Ionizable lipid compound and application thereof

Abstract

The invention provides a completely biodegradable ionizable lipid compound. The compound has a structure as shown in the specification. The compound can be used for preparing lipid nanoparticles together with other lipid compounds, has high nucleic acid drug encapsulation efficiency, can be completely degraded in a living body, has good safety, and is suitable for being applied to nucleic acid drug delivery, nucleic acid vaccines, nucleic acid therapeutic drugs and the like. # imgabs0 #

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceutical technology. Specifically, the present invention relates to a fully biodegradable ionizable lipid compound, and related compositions and applications thereof. Background Art

[0002] Lipid nanoparticles have rapidly emerged as a biological delivery platform in the field of nucleic acid or gene therapy, and certain research progress has been made in prophylactic or therapeutic vaccines and drugs. To date, the FDA has approved 4 LNP-delivered drugs and vaccines for clinical treatment and prevention of related diseases. However, there is still room for optimization in terms of the delivery efficiency, complete biodegradability, and specific targeting of the delivery lipids.

[0003] The LNP delivery components mainly include 4 lipids, namely ionizable cationic lipids, phospholipids, cholesterol, and PEG lipids. Among them, ionizable lipids are the main components, which are related to the encapsulation rate of nucleic acids, the delivery efficiency in vivo, and cytotoxicity, etc. The phospholipids and cholesterol in the existing LNP delivery components are both of natural origin or widely present in organisms, and have good biosafety; while the toxicity of the existing LNP delivery systems is mainly composed of artificially synthesized ionizable lipids and PEG lipids, among which the dosage of ionizable lipids is relatively high, posing the greatest risk of biological toxicity. Therefore, the development and design of fully biodegradable ionizable lipid compounds can better promote the clinical drug development of nucleic acid drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a series of fully biodegradable ionizable lipid compounds to enrich the types of ionizable lipid compounds, and by selecting the ionizable lipid compounds of the present invention as lipid carriers, the delivery and biodegradability of nucleic acids in vivo can be improved.

[0005] In a first aspect of the present invention, there is provided a compound or a pharmaceutically acceptable salt thereof, the compound having the structure shown in formula (I):

[0006]

[0007] Wherein, A1 is selected from hydrogen or a residue of a natural amino acid, wherein the -C=O- in the residue of the natural amino acid forms an amide bond with the -NH in formula (I); A2 is absent or selected from a hydroxyl group or R1, R2, or R3 are each independently selected from a residue of a natural fatty acid or a residue of a natural fatty acid ester; m is an integer selected from 0 to 3, and n and q are each independently an integer selected from 1 to 3.

[0008] In some embodiments, the natural amino acid is selected from non-polar hydrophobic amino acids, polar neutral amino acids, acidic amino acids, or basic amino acids.

[0009] In some embodiments, the non-polar hydrophobic amino acids are selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), methionine (Met), tryptophan (Trp), or phenylalanine (Phe).

[0010] In some embodiments, the polar neutral amino acids are selected from glutamine (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), tyrosine (Tyr), or glycine (Gly).

[0011] In some embodiments, the acidic amino acids are selected from aspartic acid (Asp) or glutamic acid (Glu).

[0012] In some embodiments, the basic amino acids are selected from lysine (Lys), arginine (Arg), or histidine (His). In some embodiments, A1 is selected from H or a group selected from the following: Denotes the attachment site of the group:

[0013]

[0014] In some embodiments, R1, R2, or R3 are each independently selected from natural fatty acids or residues obtained by removing the terminal carboxyl group from natural fatty acid esters.

[0015] In some embodiments, R1, R2, or R3 are each independently selected from residues of C12-C30 natural fatty acids or residues of C12-C30 natural fatty acid esters.

[0016] In some embodiments, the natural fatty acid or natural fatty acid ester is selected from the compounds shown below:

[0017]

[0018] Wherein, e, f, j, h, g, k are each independently selected from integers from 1 to 30, i is selected from integers from 1 to 5, L1 is selected from -C=O-, -C(=O)O-, -OC(=O)O-, or -OC(=O)-, R4, R5 are each independently selected from C1-C20 straight-chain alkyl groups or C3-C30 branched-chain alkyl groups, and R6 is selected from C3-C30 branched-chain alkyl groups.

