WO2024067639A1

WO2024067639A1 - Ionizable lipid compound having high transfection efficiency and use thereof

Info

Publication number: WO2024067639A1
Application number: PCT/CN2023/121746
Authority: WO
Inventors: 章雪晴; 滕以龙; 陈起静
Original assignee: 荣灿生物医药技术(上海)有限公司
Priority date: 2022-09-30
Filing date: 2023-09-26
Publication date: 2024-04-04

Abstract

The present invention relates to the field of biomedicine. Disclosed are an ionizable lipid compound having high transfection efficiency and a use thereof. The ionizable lipid compound is a compound having the following structure: formula (I), wherein n=0-10; G₁ and G₂ are each independently an alkylene group; R₁, R₂, R₃, and R₄ are each independently an alkane group, an olefin group, or H; G₃ is an alkylene group or formula (II). A lipid nanoparticle carrier prepared from the compound of the present invention has good cytocompatibility in a physiological environment, can promote endosomal escape in an intracellular endosome acidic environment, has unexpected progress in improving the transfection effect and has high safety, and is suitable for biomedical industrialization.

Description

An ionizable lipid compound with high transfection efficiency and its application

Technical Field

The invention relates to the field of biomedicine, in particular to an ionizable lipid compound with high transfection efficiency and application thereof.

Background technique

Gene therapy is a treatment method that corrects or compensates for diseases caused by defective or abnormal genes by introducing exogenous genes into target cells. Nucleic acid vaccines, also known as genetic vaccines, refer to the introduction of nucleic acid sequences (such as DNA, mRNA, etc.) encoding immunogenic proteins or polypeptides into the host, and the expression of immunogenic proteins or polypeptides by host cells to induce host cells to produce immune responses to the immunogens, so as to achieve the purpose of preventing and treating diseases. Among them, ensuring the smooth introduction of exogenous genes is an extremely important part of the gene therapy process and gene vaccine immunization. Among the many methods of gene introduction, the method of developing suitable lipid nanoparticles (LNP) to encapsulate nucleic acids, target them to target cells, and deliver nucleic acids of specific genes into cells has gradually been applied.

The LNP system mainly includes four components: ionizable lipids, structural lipids, co-lipids and polymer-conjugated lipids. Among them, ionizable lipids refer to lipid molecules that are positively charged at acidic pH values and neutral at physiological pH values, which can affect the surface charge of LNP under different pH conditions. This charge state can affect its immune recognition, blood clearance and tissue distribution in the blood, as well as its ability to escape from endosomes in cells, which is crucial for the intracellular delivery of nucleic acids.

When LNP enters the body through different administration routes, it becomes pH-sensitive under the influence of ionizable lipid compounds. The pH of body fluids is 7.4, and at this time, LNP is preferably electrically neutral, and needs to have the best stability in the biological system to avoid immune clearance; after LNP carrying nucleic acid (such as mRNA) enters the cell through endocytosis, it is trapped in acidic vesicles (endosomes), and the acidic medium in the endosomes makes the pH environment acidic, with a pH value of about 5.5. The core component of LNP, ionizable lipid compounds, is protonated in an acidic environment, destroying the endosomal membrane, thereby allowing mRNA to escape from the endosomal.

Therefore, there is a need in the art to develop an ionizable lipid compound for preparing LNPs that is safe and stable in a neutral environment, appropriately promotes membrane disruption in an acidic environment (pH = 3-5.5), and has better nucleic acid endosome escape ability.

Summary of the invention

In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide an ionizable lipid compound with high transfection efficiency and its application. The mRNA-LNP prepared by the ionizable lipid compound with a new structure of the present invention has good nucleic acid endosomal escape ability, high transfection efficiency and high stability.

In order to achieve the above object, the present invention adopts the following technical solution:

An ionizable lipid compound with high transfection efficiency, characterized in that the compound has the following structure:

Wherein, n=an integer from 0 to 10;

G ₁ and G ₂ are each independently C ₁ -C ₁₀ alkylene;

R ₁ , R ₂ , R ₃ , and R ₄ are each independently a C ₁ -C ₂₀ alkane group, a C ₂ -C ₂₀ alkene group, or H; when R ₁ is H, R ₂ is a C ₂ -C ₂₀ alkene group; when R ₃ is H, R ₄ is a C ₂ -C ₂₀ alkene group;

G ₃ is C ₁ -C ₁₀ alkylene, or wherein a and b are each independently an integer of 1-9, and a+b=an integer of 2-10.

In the aforementioned ionizable lipid compound with high transfection efficiency, _G1 is a _C6 alkylene group, _G2 is a _C5 - _C7 alkylene group, and preferably _G2 is a _C7 alkylene group.

The aforementioned ionizable lipid compound with high transfection efficiency, _G3 is _C2-4 alkylene, or

In the aforementioned ionizable lipid compound with high transfection efficiency, as an embodiment, _G1 is _C6 alkylene, _G2 is _C5 - _C7 alkylene, preferably _G2 is _C7 alkylene, _G3 is _C2-4 alkylene, and _R1 , _R2 , _R3 , and _R4 are each independently _C1 - _C20 alkylene.

