WO2024067639A1 - Composé lipidique ionisable ayant une efficacité de transfection élevée et son utilisation - Google Patents

Composé lipidique ionisable ayant une efficacité de transfection élevée et son utilisation Download PDF

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WO2024067639A1
WO2024067639A1 PCT/CN2023/121746 CN2023121746W WO2024067639A1 WO 2024067639 A1 WO2024067639 A1 WO 2024067639A1 CN 2023121746 W CN2023121746 W CN 2023121746W WO 2024067639 A1 WO2024067639 A1 WO 2024067639A1
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acid
group
ionizable lipid
compound
alkylene
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PCT/CN2023/121746
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Chinese (zh)
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章雪晴
滕以龙
陈起静
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荣灿生物医药技术(上海)有限公司
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Priority claimed from CN202310086372.XA external-priority patent/CN115784920B/zh
Priority claimed from CN202310862227.6A external-priority patent/CN116947669B/zh
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Publication of WO2024067639A1 publication Critical patent/WO2024067639A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons

Definitions

  • the invention relates to the field of biomedicine, in particular to an ionizable lipid compound with high transfection efficiency and application thereof.
  • 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.
  • nucleic acid sequences such as DNA, mRNA, etc.
  • ensuring the smooth introduction of exogenous genes is an extremely important part of the gene therapy process and gene vaccine immunization.
  • 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.
  • 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.
  • LNP 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.
  • 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.
  • 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:
  • 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;
  • G1 is a C6 alkylene group
  • G2 is a C5 - C7 alkylene group
  • preferably G2 is a C7 alkylene group
  • G3 is C2-4 alkylene, or
  • G1 is C6 alkylene
  • G2 is C5 - C7 alkylene, preferably G2 is C7 alkylene
  • G3 is C2-4 alkylene
  • R1 , R2 , R3 , and R4 are each independently C1 - C20 alkylene.
  • 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
  • R4 is C2 - C20 alkene.
  • 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:
  • ionizable lipid compound with high transfection efficiency is used as an example, wherein Each is independently a structure selected from the following group:
  • ionizable lipid compound with high transfection efficiency is used as an example, wherein Each is independently a structure selected from the following group:
  • 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.
  • 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.
  • the pharmaceutical composition may comprise: a carrier containing the ionizable lipid compound, a pharmaceutical agent carried therein, a pharmaceutical adjuvant, or a combination thereof.
  • 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, ⁇ -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.
  • 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.
  • PEG-modified 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.
  • 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( ⁇ -amino ester) (PBAE) or polyethylene glycol (PEG) modified amphiphilic block copolymers.
  • PLGA polylactic acid-polyglycolic acid copolymer
  • PLA polylactic acid
  • PCL polycaprolactone
  • PEG polyethylene glycol
  • the molar ratio of the ionizable lipid compound of the present invention to the lipid aid is 0.5:1-10:1.
  • the molar ratio of the ionizable lipid compound of the present invention to the structural lipid is 0.5:1-5:1.
  • the molar ratio of the ionizable lipid compound of the present invention to the polymer-conjugated lipid is 10:1-250:1.
  • the molar ratio of the ionizable lipid compound of the present invention to the amphiphilic block copolymer is 0.5:1-80:1.
  • 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.
  • LNP lipid nanoparticle
  • 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 (Poly ⁇ -amino acid), etc., which are not listed here.
  • 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.
  • 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 ⁇ -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.
  • compositions 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.
  • the present invention is beneficial in that:
  • Such a structure has low destructive effect on cell membranes in a neutral environment and is highly safe.
  • the ionizable lipid compound of the present invention 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;
  • the ionizable lipid compound of the present invention has high biocompatibility.
  • the synthesis steps of the ionizable lipid compound of the present invention are simple and suitable for biopharmaceutical industrialization.
  • 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.
  • 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.
  • 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.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • 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
  • 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.
  • C1-C20 alkane group 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.
  • C2-C20 olefin group 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.
  • the olefin group can be a monoolefin or a polyolefin (such as a diene).
  • C1-C10 alkylene 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,
  • 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,
  • 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:
  • SM Chinese name: sphingomyelin (SM); English name: sphingomyelin.
  • PEG Chinese name: polyethylene glycol; English name: Polyethylene glycol.
  • the ionizable lipid compound was prepared by the preparation method of Example 1 below.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • 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.
  • LNP lipid nanoparticle
  • 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).
  • mice Male ICR mice (6-8 weeks, Shanghai Jiesijie Experimental Animal Co., Ltd.) were housed under experimental conditions of 22 ⁇ 2°C 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.
  • 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 150 ⁇ g/kg mRNA, with 2 mice per group of samples, two legs.
  • luciferin (20 ⁇ g/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.
  • 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.
  • 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.
  • mice Four-week-old female ICR mice, weighing 15-20g, were kept in an experimental environment with a temperature of 22 ⁇ 2°C, 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.
  • PBS pH 7.4
  • 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.
  • 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
  • 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.
  • 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 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 increased significantly with time before 60 minutes, and began to remain stable after 60 minutes;
  • 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.
  • 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.
  • Cell viability was determined using the CCK-8 (cell counting kit-8) kit.
  • a suspension of Hep3B cells (100 ⁇ L, 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 ⁇ L of fresh cell culture medium containing 20 ⁇ g/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.
  • A1 is the absorbance of the drug-added group
  • A0 is the absorbance of the blank group
  • A2 is the absorbance of the control group.
  • 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.
  • mice Materials 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), 10 ⁇ g 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), 10 ⁇ g mRNA, and PBS in the hind leg muscle
  • the fourth group was injected with a mixture of sample H-3 (experimental group 1), 10 ⁇ g mRNA, and PBS in the hind leg muscle
  • the fifth group was injected with a mixture of sample H-11 (experimental group 2), 10 ⁇ g 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 0.25 ⁇ g to each well, and place it at 4°C 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°C. Then discard the liquid in the wells, wash 3 times with 200ul PBST washing solution, 3min each time, and shake the plate to dry.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention se rapporte au domaine de la biomédecine. L'invention concerne un composé lipidique ionisable ayant une efficacité de transfection élevée et son utilisation. Le composé lipidique ionisable est un composé ayant la structure suivante : formule (I), dans laquelle n = 0-10 ; G1 et G2 représentent chacun indépendamment un groupe alkylène ; R1, R2, R3 et R4 représentent chacun indépendamment un groupe alcane, un groupe oléfine ou H ; G3 représente un groupe alkylène ou la formule (II). Un support de nanoparticules lipidiques préparé à partir du composé selon la présente invention a une bonne cytocompatibilité dans un environnement physiologique, peut favoriser l'échappement endosomal dans un environnement acide d'endosome intracellulaire, présente une progression inattendue dans l'amélioration de l'effet de transfection et a une sécurité élevée, et est approprié pour une industrialisation biomédicale.
PCT/CN2023/121746 2022-09-30 2023-09-26 Composé lipidique ionisable ayant une efficacité de transfection élevée et son utilisation WO2024067639A1 (fr)

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CN202310086372.XA CN115784920B (zh) 2023-02-09 2023-02-09 一种转染效率高的可电离脂质化合物及其应用
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