[0019] In some embodiments, the natural fatty acid or natural fatty acid ester is selected from the compounds shown below:

[0020]

[0021]

[0022] In some embodiments, each of R1, R2 or R3 is independently selected from the following groups: indicating the attachment site of the group:

[0023]

[0024]

[0025] In some preferred embodiments, each of said R1, R2 or R3 is independently selected from the residues of linoleic acid, oleic acid, stearic acid or monooctyl sebacate.

[0026] In some embodiments, m is 0, 1, 2 or 3.

[0027] In some embodiments, n is 1, 2 or 3.

[0028] In some embodiments, p is 1, 2 or 3.

[0029] In some embodiments, e is an integer selected from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any value therebetween. Preferably, e is an integer selected from 11 to 30.

[0030] In some embodiments, f is an integer selected from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any value therebetween. Preferably, f is an integer selected from 1 to 10.

[0031] In some embodiments, j is an integer selected from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any value therebetween. In some embodiments, j is an integer selected from 5 to 30.

[0032] In some embodiments, h is an integer selected from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any value therebetween. Preferably, h is an integer selected from 1 to 10.

[0033] In some embodiments, g is selected from integers from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any value therebetween. Preferably, g is selected from integers from 5 to 30.

[0034] In some embodiments, k is selected from integers from 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any value therebetween. Preferably, k is selected from integers from 1 to 10.

[0035] In some embodiments, A1 is hydrogen, A2 is hydroxyl, and m, n, and q are each independently 1, 2, or 3.

[0036] In some embodiments, A1 is hydrogen, A2 is and m, n, and q are each independently 1, 2, or 3.

[0037] In some embodiments, A1 is a residue of a natural amino acid, A2 is hydroxyl, and m, n, and q are each independently 1, 2, or 3.

[0038] In some embodiments, A1 is a residue of a natural amino acid, A2 is and m, n, and q are each independently 1, 2, or 3.

[0039] In some embodiments, A1 is A2 is absent, and m is 0, and n and q are each independently 1, 2, or 3.

[0040] In some embodiments, the compound has the structure shown in formula (II):

[0041]

[0042] wherein, A1 is defined as A1 as defined in formula (I).

[0043] In some preferred embodiments, A1 is selected from hydrogen, a histidine residue, an arginine residue, or a lysine residue, a glycine residue, a leucine residue, an isoleucine residue, or a serine residue.

[0044] In some embodiments, the compound is selected from:

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] The compound with the structure shown in formula (I) provided by the present invention has a natural amino acid head and a natural fatty acid or fatty acid ester tail connected by a biodegradable linker (connected by an ester bond or an amide bond that is easily degradable in vivo), and can achieve complete biodegradation.

[0055] The second aspect of the present invention provides a biodegradable lipid nanoparticle, which includes the compound described in the first aspect or a pharmaceutically acceptable salt thereof, a sterol, a polyethylene glycol lipid, and a neutral auxiliary phospholipid.

[0056] In some embodiments, the sterol includes at least one of cholesterol, coprosterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, corticosteroids, and their derivatives. Preferably, the sterol is cholesterol.

[0057] In some embodiments, the polyethylene glycol lipid includes at least one of phosphatidylethanolamine modified with polyethylene glycol, phosphatidic acid modified with polyethylene glycol, ceramide modified with polyethylene glycol, dialkylamine modified with polyethylene glycol, diacylglycerol modified with polyethylene glycol, and dialkylglycerol modified with polyethylene glycol. Preferably, the polyethylene glycol lipid is DMG-PEG 2000 .

[0058] In some embodiments, the neutral helper phospholipids include at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin (SM), ceramide, sterol and its derivatives. Preferably, the neutral helper phospholipids are DSPC, DOPC or DOPE.

[0059] In some embodiments, the molar ratio of the compound or its pharmaceutically acceptable salt, steroid compound, neutral helper phospholipid and polyethylene glycol lipid is (40 - 60):(22 - 60):(5 - 30):1.5, preferably (45 - 50):(25 - 55):(10 - 20):1.5. In some specific embodiments, the molar ratio of the compound or its pharmaceutically acceptable salt, steroid compound, neutral helper phospholipid and polyethylene glycol lipid is 50:38.5:10:1.5, 40:38.5:20:1.5, 45:33.5:20:1.5, 50:28.5:20:1.5 or 45:43.5:10:1.5.