In the aforementioned ionizable lipid compound with high transfection efficiency, as an embodiment, _G1 is _C6 alkylene, _G2 is _C5 - _C7 alkylene, preferably _G2 is _C7 alkylene, _G3 is _C2-4 alkylene, _R1 and _R2 are each independently _C1 - _C20 alkane, _R3 is H, and _R4 is _C2 - _C20 alkene.

The above-mentioned ionizable lipid compound with high transfection efficiency is, as an embodiment, _G1 is _C6 alkylene, _G2 is _C5 - _C7 alkylene, preferably _G2 is _C7 alkylene, _G3 is R ₁ , R ₂ , R ₃ and R ₄ are each independently a C ₁ -C ₂₀ alkane group.

The aforementioned ionizable lipid compound with high transfection efficiency is, as a preferred embodiment, a compound structure as shown below:

The aforementioned ionizable lipid compound with high transfection efficiency is used as an example, wherein Each is independently a structure selected from the following group:

The aforementioned ionizable lipid compound with high transfection efficiency is used as an example, wherein in Formula I Each is independently a structure selected from the following group:

It should be noted that the structure of the hydrophobic group is not limited, and the number and position of the substituents on the alkane group are also not limited. As long as the compound uses the N atom as the charge center, the hydrophilic group as the head, and the two hydrophobic groups as the tail, and introduces -(C＝O)O- and a carbonate bond as a linker between the N atom and the two hydrophobic groups, it can be used to achieve the purpose of the present invention.

In a preferred embodiment, the ionizable lipid compound with high transfection efficiency, its stereoisomers, its tautomers or pharmaceutically acceptable salts thereof can be used to prepare a pharmaceutical composition.

In a more preferred embodiment, the pharmaceutical composition may comprise: a carrier containing the ionizable lipid compound, a pharmaceutical agent carried therein, a pharmaceutical adjuvant, or a combination thereof.

In the application of the aforementioned ionizable lipid compound with high transfection efficiency, the carrier further includes: one or a combination of auxiliary lipids, structural lipids, polymer-conjugated lipids or amphiphilic block copolymers; it should be noted that: the components of the carrier composition are not limited, and can be a composition composed of existing known substances or a composition composed of unknown substances. As long as the ionizable lipid compound adopts the structure of the present invention, it is within the protection scope of the present invention and is inspired by the present invention.

Lipid aids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), ceramide, A combination of one or more of the charged lipids; phosphatidylcholine as a preferred one includes: DSPC, DPPC, DMPC, DOPC, POPC; phosphatidylethanolamine as a preferred one is DOPE; charged lipids refer to a class of lipid compounds that exist in the form of positive or negative charges; their charge does not depend on the pH within the physiological range, such as pH 3-9, and is not affected by pH. Charged lipids can be synthetic or naturally derived. Examples of charged lipids include, but are not limited to, DOTAP, DOTMA, 18PA. The examples here are not exhaustive, and any co-lipid can be applied to the present invention.

The structural lipids include, but are not limited to, one or more of sterols and their derivatives, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, α-tocopherol or corticosteroids. Sterols are preferably cholesterol; this is not exhaustive, the selection of structural lipids is not limited, and any structural lipid can be applied to the present invention.

As an example, the polymer-conjugated lipid is a PEGylated lipid; the PEGylated lipid includes: one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol. This is not exhaustive, the selection of polymer-conjugated lipids is not limited, and any polymer-conjugated lipid can be applied to the present invention.

As an embodiment, the amphiphilic block copolymer may include: a combination of one or more of polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyorthoester, polyanhydride, poly(β-amino ester) (PBAE) or polyethylene glycol (PEG) modified amphiphilic block copolymers. It should be noted that the examples here are not exhaustive, and any amphiphilic block copolymer can be applied to the present invention.

As an example, when used to prepare a composition containing an ionizable lipid compound, the molar ratio of the ionizable lipid compound of the present invention to the lipid aid is 0.5:1-10:1.

As an example, when used to prepare a preparation containing an ionizable lipid compound, the molar ratio of the ionizable lipid compound of the present invention to the structural lipid is 0.5:1-5:1.

As an example, when used to prepare a composition containing an ionizable lipid compound, the molar ratio of the ionizable lipid compound of the present invention to the polymer-conjugated lipid is 10:1-250:1.

As an example, when used to prepare a composition containing an ionizable lipid compound, the molar ratio of the ionizable lipid compound of the present invention to the amphiphilic block copolymer is 0.5:1-80:1.

As an embodiment, the carrier is a lipid nanoparticle (LNP), the average particle size of the lipid nanoparticle is 30-200nm, and the polydispersity index of the nanoparticle preparation is ≤0.5. It should be noted that any nanoparticles prepared from one or more ionizable lipid compounds are within the scope of this patent and are inspired by the present invention; for example, in addition to lipid nanoparticles, there may also be hybrid nanoparticles formed by one or more ionizable lipid compounds and polymers, such as PLGA-PEG, PLA-PEG, PCL, PBAE (Polyβ-amino acid), etc., which are not listed here.

