of nucleic acid LNP drug was intravenously administered to each mouse, blood samples were collected 6 hours after administration, and the protein content in the serum was detected using an Elisa reagent kit.

[0192]

Table 6

[0193] This specific example is only for interpreting the present application and does not limit the present application. After reading this specification, those skilled in the art can make changes to this example that do not contribute to the inventiveness as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A cationic lipid compound, The compound is represented by formula I, 【Chemistry 1】 In equation I, n = 1, 2, 3, or 4, R １ and R ２ Each is independently selected from the secondary long-chain alkyl esters shown in formula II, 【Chemistry 2】 In equation II, n １ = 2, 4 or 6, n ２ = 5, 7, 9 or 11, n ３ A cationic lipid compound characterized in that the ratio is 5, 7, 9, or 11, and the dashed line represents the bond point between formula II and the other part of formula I.

2. The cationic lipid compound according to claim 1, wherein n = 1, 2, or 3.

3. The cationic lipid compound according to claim 1, wherein n = 2.

4. R １ n1 in R ２ The cationic lipid compound according to claim 1, wherein the value of n1 is the same as in the above.

5. The cationic lipid compound includes compounds represented by formula III, formula IV, formula V, formula VI, formula VII, formula VIII, or formula IX. 【Transformation 3】 【Chemistry 4】 【Transformation 5】 The cationic lipid compound according to claim 1, characterized in that...

6. A method for producing a cationic lipid compound according to any one of claims 1 to 5, The process involves sequentially carrying out alkylation and reduction reactions using a bromoester compound as a starting material. A method characterized by comprising the step of further reacting with an acid chloride compound in a condensation reaction, carrying out two sequential substitution reactions and one hydrolysis reaction, and finally reacting with a monovalent secondary alcohol compound in an esterification reaction to produce the cationic lipid compound.

7. LNP carrier, An LNP carrier characterized by comprising any one or at least two combinations of the cationic lipid compounds described in claim 1.

8. In mole fraction, the LNP carrier contains 15% to 70% cationic lipid compounds. Preferably, the LNP carrier according to claim 7, characterized in that the LNP carrier further contains 5% to 40% phospholipids and 10% to 65% cholesterol in mole fraction.

9. It is a drug, A drug characterized by comprising the LNP carrier described in claim 7 or 8.

10. The drug further comprises a nucleic acid drug, Preferably, the drug according to claim 9, characterized in that the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is 2:1 to 15:

1.

11. The drug according to claim 10, wherein the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is 4:1 to 6:

1.

12. The drug further comprises a nucleic acid drug, Preferably, in the drug, the mass parts (drug-carrying amount) of the nucleic acid drug in the drug-containing LNP is between 1.0 and 15.0%, and particularly preferably, the drug-carrying amount of the nucleic acid drug is between 3.0 and 8.0 wt.%, characterized in that the drug according to claim 9.

13. The method for producing the aforementioned drug is: The steps include weighing and dissolving the other components, excluding the cationic lipid compounds, to produce the stock solution, The steps include adding a cationic lipid compound to the stock solution, mixing, and obtaining a lipid alcohol phase, The steps include diluting the nucleic acid drug to produce the nucleic acid aqueous phase, A method for producing a drug according to claim 9, characterized by comprising the steps of mixing the lipid alcohol phase and the nucleic acid aqueous phase to produce a drug intermediate product, dialyzing it, and obtaining the drug.

14. It is a vaccine, A vaccine characterized by comprising the LNP carrier described in claim 7 or 8.

15. An application of a cationic lipid compound according to any one of claims 1 to 5 in the manufacture of an LNP carrier, a drug, or a vaccine, or an application of an LNP carrier according to claim 7 or 8 in the manufacture of a drug and / or a vaccine.

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