[0060] In some embodiments, the average particle size of the lipid nanoparticles is 50 nm - 200 nm, preferably 70 nm - 150 nm. In some specific embodiments, the average particle size of the lipid nanoparticles is 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm or any value therebetween.

[0061] In some embodiments, the polydispersity index (PDI) of the lipid nanoparticles is 0.05 - 0.3, preferably 0.1 - 0.25. In some specific embodiments, the polydispersity index (PDI) of the ionizable lipid nanoparticles is 0.05, 0.06, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3 or any value therebetween.

[0062] The present invention also provides a method for preparing the degradable lipid nanoparticles, comprising the following steps:

[0063] (1) Dissolve the compound, cholesterol, neutral co - phospholipid and PEG lipid in an alcohol organic solvent such as anhydrous ethanol in proportion to prepare an oil - phase solution until the total lipid concentration is 10 - 20 mM;

[0064] (2) Dissolve a drug active ingredient such as nucleic acid (mRNA, DNA, siRNA, etc.) in a citric acid buffer solution with pH = 3 - 6 to form an aqueous - phase solution;

[0065] (3) Using a microfluidic device or an impinging mixer, mix the oil phase and the aqueous phase by collision at a flow rate ratio of 1:3 - 1:7 to form lipid nanoparticles encapsulating the nucleic acid; wherein, the molar ratio of the P element of the nucleic acid to the N element in the compound is 4:1 - 8:1;

[0066] (4) Dialyze through a dialysis bag with a molecular weight cut - off of 3 kDa - 50 kDa in Tris - HCl buffer or PBS buffer for 4 - 16 hours to obtain completely degradable lipid nanoparticles.

[0067] The third aspect of the present invention provides the use of the compound described in the first aspect or its pharmaceutically acceptable salt or the lipid nanoparticles described in the second aspect in the delivery of a drug active ingredient.

[0068] In some embodiments, the drug active ingredient is selected from nucleic acids, small molecule compounds, polypeptides, and / or proteins.

[0069] In some embodiments, the nucleic acid is selected from one or more of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA, and plasmids.

[0070] In some embodiments, the drug active ingredient is encapsulated within the degradable lipid nanoparticles or electrostatically bound to the degradable lipid nanoparticles.

[0071] The fourth aspect of the present invention provides a pharmaceutical composition, comprising the degradable lipid nanoparticles according to the second aspect and a drug active ingredient.

[0072] In some embodiments, the drug active ingredient is selected from nucleic acids, small molecule compounds, polypeptides, and / or proteins.

[0073] In some embodiments, the nucleic acid is selected from one or more of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA, and plasmids.

[0074] In some embodiments, the pharmaceutically active ingredient is encapsulated within the degradable lipid nanoparticles or electrostatically bound to the degradable lipid nanoparticles.

[0075] In some embodiments, the pharmaceutical composition further comprises an adjuvant.

[0076] In some embodiments, the adjuvant comprises any one or a combination of at least two of a pharmaceutical carrier, an excipient, or a diluent.

[0077] The fifth aspect of the present invention provides the use of the compound or its pharmaceutically acceptable salt according to the first aspect, the degradable lipid nanoparticles according to the second aspect, or the pharmaceutical composition according to the third aspect in the preparation of a vaccine or a non-viral gene vector.

[0078] In some embodiments, the compound or its pharmaceutically acceptable salt, the degradable lipid nanoparticles, or the pharmaceutical composition is used for preparing a non-viral gene vector, and has good cellular immune response and humoral immune response.

[0079] The compound or its pharmaceutically acceptable salt provided by the present invention can be used together with other lipid compounds to prepare lipid nanoparticles, which have pH-dependent ionizability, high encapsulation efficiency for nucleic acid drugs, can be completely degraded in vivo, have good safety, and are suitable for applications such as nucleic acid drug delivery, nucleic acid vaccines, and nucleic acid therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 1H NMR spectrum of the compound SV-101 prepared in Example 1 of the present invention.

[0081] Figure 2 Mass spectrum of the compound SV-101 prepared in Example 1 of the present invention.

[0082] Figure 3 1H NMR spectrum of the compound SV-201 prepared in Example 1 of the present invention.