In the technical solution of the present invention, there is no specific limitation on the drug agents contained therein, including but not limited to: one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins; the selection and combination formula of the drugs contained therein are not limited, as long as the ionizable lipid compounds using the structure of the present invention are within the protection scope of the present invention and are inspired by the present invention.

Small molecule compounds can be active ingredients in therapeutic or preventive agents, such as anti-tumor drugs, anti-infective drugs, local anesthetics, antidepressants, anticonvulsants, antibiotics/antibacterial agents, antifungal drugs, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents, etc., which are not exhaustive.

Polypeptides are compounds formed by α-amino acids linked together by peptide bonds and are intermediate products of protein hydrolysis.

Protein is a substance with a certain spatial structure formed by the coiling and folding of polypeptide chains composed of amino acids in a "dehydration condensation" manner; protein can be interferon, protein hormone, cytokine, chemokine or enzyme, etc.

Pharmaceutical adjuvants include, but are not limited to: one or more of diluents, stabilizers, preservatives or freeze-drying protective agents. This is not an exhaustive list, as long as the ionizable lipid compound of the structure of the present invention is used, no matter which pharmaceutical adjuvant is used for compounding, it is within the protection scope of the present invention and is inspired by the present invention.

The diluent is any pharmaceutically acceptable water-soluble excipient known to those skilled in the art, including but not limited to: amino acids, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, other oligosaccharides, mannitol, dextran, sodium chloride, sorbitol, polyethylene glycol, phosphates, or derivatives thereof.

The stabilizer can be any pharmaceutically acceptable excipient known to those skilled in the art, including but not limited to Tween-80, sodium lauryl sulfate, sodium oleate, mannitol, mannose or sodium alginate.

The preservative can be any pharmaceutically acceptable preservative known to those skilled in the art, an exemplary representative of which is thimerosal.

The lyoprotectant can be any pharmaceutically acceptable lyoprotectant known to those skilled in the art, and exemplary representatives include glucose, mannitol, sucrose, lactose, trehalose, maltose, and the like.

Compared with the prior art, the present invention is beneficial in that:

1. The ionizable lipid compound of the present invention has a novel structure, with N atom as the charge center, a hydrophilic group as the head, and two hydrophobic groups as the tail. -(C=O)O- and a carbonate bond are introduced as linkers between the N atom and the two hydrophobic groups. Such a structure has low destructive effect on cell membranes in a neutral environment and is highly safe.

2. After the ionizable lipid compound of the present invention enters the cell, it has a higher effect of destroying the endosomal membrane in the acidic environment of the endosomal membrane. Compared with commercial products, it has a stronger endosomal escape ability and a faster escape rate, thereby producing a higher transfection efficiency;

3. The ionizable lipid compound of the present invention has high biocompatibility.

4. The synthesis steps of the ionizable lipid compound of the present invention are simple and suitable for biopharmaceutical industrialization.

5. The ionizable lipid compound of the present invention can be stored stably for a long time, with little change in key parameters, and can be transported and commercialized. Low storage cost.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a fluorescence schematic diagram of LNP composed of compounds H1-H16 and h1-1, h1-2, h2-1, and h2-2 after transfection of Luciferase mRNA in Experiment 2 of the present invention;

FIG2 is a schematic diagram of the experimental results of samples in a neutral pH environment in the endosomal escape ability experiment of the present invention;

FIG3 is a schematic diagram of the experimental results of samples in an acidic pH environment in the endosomal escape ability experiment of the present invention;

FIG4 is a schematic diagram of the experimental results of the sample in the endosomal escape rate experiment in the present invention in an acidic pH environment;

FIG5 is an electron micrograph of lipid nanoparticles prepared using the compound of sample H-3 of the present invention;

FIG6 is a schematic diagram showing the results of an experimental comparison of the immune effects of LNPs prepared from the ionizable lipid compound of the present invention and LNPs on the market.

Explanation of terms and English abbreviations:

Nucleic acid is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is a biological macromolecule composed of multiple nucleotide monomers; nucleic acid is composed of nucleotides, and nucleotide monomers are composed of pentose, phosphate, nitrogenous base, or any modified group. If the pentose is ribose, the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA.

The "nucleic acid" includes but is not limited to single-stranded DNA, double-stranded DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA (Telomerase RNA Component), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense RNA, ribozyme, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotide, morpholino oligonucleotide or biological custom oligonucleotide one or more combinations. The examples here are not exhaustive, as long as they are polymerized from nucleotide monomers, they can be applied to the present invention.

mRNA, messenger RNA, Chinese translation: messenger ribonucleic acid, is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis. mRNA can be monocistronic mRNA or polycistronic mRNA. mRNA can also contain one or more functional nucleotide analogs, examples of which include: pseudouridine, 1-methyl-pseudouridine or 5-methylcytosine. The examples here are not exhaustive, and any modified mRNA or its derivatives can be applied to the present invention.

In the claims of the present invention, when describing "C1-C20 alkane group", it means that the group may be an alkane group having 1-20 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms), and the alkane group is a saturated alkane group, which may be a straight chain or have a branched structure, satisfying All alkane groups having the aforementioned number of carbon atoms are within the scope of the description of this term.

When describing "C2-C20 olefin group", it means that the group can be an olefin group with 2-20 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms), and the olefin group can be a straight chain or have a branched structure. The olefin groups satisfying the aforementioned number of carbon atoms are all within the scope of the term description. In different embodiments of the present invention, the olefin group can be a monoolefin or a polyolefin (such as a diene).

When describing "C1-C10 alkylene", it means that the group may be an alkylene group having 1 to 10 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), and the alkylene group may be a linear or branched structure.

Pharmaceutically acceptable salts refer to acid addition salts or base addition salts.

The acid of the acid addition salt includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acid phosphate, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, gluconic acid, glucanic acid, gluconic ... Uronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-dicarboxylic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, quaternary ammonium acid, and undecylenic acid.

Examples of base addition salts include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts; organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins; preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

DSPC: English name: Distearoyl Phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine; Chinese name: Distearoyl Phosphatidylcholine, CAS number: 816-94-4.

DPPC: Chinese name: Dipalmitoylphosphatidylcholine; English name: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHOCHOLINE, CAS number: 63-89-8.

DMPC: Chinese name: Dimyristoylphosphatidylcholine; English name: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine, CAS number: 18194-24-6.

DOPC: Chinese name: 1,2-dioleoyl-sn-glycero-3-phosphocholine; English name: 1,2-dioleoyl-sn-glycero-3-phosphocholine, CAS number: 4235-95-4.

POPC: Chinese name: 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine; English name: 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, CAS number: 26853-31-6.

DOPE: Chinese name: 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine; English name: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE, CAS number: 4004-05-1.

DOTAP: Chinese name: (1,2-dioleoylpropyl) trimethylammonium chloride; English name: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt), CAS number: 132172-61-3; chemical structure is as follows:

DOTMA: Chinese name: N,N,N-trimethyl-2,3-bis(octadec-9-en-1-yloxy)propan-1-ammonium chloride, CAS number: 1325214-86-5, chemical structure is as follows:

18PA: CAS No.: 108392-02-5, chemical structure is as follows:

SM: Chinese name: sphingomyelin (SM); English name: sphingomyelin.

PEG: Chinese name: polyethylene glycol; English name: Polyethylene glycol.

Detailed ways

The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available.

The ionizable lipid compound was prepared by the preparation method of Example 1 below.

Embodiment 1:

Synthesis of compound a: 6-bromohexanoic acid (10.00 g, 51.27 mmol), dicyclohexylcarbodiimide (DCC, 12.69 g, 61.52 mmol), 4-dimethylaminopyridine (1.25 g, 10.25 mmol) were dissolved in 200 mL of dichloromethane (DCM), 6-2-hexyldecanol (12.43 g, 51.27 mmol) was added, and the reaction was stirred at room temperature for 12 h. After TLC monitoring of the reaction was complete, the solvent was removed by vacuum distillation using a rotary evaporator. 300 mL of ethyl acetate was added, and the mixture was washed twice with an equal volume of saturated sodium bicarbonate solution, washed once with an equal volume of saturated sodium chloride solution, dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by vacuum distillation using a rotary evaporator. Column separation and purification (silica gel column, eluent PE: EA = 100: 1 (volume ratio)) was obtained to obtain 19.78 g of colorless liquid with a yield of 92%.

Synthesis of compound b: Compound a (10.00 g, 23.84 mmol), 2-(2-aminoethoxy)ethanol (5.01 g, 47.68 mmol), N,N-diisopropylethylamine (DIPEA, 3.70 g, 28.61 mmol) were dissolved in 100 mL of ethanol (EtOH), and the mixture was stirred at 60 °C for 12 h. After the reaction was complete as monitored by TLC, the solvent was removed by vacuum distillation using a rotary evaporator. 200 mL of ethyl acetate was added, and the mixture was washed three times with an equal volume of saturated sodium chloride solution, dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by vacuum distillation using a rotary evaporator. The mixture was purified by column separation (silica gel column, eluent DCM: MeOH = 20: 1 (volume ratio)), and 7.51 g of light yellow liquid was obtained, with a yield of 71%.

Synthesis of compound c: 6-bromohexanol (10 g, 55.23 mmol) and 4-dimethylaminopyridine (6.75 g, 55.23 mmol) were dissolved in 200 mL of dichloromethane (DCM), nitrogen protection, ice bath stirring for 10 min, and then p-nitrochloroformic acid phenyl ester (13.36 g, 66.27 mmol) was added in batches, and the mixture was gradually restored to room temperature. After stirring at room temperature for 3 h, 2-hexyldecanol (14.73 g, 60.75 mmol) was added under ice bath conditions, and the mixture was stirred at room temperature for 12 h. After the reaction was complete under TLC monitoring, the solvent was removed by reduced pressure distillation using a rotary evaporator. Add 400 mL of ethyl acetate, wash twice with an equal volume of saturated sodium bicarbonate solution, wash once with an equal volume of saturated sodium chloride solution, dry over anhydrous sodium sulfate for 30 min, use a rotary evaporator to distill off the solvent under reduced pressure, and purify by column separation (silica gel column, eluent PE:EA=100:1 (volume ratio)) to obtain 20.01 g of colorless liquid, with a yield of 82%.