[0083] Figure 4 Mass spectrum of the compound SV-201 prepared in Example 1 of the present invention.

[0084] Figure 5 1H NMR spectrum of the compound SV-202 prepared in Example 1 of the present invention.

[0085] Figure 6 Mass spectrum of the compound SV-202 prepared in Example 1 of the present invention.

[0086] Figure 71H NMR spectrum of compound SV-203 prepared in Example 1 of the present invention.

[0087] Figure 8 Mass spectrum of compound SV-203 prepared in Example 1 of the present invention.

[0088] Figure 9 1H NMR spectrum of compound SV-204 prepared in Example 1 of the present invention.

[0089] Figure 10 Mass spectrum of compound SV-204 prepared in Example 1 of the present invention.

[0090] Figure 11 Fluorescence distribution in small animals in Example 3 of the present invention.

[0091] Figure 12 Humoral immune results of the varicella mRNA vaccine in Example 4 of the present invention.

[0092] Figure 13 Cellular immune results of the varicella mRNA vaccine in Example 4 of the present invention. Detailed implementation manners

[0093] To make the objectives, technical solutions and advantages of the present disclosure clearer and more understandable, the following further details the present disclosure in combination with embodiments. The specific embodiments described herein are only used to explain the present disclosure and do not constitute any limitation to the present disclosure. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0094] Terms

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0096] In the present application, the term "alkyl" refers to an aliphatic hydrocarbon group, which can be a branched or straight-chain alkyl. In the present application, for example, "C1-C20 straight-chain alkyl" includes, but is not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl, etc. "C3-C30 branched-chain alkyl" includes, but is not limited to, isopropyl, neopentyl, isopentyl, etc.

[0097] Lipid nanoparticles (LNPs) have a structure that includes a single monolayer or bilayer of lipids encapsulating a compound in a solid phase. Different from liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phases inside, but the lipids from the bilayer or monolayer shell directly complex with the internal compound, thus encapsulating it in a solid core. Lipid nanoparticles are generally spherical vesicles with a relatively uniform shape and size distribution. It is generally believed that the diameter of lipid nanoparticles can be in the range of 10 nm to 1000 nm.

[0098] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include ionizable lipids, which can complex and associate with the negatively charged backbone of the nucleic acid core. At physiological pH values, the lipid nanoparticles can adopt a relatively neutral exterior, thus significantly increasing the circulating half-life of the particles after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles have many advantages compared to other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0099] Drug compositions can be formulated for administration by specific routes of administration. For example, drug compositions can be formulated for intravenous, intratumoral, intraperitoneal, intradermal, subcutaneous, intranasal, or other routes of administration.

[0100] Dosage forms of the drug compositions of the present application include (but are not limited to): injections, aerosols, dripping pills, topical liniments, controlled-release or sustained-release or nano-formulations.

[0101] As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, delivery of mRNA encompasses cases where mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and cases where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), and is systemically distributed and absorbed by other tissues (also referred to as "systemic delivery"). "Local delivery" refers to tissue-specific delivery or distribution. Generally, local delivery requires the protein or peptide encoded by the mRNA to be translated and expressed intracellularly or with limited secretion, which avoids entering the patient's circulatory system. The term "target tissue" refers to any tissue affected by the disease to be treated. In some embodiments, the target tissues include those that exhibit disease-related pathology, symptoms, or characteristics.

[0102] In the present application, "pharmaceutical carrier, excipient or diluent" refers to any component other than the compounds described herein (e.g., a medium capable of suspending, complexing or dissolving the active compound) and having substantially non-toxic and non-inflammatory properties in a patient. Excipients may include, for example: anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose sodium, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0103] The lipid compounds of the present invention will be further described below in conjunction with examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each implementation manner of the present invention can be combined with each other as long as they do not conflict with each other. In particular, those skilled in the art can make corresponding modifications and optimizations to the present invention, and these equivalent forms are also included within the scope of protection of the appended claims of this application.

[0104] In the specific examples of the present invention, the raw materials used can all be obtained commercially.

[0105] The lipid compound prepared by the preparation method of Example 1 below.

[0106] Synthesis route of the fully biodegradable ionizable lipid in Example 1

[0107] 1: Synthesis route of compound SV-101

[0108]

[0109] (1) Dissolve tris(hydroxymethyl)aminomethane (1 mmol) and di-tert-butyl dicarbonate (1.2 mmol) in a mixed solvent of MeOH and H2O, react at room temperature for 12 h, concentrate under reduced pressure, extract, and separate by column chromatography to obtain SV-101-1 with a yield of 89%.

[0110] (2) Dissolve SV-101-1 (1 mmol), linoleic acid (2 mmol), EDCI (1.1 mmol), and DMAP (1.2 mmol) in DCM, react at room temperature, filter, extract, and separate by column chromatography to obtain SV-101-2 with a yield of 78%.

[0111] (3) Dissolve SV-101-2 (1 mmol) in DCM, add TFA (1 mL) dropwise thereto under an ice bath, stir the mixture at room temperature for 5 h, extract, and separate by column chromatography (dichloromethane:methanol = 10:1) to obtain SV-101 with a yield of 92%.

[0112] After testing, the 1H NMR spectrum of SV-101 is as shown in Figure 1 and the mass spectrum is as shown in Figure 2 .

[0113] 2: Synthetic route of compound SV-201

[0114]

[0115] (1) Dissolve SV-101-1 (1 mmol), linoleic acid (4 mmol), EDCI (1.1 mmol), and DMAP (1.2 mmol) in DCM, react at room temperature, filter, extract, and separate by column chromatography to obtain SV-201-1 with a yield of 75%.

[0116] (2) Dissolve SV-201-1 (1 mmol) in DCM, add TFA (1 mL) dropwise thereto under an ice bath, stir the mixture at room temperature for 5 h, extract, and separate by column chromatography (dichloromethane:methanol = 20:1) to obtain SV-201 with a yield of 90%.

[0117] After testing, the 1H NMR spectrum of SV-201 is as shown in Figure 3 and the mass spectrum is as shown in Figure 4 .

[0118] 3: Synthetic route of compound SV-202

[0119]

[0120] (1) Add TBDMS-Cl (1.1 mmol) to a reaction flask containing tris(hydroxymethyl)aminomethane (1 mmol), imidazole (2 mmol) and DMF (40 mL), stir overnight at room temperature, pour into water, extract and separate by column chromatography to obtain SV-202-1 with a yield of 75%.

[0121] (2) Dissolve SV-202-1, boc-protected arginine (1.5 mmol), HATU (1.5 mmol), and DIPEA (4 mmol) in DCM and react at room temperature for 5 hours. Dilute with ice-cold water, extract and separate by column chromatography to obtain SV-202-2 with a yield of 62%.

[0122] (3) Add tetrabutylammonium fluoride (TBAF, 1 M THF solution) (2 mmol) to a solution of SV-202-2 (1 mmol) in THF, stir the mixture at room temperature for 18 hours, concentrate the reaction solution under reduced pressure, and separate by column chromatography to obtain SV-202-3 with a yield of 85%.

[0123] (4) Dissolve SV-202-3 (1 mmol), linoleic acid (4 mmol), EDCI (1.1 mmol), and DMAP (1.2 mmol) in DCM, react at room temperature, filter, extract and separate by column chromatography to obtain SV-202-4 with a yield of 81%.

[0124] (5) Dissolve SV-202-4 (1 mmol) in DCM, add TFA (1 mL) dropwise thereto under an ice bath, stir the mixture at room temperature for 5 hours, extract and separate by column chromatography (dichloromethane:methanol = 20:1) to obtain SV-202 with a yield of 93%.

[0125] After testing, the 1H NMR spectrum of SV-202 is as Figure 5 shown, and the mass spectrum is as Figure 6 shown.

[0126] 4: Synthetic route of compound SV-203

[0127]

[0128] Synthesis steps of SV-203:

[0129] (1) Dissolve SV-201 (1 mmol), boc-protected histidine (1.5 mmol), HATU (1.5 mmol), and DIPEA (4 mmol) in DCM and react at room temperature for 4 hours. Dilute with ice-cold water, extract and separate by column chromatography to obtain SV-203-1 with a yield of 62%.

[0130] (2) Dissolve SV-203-1 (1 mmol) in dry DCM, add 6 mol / L hydrogen chloride dioxane solution (6 mmol), react at room temperature for 5 hours, concentrate the reaction system, and separate by column chromatography (methylene chloride: methanol = 20:1) to obtain SV-203 with a yield of 93%.