Synthesis of compound H-1: Compound b (2.04 g, 4.59 mmol), compound c (2 g, 4.59 mmol), and N,N-diisopropylethylamine (DIPEA, 0.71 g, 5.51 mmol) were dissolved in 50 mL of ethanol (EtOH), heated at 60°C with stirring and reacted. After 12 hours, the reaction was completed by TLC monitoring, and the solvent was removed by vacuum distillation using a rotary evaporator. 200 mL of ethyl acetate was added, and the mixture was washed three times with an equal volume of saturated sodium chloride solution, dried over anhydrous sodium sulfate for 30 minutes, and the solvent was removed by vacuum distillation using a rotary evaporator. Column separation and purification (silica gel column, eluent DCM: MeOH = 50: 1 (volume ratio)) was performed to obtain 3.01 g of light yellow liquid, with a yield of 82%. ¹ H NMR (400 MHz, Chloroform-d) δ 4.43 (s, 1H), 4.09 (t, J = 6.8 Hz, 2H), 4.00 (d, J = 5.9 Hz, 2H), 3.94 (d, J = 5.8 Hz, 2H), 3.70–3.63 (m, 2H), 3.64–3.55 (m, 4H), 2.63 (s, 2H), 2.47 (s, 4H), 2.28 (t, J = 7.5 Hz, 2H), 1.70–1.18 (m, 64H), 0.86 (t, J = 6.5 Hz, 12H). MS m/z (ESI): 812.8 [M+H] ⁺ .

By adopting the method of Example 1, compounds H-3 to H-12, comparative sample h1-2, comparative sample h2-1, and comparative sample h2-2 can also be prepared, which will not be described one by one here.

The hydrogen spectrum data of some compounds are shown below:

experiment one:

The mRNA-LNP was prepared for the following experiments. The preparation method was as follows:

Step 1: The ionizable lipid compounds corresponding to H-1-H-16 and comparative samples h1-2, h2-1, and h2-2 in Table 1, DOPE, cholesterol, and PEG-lipid are prepared in a designed formula ratio (Lipid/DOPE/Cholesterol/lipid-PEG is 35/25/38.5/1.5 (molar ratio)) to prepare lipid nanoparticles. The ionizable compound corresponding to the commercial comparative sample h1-1 in Table 1 (Pfizer vaccine BNT162b2) is prepared by its optimal ratio of Lipid/DSPC/Cholesterol/lipid-PEG is 46.3/9.4/42.7/1.6 (molar ratio)) to prepare lipid nanoparticles. The commercial comparative sample MC3 The lipid nanoparticles prepared by the formula ratio (Lipid/DSPC/Cholesterol/DMG-PEG is 50/10/38.5/1.5 (molar ratio)) were dissolved in ethanol (Lipid concentration 20 mg/mL) and mixed thoroughly to obtain an ionizable lipid ethanol solution.

Step 2: Prepare mRNA at a lipid nanoparticle (LNP) to mRNA mass ratio of 10:1 to 30:1, and dilute the mRNA to 0.2 mg/mL using citrate or sodium acetate buffer (pH = 3 or 5).

Step 3: The ionizable lipid ethanol solution obtained in step 1 and the mRNA solution were mixed thoroughly at a volume ratio of 1:5 to 1:1. The obtained nanoparticles were purified by ultrafiltration and dialysis. After filtration and sterilization, the particle size and PDI of mRNA-LNP (lipid nanoparticles encapsulating mRNA) were characterized using Malvern Zetasizer Nano ZS, and the mRNA encapsulation efficiency was determined using Ribogreen RNA quantification kit (Thermo Fisher).

Table 1

Experiment 2: Transfection efficiency verification experiment:

Male ICR mice (6-8 weeks, Shanghai Jiesijie Experimental Animal Co., Ltd.) were housed under experimental conditions of 22±2℃ and relative humidity of 45–75%, with a light/dark cycle of 12h. mRNA encoding luciferase (luciferase mRNA) was used as a reporter gene. Luciferase catalyzes luciferin to produce bioluminescence, and the transfection efficiency of LNP is reflected by detecting the intensity of bioluminescence per unit time. Taking luciferase mRNA as an example, the mRNA-LNP sample H1-16 obtained in Experiment 1 was prepared, and the comparison samples MC3, h1-2, h2-1, and h2-2 were prepared; the above samples were administered by intramuscular injection at a dose of 150μg/kg mRNA, with 2 mice per group of samples, two legs. At a specific time point, luciferin (20μg/mL) was injected intraperitoneally into the mice. After 5 minutes, the mice were placed in a small animal in vivo imager to measure the fluorescence intensity. The final results were expressed as the average fluorescence intensity. The experimental results of fluorescence intensity after intraperitoneal injection of mice are shown in Figure 1 and Table 2.