[0131] After testing, the 1H NMR spectrum of SV-203 is as shown in Figure 7 the following figure, and the mass spectrum is as shown in Figure 8 the following figure.

[0132] 5: Synthetic route of compound SV-204

[0133]

[0134] Synthesis steps of SV-204:

[0135] (1) Dissolve SV-202-1 (1 mmol), boc-protected lysine (1.2 mmol), EDC (1.5 mmol), and DMAP (1.1 mmol) in DCM, react at room temperature for 4 hours. Dilute with ice-cold water, extract, and separate by column chromatography to obtain SV-204-1 with a yield of 76%.

[0136] (2) Add tetrabutylammonium fluoride (TBAF, 1 M THF solution) (2 mmol) to the solution of SV-204-1 (1 mmol) in THF, and stir the mixture at room temperature for 20 hours. After concentrating the reaction solution under reduced pressure, separate by column chromatography to obtain SV-204-2 with a yield of 93%.

[0137] (3) Dissolve SV-204-2 (1 mmol), linoleic acid (4 mmol), EDCI (1.1 mmol), and DMAP (1.2 mmol) in DCM, react at room temperature, filter, extract, and separate by column chromatography to obtain SV-204-3 with a yield of 85%.

[0138] (4) Dissolve SV-204-3 (1 mmol) in DCM, add TFA (1 mL) dropwise thereto under ice bath, stir the mixture at room temperature for 5 hours, extract, and separate by column chromatography (methylene chloride: methanol = 20:1) to obtain SV-204 with a yield of 87%.

[0139] After testing, the 1H NMR spectrum of SV-204 is as shown in Figure 9 the following figure, and the mass spectrum is as shown in Figure 10 the following figure.

[0140] The synthesis processes of other compounds are basically the same as those of the above compounds, only some process conditions need to be adjusted and some substrates need to be replaced.

[0141] Example 2 Preparation and Characterization of Nanoliposome Particles

[0142] Step 1: Prepare completely biodegradable mRNA liposome nanoparticles. The ionizable lipid compound purified in Example 1 is dissolved in ethanol with DSPC (Avitide (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Avitide (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG 2000 (Avitide (Shanghai) Pharmaceutical Technology Co., Ltd.) according to a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution, and the total lipid concentration in the solution is 3.64 mg / mL.

[0143] Step 2: Dilute mRNA in 10 - 50 mM citrate buffer (pH = 4) to obtain an mRNA aqueous solution according to the mass ratio of lipid nanoparticles (LNP) to mRNA encoding the extracellular domain of varicella-zoster virus gE (Wang Y, et al., Vaccines (Basel). 2023 Sep 11;11(9):1475.) of 10 - 30:1. The final concentration of mRNA in the solution is 0.133 mg / mL.

[0144] Step 3: Use a microfluidic device (Myanna (Shanghai) Instrument Technology Co., Ltd.) to mix the ethanol lipid solution and the mRNA aqueous solution at a total flow rate of 12 mL / min and a volume ratio of 1:3. The prepared LNP sample is dialyzed for 12 - 24 h to remove ethanol and replaced with 10 mM Tris-HCl (PH = 7.5). Finally, the lipid nanoparticles are sterilized and filtered through a 0.2 μm filter.

[0145] Step 4: Use Malvern Zetasizer Nano ZS to characterize the particle size and polydispersity index (PDI) of mRNA-LNP (lipid nanoparticles encapsulating mRNA), and then use Ribogreen RNA Quantitation Kit (Thermo Fisher) to measure the encapsulation efficiency of mRNA. The results are shown in Table 1, indicating that the ionizable lipid compound of the present invention can form a stable nanoparticle structure with a narrow size distribution.