Table 2

Result analysis:

It can be seen intuitively from the fluorescence intensity of Figure 1 combined with Table 2 that the transfection efficiency of the LNP samples containing H1-H16 of the present invention is improved by 2-3 orders of magnitude compared with the commercially available MC3 sample, and compared with compounds with similar structures (compared with the comparison samples h1-2, h2-1, and h2-2, there are slight differences in the degradable groups), the transfection effect is also significantly different, which can be improved by 1 order of magnitude.

In summary, the mRNA-LNP sample prepared from the ionizable lipid compound of the specific structure of the present invention has a very excellent effect on transfection efficiency.

Experiment 3: Endosomal escape ability experiment

The mRNA escape is mainly achieved because pH-sensitive liposomes promote membrane fusion in the intracellular acidic environment (pH3-5.5). The following experiment simulates the interaction between LNP and cell membrane in a neutral pH environment; and the interaction between LNP and endosomal membrane in the acidic pH environment of intracellular endosomes; thereby verifying the safety and endosomal escape ability of LNP prepared by ionizable lipid compounds.

The experimental process is as follows: Four-week-old female ICR mice, weighing 15-20g, were kept in an experimental environment with a temperature of 22±2℃, a relative humidity of 45-75%, and a light/dark cycle of 12h. After the mice were purchased, they were first adapted to the animal room for one week before formal animal experiments could be carried out. After taking the whole blood of the mice, the mouse blood was centrifuged at 10000g for 5min in a centrifuge, and the mouse red blood cells were separated and rinsed with PBS (pH 7.4) five times. Then, the separated red blood cells were suspended in pH 7.4 and pH5.5 respectively. PBS solution and added to a 96-well plate. Then add the LNP prepared in Experiment 1 with a concentration gradient, sample H-1, comparison sample h1-1, and comparison sample h1-2. Incubate at 37°C for 1 hour, take the sample in the well plate and centrifuge it at 10,000g for 5 minutes in a centrifuge, and take the supernatant containing hemoglobin. Use a multifunctional microplate reader to detect the absorbance of each well at 540nm (no bubbles should appear in the well plate during the detection process), and use cells not treated with LNP as a negative control group.

The experimental results are shown in Figures 2 and 3.

Result analysis: As shown in Figure 2, the LNP prepared by the ionizable lipids with structural characteristics of the present invention has a very low erythrocyte lysis rate in a neutral pH environment, indicating that the damage to the cell membrane in a neutral environment is very low, showing safety. In contrast, the control samples (Pfizer samples and samples prepared by compounds that do not meet the structural characteristics of the present invention) have a serious effect of damaging the cell membrane at high concentrations (0.12-0.24mM), indicating that they have potential cytotoxicity at high concentrations. As shown in Figure 3, the LNP prepared by the ionizable lipids with structural characteristics of the present invention has a significantly higher erythrocyte lysis rate in an acidic pH environment than the control samples, indicating that the ionizable lipids with structural characteristics of the present invention can have a higher effect of damaging the endosomal membrane in the endosomal body after entering the cell, showing a stronger endosomal escape effect than the control samples, thereby producing a stronger transfection efficiency.

Experiment 4: Endosomal escape rate experiment

The red blood cells isolated in Experiment 3 were suspended in a pH 5.5 PBS solution and added to a 96-well plate. Then a fixed concentration of LNP prepared in Experiment 2, sample H-1, commercially available comparison sample h1-1 (Pfizer), and comparison sample h1-2 were added. The samples were incubated at 37°C for 10 min, 20 min, 40 min, 60 min, and 80 min, respectively. The samples in the well plate were centrifuged at 10,000 g for 5 min in a centrifuge, and the supernatant containing hemoglobin was taken. The absorbance of each well at 540 nm was detected using a multifunctional microplate reader (no bubbles should appear in the well plate during the detection process), and the cells not treated with LNP were used as the negative control group.

The experimental results are shown in Figure 4.

Analysis of results: As shown in Figure 4, the erythrocyte lysis rate of the LNP prepared from the ionizable lipids with the structural characteristics of the present invention increased significantly with time before 40 minutes, and began to remain stable after 40 minutes; the erythrocyte lysis rate of the LNP prepared from the comparison samples (Pfizer samples and compounds not meeting the structural characteristics of the present invention) increased significantly with time before 60 minutes, and began to remain stable after 60 minutes; it can be seen that the LNP prepared from the ionizable lipids of the present invention promotes the endosomal membrane fusion faster under acidic conditions, thereby the endosomal escape rate is faster, allowing more biologically active mRNA to reach the cytoplasm, thereby having a higher translation efficiency and a better transfection efficiency.

Experiment 5: Lipid nanoparticle structure morphology characterization experiment

Preparation and characterization of transmission electron microscopy samples (taking sample H-1 as an example). Drop 10μ of the prepared sample 15L on the copper grid, leave it for 10 minutes, then blot and air dry the sample. Dye with uranyl acetate for 5 minutes, blot the dye with filter paper, and dry overnight. Observe its morphology using a transmission electron microscope (TEM).