[0146] Table 1: Particle Size, PDI, and Encapsulation Efficiency of Different Lipid Samples

[0147]

[0148]

[0149] Example 3 Influence of Lipid Structure on In Vivo Transfection Efficiency of Lipid Nanoparticles

[0150] Female BALB / c mice (6 - 8 weeks old, Spf (Beijing) Biotechnology Co., Ltd.) were housed under experimental conditions of 22 ± 2 °C and a relative humidity of 45 - 75%, with a 12-hour light / dark cycle. Complete biodegradable luci-mRNA lipid nanoparticles were prepared in the same manner as in Example 2, where the mRNA was replaced with mRNA encoding luciferase (genbank accession number: KJ561464). They were injected into BALB / c mice via intramuscular injection at a dose of 0.25 mg / kg. Six hours later, 100 μL of a 40 mg / mL D-luciferin solution was injected intraperitoneally; imaging was performed using a small animal in vivo imager to detect the in vivo delivery efficiency.

[0151] Figure 11 For the completely biodegradable luci-mRNA lipid nanoparticles SV-201, SV-202, SV-203, SV-204, SV-211, SV-212, SV-213, and SV-217, with a dose of 0.25 mg / kg, the imaging results in BALB / c mice after intramuscular injection into the hind limb are shown. The results indicate that Figure 11 As can be seen, the lipid nanoparticles of the present invention can efficiently deliver nucleic acids into animals, and basically do not have liver targeting, and the compound SV-203 has a better in vivo delivery efficiency than the commercially available SM102 in Example 2.

[0152] Example 4 Effect of Lipid Structure on the Immunogenicity of Herpes Zoster Virus mRNA Vaccine

[0153] mRNA encoding the gE protein of herpes zoster virus was synthesized, and the herpes zoster virus mRNA vaccine was prepared in the same manner as in Example 2 (Wang Y, et al., Vaccines (Basel). 2023 Sep 11;11(9):1475.). Female C57BL / 6 mice at 6 - 8 weeks old were selected, with an immunization dose of 10 μg / mouse and an immunization schedule of 0 / 21 days. Mouse sera were collected on the 49th day after immunization to detect the binding antibody titer; on the 49th day, mouse spleens were collected, splenocytes were isolated, and the splenocytes were stimulated with a peptide library for 24 hours, and the cellular immune levels (IFN-γ and IL-2) were detected by ELISPOT.

[0154] Figure 12The results showed that both the SM-102 control group and other experimental groups could produce specific binding antibodies against the gE protein, and the binding antibody titers of the experimental groups were significantly higher than those of the negative control group, indicating that the experimental groups could efficiently deliver mRNA into cells, express antigens, and then stimulate an immune response in vivo, produce corresponding antibodies, and play a protective function. Moreover, after immunizing mice with the same dose, the level of humoral immune response induced by the compound SV-203 was significantly higher than that of the commercially available SM102 control group, and there was no statistical difference in the binding antibody titers produced by the other compounds and the control group.

[0155] Figure 13 The results showed that after immunizing mice with the same dose, both the SM-102 control group and other experimental groups could induce the production of high levels of IFN-γ cytokine and IL-2 cytokine. And there was no statistical difference in the level of IFN-γ cytokine induced by the compound SV-203 and the commercially available SM102 control group; the level of IL-2 cytokine induced by the compound SV-203 was significantly higher than that of the commercially available SM102 control group. Therefore, the compound SV-203 could simultaneously stimulate a higher humoral immune and cellular immune response.

[0156] The technical solutions of the present disclosure are not limited to the limitations of the above specific embodiments, and any technical deformation made according to the technical solutions of the present disclosure falls within the protection scope of the present disclosure.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, said compound having the structure shown in formula (I): Wherein, A1 is selected from hydrogen or a residue of a natural amino acid, wherein the -C=O- in the residue of the natural amino acid is bonded to the -NH in formula (I) to form an amide bond; A2 does not exist, or is selected from a hydroxyl group or R1, R2 or R3 are each independently selected from a residue of a natural fatty acid or a residue of a natural fatty acid ester; m is selected from an integer of 0 to 3, n and q are each independently selected from an integer of 1 to 3.

2. The compound according to claim 1, wherein The natural amino acid is selected from non-polar hydrophobic amino acids, polar neutral amino acids, acidic amino acids or basic amino acids; Preferably, the non-polar hydrophobic amino acids are selected from alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), methionine (Met), tryptophan (Trp) or phenylalanine (Phe); Preferably, the polar neutral amino acids are selected from glutamine (Gln), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn), tyrosine (Tyr) or glycine (Gly); Preferably, the acidic amino acids are selected from aspartic acid (Asp) or glutamic acid (Glu); Preferably, the basic amino acids are selected from lysine (Lys), arginine (Arg) or histidine (His).