As shown in FIG5 , the lipid nanoparticles of the present invention can form a stable nanostructure with a narrow size distribution. The structures of different lipid nanoparticles vary, with an average particle size ranging from 30 to 200 nm.

Experiment 6: Biocompatibility Experiment

Cell viability was determined using the CCK-8 (cell counting kit-8) kit. A suspension of Hep3B cells (100 μL, cell density of 2×10 ⁴ /ml) in the exponential growth phase was added to a 96-well plate and incubated in a cell culture incubator for 24 h. The cell culture medium was then removed from each well, and 100 μL of fresh cell culture medium containing 20 μg/mL of LNP mRNA was added and incubated with the cells for 4 h. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and the incubation continued for 20 h. Then, the supernatant was removed, 100 μL of fresh cell culture medium containing CCK-8 working solution (10 μL/mL) was added, and incubated for 2 h. The blank well was set up as follows: cell culture medium containing CCK-8 working solution was added. The absorbance of each well at 450 nm was detected using a multifunctional microplate reader (no bubbles should appear in the well plate during the detection process), and cells not treated with LNP were used as the control group, and their cell viability was set to 100%.

Cell viability (%) = [A1-A0]/[A2-A0] × 100;

A1 is the absorbance of the drug-added group, A0 is the absorbance of the blank group, and A2 is the absorbance of the control group. The experimental results are shown in Table 3.

table 3

The experimental results showed that within the limited LNP concentration range, the viability of most cells was greater than 95% and no obvious cytotoxicity was observed.

Experiment 7: Experiment on the stability of lipid nanoparticles under low temperature storage

Taking sample H-3 as an example, the lipid nanoparticles prepared according to the formula were stored at 4°C. The particle size (Size) and PDI of mRNA-LNP (lipid nanoparticles encapsulating mRNA) were characterized by Malvern Zetasizer Nano ZS at different time points (0 days, 6 days, 10 days, 15 days, 30 days, 45 days, 60 days, and 90 days). The encapsulation efficiency of mRNA was determined using Ribogreen RNA quantification kit (Thermo Fisher).

The results show that the LNP formed by the lipid molecules of the present invention can be stored at low temperature for 90 days without any change in particle size, PDI and encapsulation efficiency, which further illustrates that the LNP formed by the lipid molecules of the present invention is easy to transport and store and is suitable for industrial production.

Experiment 8: Animal test on immune effect

Material preparation: 30 six-week-old female Balb/c mice, weighing 15-20 g, housed at 22±2°C, relative humidity The experimental environment was 45-75% and the light/dark cycle was 12h. After the mice were purchased, they were first adapted to the animal room for one week before formal animal experiments could be carried out. 30 mice were randomly divided into 5 groups. The first group was injected with an equal volume of PBS (negative control group) in the hind leg muscle, the second group was injected with a mixture of commercially available comparison sample h1-1 (positive control group 1), 10μg mRNA, and PBS in the hind leg muscle, the third group was injected with a mixture of comparison sample h1-2 (positive control group 2), 10μg mRNA, and PBS in the hind leg muscle, the fourth group was injected with a mixture of sample H-3 (experimental group 1), 10μg mRNA, and PBS in the hind leg muscle, and the fifth group was injected with a mixture of sample H-11 (experimental group 2), 10μg mRNA, and PBS in the hind leg muscle. The above mRNAs were synthesized by in vitro transcription based on independently designed templates and can express the full length of Spike.

The experimental process is as follows: On days 0 and 14, the LNP mixture containing mRNA was injected intramuscularly into Balb/c mice according to the above five groups. Blood was collected from the eyes on days 13 and 21. After incubation at 37°C for 1 hour, the blood samples were centrifuged at 3500rpm for 15 minutes, and the supernatant was analyzed. The specific antibody titer of the S1 protein of the Delta variant in the serum of the first and second immunized mice was detected by a homemade ELISA kit.

The specific operation process of detecting the specific antibody titer of the S1 protein of the Delta variant strain in the serum of the first and second immunized mice is as follows: add the Spike S1 recombinant protein to a 96-well plate, add 0.25μg to each well, and place it at 4℃ overnight. On the second day, discard the liquid in the wells and use 5% BSA in PBST solution (200ul) to block for 1h at 37℃. Then discard the liquid in the wells, wash 3 times with 200ul PBST washing solution, 3min each time, and shake the plate to dry. Dilute the mouse serum with PBS (dilution ratio of 1:20000), or dilute the standard with PBS to a series of concentrations (the mother solution is 1ug/ul, half-diluted, a total of 14 standard curves). Add 100μL of the diluted sample and standard to the air, incubate at 37℃ for 2h. Discard the liquid in the wells, wash 3 times with 200ul PBST washing solution, 3min each time, and shake the plate to dry. Add Goat anti-mouse IgG HRP (PBS 1:5000 dilution), 100ul per well, 37℃, 1h. Discard the liquid in the wells, wash 3 times with 200ul PBST washing solution, 3min each time, shake the plate to dry. Mix TMB substrate A solution and B solution in equal proportions, 100μL per well, place at 37℃ away from light for several minutes (3-5mins). Measure the absorbance at 650nm, and when the highest absorbance value is around 1.5, add 100ul stop solution. Detect the absorbance value at 450nm within 15min after adding the stop solution. Calculate the IgG content of each group according to the standard curve formula.

The experimental results are shown in Figure 6, which show that the four groups of mRNA, namely the positive control group 1, 2 and the experimental group 1, 2, can produce specific antibodies against the S1 protein, and the antibody titers of the experimental group 1 and the experimental group 2 are significantly higher than those of the positive control group 1 and 2. The experimental group 1 and the experimental group 2 can efficiently deliver mRNA into cells, express antigens, and then stimulate the immune response in the body, produce corresponding antibodies, and exert a protective function.

In summary, the structure of the novel ionizable lipid compound of the present invention has outstanding substantial characteristics, and the above experiments prove that the ionizable lipid compound of the present invention has N atom as the charge center, a hydrophilic group as the head, two hydrophobic groups as the tail, and -(C=O)O- is introduced on one side of the N atom and the two hydrophobic groups, and a carbonate bond is introduced on the other side as a degradable functional group. Such a structure enables LNP to promote endosomal escape in the intracellular acidic endosomal environment; and the endosomal escape rate is faster than that of commercial LNP (Pfizer sample) already in use in the market, thereby making nucleic acid nanopharmaceuticals The transfection efficiency is better: From the fluorescence results, it can be seen that the LNP prepared from the polynucleotide with the structural characteristics of the present invention has a significantly higher fluorescence intensity than the LNP prepared from the comparative compound, and the fluorescence intensity is increased by at least 3 times. The above-mentioned characteristics have a synergistic effect in improving the transfection effect, have unexpected technical effects, are non-obvious, and are creative.

It should be noted here that the ionizable lipid compound of the present invention is a drug raw material and a drug product, and does not involve any method for treating or diagnosing any disease, and is within the scope of patent rights. The scope of application of the present invention is not limited, and can be applied to the field of vaccines, protein replacement therapy, gene editing, cell therapy and other fields, and the examples here are not exhaustive, as long as the ionizable lipid compound adopts the structural characteristics of the present invention, it is within the protection scope of the present invention.

The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

An ionizable lipid compound, characterized in that the compound has a structure shown in the following formula:

Wherein, n=an integer from 0 to 10;

G 1 and G 2 are each independently C 1 -C 10 alkylene;

R 1 , R 2 , R 3 , and R 4 are each independently a C 1 -C 20 alkane group, a C 2 -C 20 alkene group, or H; when R 1 is H, R 2 is a C 2 -C 20 alkene group; when R 3 is H, R 4 is a C 2 -C 20 alkene group;

G 3 is C 1 -C 10 alkylene, or wherein a and b are each independently an integer of 1-9, and a+b=an integer of 2-10.
The ionizable lipid compound according to claim 1, characterized in that G1 is a C6 alkylene group, G2 is a C5 - C7 alkylene group, and preferably G2 is a C7 alkylene group.
The ionizable lipid compound according to claim 1, characterized in that G1 is C6 alkylene, G2 is C5 - C7 alkylene, preferably G2 is C7 alkylene, G3 is C2-4 alkylene, and R1 , R2 , R3 , and R4 are each independently C1 - C20 alkane groups.
The ionizable lipid compound according to claim 1, characterized in that G1 is a C6 alkylene group, G2 is a C5 - C7 alkylene group, preferably G2 is a C7 alkylene group, and G3 is R 1 , R 2 , R 3 and R 4 are each independently a C 1 -C 20 alkane group.
The ionizable lipid compound according to claim 1, characterized in that the compound has a structure selected from the group consisting of:
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: the ionizable lipid compound according to any one of claims 1 to 5, its stereoisomers, its tautomers or pharmaceutically acceptable salts thereof.
The pharmaceutical composition according to claim 6, characterized in that the pharmaceutical composition comprises: a carrier containing the ionizable lipid compound, a pharmaceutical agent carried therein, a pharmaceutical adjuvant, or a combination thereof.
The pharmaceutical composition according to claim 7, characterized in that the carrier further comprises: a co-lipid, a structural lipid, a polymer-conjugated lipid or an amphiphilic block copolymer, or a combination thereof.
The pharmaceutical composition according to claim 8, characterized in that the molar ratio of the ionizable lipid compound to the lipid enhancer is 0.5:1-10:1.
The pharmaceutical composition according to claim 8, characterized in that the molar ratio of the ionizable lipid compound to the structural lipid is 0.5:1-5:1.
The pharmaceutical composition according to claim 8, characterized in that the molar ratio of the ionizable lipid compound to the polymer-conjugated lipid is 10:1-250:1.
The pharmaceutical composition according to claim 8, characterized in that the molar ratio of the ionizable lipid compound to the amphiphilic block copolymer is 0.5:1-80:1.
Use of the ionizable lipid compound as described in any one of claims 1 to 5, its stereoisomer, its tautomer or its pharmaceutically acceptable salt for preparing a pharmaceutical composition.