3. The compound according to claim 1 or 2, characterized in that, R1, R2 or R3 are each independently selected from a residue obtained by removing the terminal carboxyl group from a natural fatty acid or a natural fatty acid ester; Preferably, R1, R2 or R3 are each independently selected from a residue of a C12-C30 natural fatty acid or a residue of a C12-C30 natural fatty acid ester; Preferably, the natural fatty acid or natural fatty acid ester is selected from the compounds shown below: Wherein, e, f, j, h, g, k are each independently selected from an integer of 1 to 30, i is selected from an integer of 1-5, L1 is selected from -C=O-, -C(=O)O-, -OC(=O)O- or -OC(=O)-, R4 and R5 are each independently selected from a C1-C20 straight-chain alkyl group or a C3-C30 branched-chain alkyl group, and R6 is selected from a C3-C30 branched-chain alkyl group; Preferably, the natural fatty acid or natural fatty acid ester is selected from the compounds shown below: Preferably, R1, R2 or R3 are each independently selected from a residue of linoleic acid, oleic acid, stearic acid or monooctyl sebacate.

4. The compound according to any one of claims 1-3, characterized in that, The compound has the structure shown in formula (II): The definition of A1 is as described in claim 1 or 2. Preferably, A1 is selected from hydrogen, a histidine residue, an arginine residue or a lysine residue, a glycine residue, a leucine residue, an isoleucine residue or a serine residue.

5. The compound according to any one of claims 1-4, characterized in that, The compound is selected from:

6. A lipid nanoparticle, comprising the compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, a steroidal compound, a polyethylene glycol lipid and a neutral helper phospholipid, Preferably, the average particle size of the lipid nanoparticle is 50nm - 200nm, preferably 70nm - 150nm; Preferably, the polydispersity index (PDI) of the lipid nanoparticle is 0.05 - 0.3, preferably 0.1 - 0.

25.

7. The lipid nanoparticle according to claim 6, wherein, The steroidal compounds include at least one of cholesterol, coprosterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids and their derivatives, preferably cholesterol; and / or The polyethylene glycol lipids include at least one of phosphatidylethanolamine modified with polyethylene glycol, phosphatidic acid modified with polyethylene glycol, ceramide modified with polyethylene glycol, dialkylamine modified with polyethylene glycol, diacylglycerol modified with polyethylene glycol, and dialkylglycerol modified with polyethylene glycol, preferably DMG-PEG 2000 ; and / or The neutral auxiliary phospholipids include at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin (SM), ceramide, sterol and their derivatives, preferably DSPC, DOPC or DOPE; Preferably, the molar ratio between the compound or its pharmaceutically acceptable salt, the steroidal compound, the neutral auxiliary phospholipid and the polyethylene glycol lipid is (40-60):(22-60):(5-30):1.5, preferably (45-50):(25-55):(10-20):1.

5.

8. Use of the compound or its pharmaceutically acceptable salt according to any one of claims 1-5 or the lipid nanoparticle according to claim 6 or 7 in the delivery of a drug active ingredient, Preferably, the drug active ingredient is selected from nucleic acids, small molecule compounds, polypeptides and / or proteins; Preferably, the nucleic acid is selected from one or more of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA and plasmid; Preferably, the drug active ingredient is encapsulated in the lipid nanoparticle or electrostatically bound to the lipid nanoparticle.

9. A pharmaceutical composition comprising the lipid nanoparticle according to claim 6 or 7, and a drug active ingredient; Preferably, the pharmaceutical composition further comprises an adjuvant; More preferably, the adjuvant comprises any one or a combination of at least two of a pharmaceutical carrier, an excipient or a diluent; More preferably, the drug active ingredient is selected from nucleic acids, small molecule compounds, polypeptides and / or proteins; Even more preferably, the nucleic acid is selected from one or more of DNA, RNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, cicleRNA and plasmid; Further preferably, the pharmaceutically active ingredient is encapsulated within the lipid nanoparticles or electrostatically bound to the lipid nanoparticles.

10. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, a lipid nanoparticle according to claim 6 or 7, or a pharmaceutical composition according to claim 9 for the preparation of a vaccine or a non-viral gene vector.

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