WO2023216423A1 - Lipid compound, and composition, preparation and use thereof - Google Patents

Lipid compound, and composition, preparation and use thereof Download PDF

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WO2023216423A1
WO2023216423A1 PCT/CN2022/107846 CN2022107846W WO2023216423A1 WO 2023216423 A1 WO2023216423 A1 WO 2023216423A1 CN 2022107846 W CN2022107846 W CN 2022107846W WO 2023216423 A1 WO2023216423 A1 WO 2023216423A1
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acid
lipid
compound
sodium
composition
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PCT/CN2022/107846
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French (fr)
Chinese (zh)
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赵凤翔
路青青
胡洪鹏
耿亦程
蒋剑豪
钟天翼
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苏州慧疗生物医药科技有限公司
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • 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
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • the present invention specifically relates to a type of lipid compound and its composition, preparation and use, especially its application in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  • Nucleic acid drugs refer to artificially designed DNA or RNA with disease prevention or treatment functions. They act on disease-causing target genes or target mRNA to fundamentally regulate the expression of disease-causing genes to achieve the purpose of disease prevention or treatment. Nucleic acid drugs mainly include antisense oligonucleotide (ASO), small interference RNA (siRNA), microRNA (miRNA), messenger RNA (message, mRNA), etc. As of the end of 2021, the FDA has approved more than 10 nucleic acid drugs, and multiple drug candidates are in clinical trials or preclinical trials.
  • ASO antisense oligonucleotide
  • siRNA small interference RNA
  • miRNA microRNA
  • messenger RNA messenger RNA
  • RNA vaccines BNT162b2 Pfizer/BioNTech
  • mRNA-1273 Moderna
  • my country also has multiple new coronavirus vaccines based on mRNA technology in clinical trials or preclinical trials.
  • mRNA technology has proven its unique advantages over traditional biopharmaceutical and vaccine technologies.
  • Therapeutic nucleic acids have the potential to revolutionize vaccinations, gene therapies, protein replacement therapies and the treatment of other genetic diseases.
  • RNA degradation determines the need for a high-quality delivery system to deliver it into the body.
  • the present invention focuses on providing a new delivery carrier for RNA drugs.
  • the present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives, which enriches the types of lipid compounds and can be used for nucleic acid drugs, gene vaccines, polypeptides, and proteins.
  • lipids based on cholic acid or its derivatives which enriches the types of lipid compounds and can be used for nucleic acid drugs, gene vaccines, polypeptides, and proteins.
  • the delivery of antibodies, small molecule drugs, etc. provides more options.
  • the present invention provides a lipid compound represented by the following general formula 1, general formula 2, or a pharmaceutically acceptable salt thereof, including stereoisomers, tautomers, solvates, chelates, and non-covalent compounds.
  • a pharmaceutically acceptable salt thereof refers to an acid addition salt or a base addition salt.
  • the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes one or more of an ester group, an amide group, a carbamate group, a carbonate group or a urea group. kind.
  • the lipid compound, wherein cholic acid or its derivatives is optionally selected from cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3 ⁇ -hydroxy-D5-cholenoic acid, Chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5 ⁇ -cholic acid, dehydrocholic acid, hyocholic acid, cholic acid, glycinechenodeoxycholic acid, tauroursodeoxycholic acid Acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, hyodeoxycholic acid methyl ester, taurohodeoxycholic acid sodium, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate , sodium taurodeoxycholate, sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate diso
  • the lipid compound is one or more lipid compounds selected from ursodeoxycholic acid derivatives, or one or more obeticholic acid derivative lipid compounds. kind.
  • a composition of lipid compounds includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, and the carrier includes one or more of the aforementioned lipid compounds.
  • the composition, therapeutic or preventive agent includes one or more of nucleic acid molecules, polypeptides, proteins, antibodies and small molecule drugs.
  • the mass ratio of the carrier to the therapeutic or preventive agent is 1:1 to 100:1.
  • the composition is a nanoparticle preparation
  • the average size of the nanoparticle preparation is 20 nm to 1000 nm
  • the polydispersity coefficient of the nanoparticle preparation is ⁇ 0.5.
  • the carrier contains three different lipid components, one of which is a lipid based on cholic acid or its derivatives.
  • the composition is characterized in that the carrier further includes a charge-assisted lipid with neutral charge, negative charge or bipolar charge.
  • the composition is characterized in that the charge-auxiliary lipid is one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphati
  • the carrier further includes a structurally modified lipid.
  • the structurally modified lipid includes polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, One or more dialkylglycerols.
  • the composition and the carrier also include but are not limited to lipids of cholic acid or its derivatives, charge-assisted lipids, and structurally modified lipids.
  • the cholic acid lipids, the charge-assisted lipids And the molar ratio of the structurally modified lipid is (30-80): (5-50): (0.5-10).
  • the composition further includes one or more pharmaceutically acceptable excipients or diluents.
  • the lipid compound or composition of the present invention can be used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  • the lipid compound or composition of the present invention is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticles have a particle size of 20 to 1000 nm.
  • compositions for preparing nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs which include nucleic acids and lipid nanoparticles encapsulating the nucleic acids, wherein each individual lipid nanoparticle contains a variety of lipids A lipid component, wherein one of the lipid components is a cholic acid-based lipid compound, including compounds thereof or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non- A covalent compound or prodrug, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
  • the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution of any lipid compound described in this patent, wherein the medium of the mRNA solution is HEPES, sodium phosphate , sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate; the medium of the lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide; wherein the lipid nanoparticles are further purified by dialysis or ultrafiltration.
  • HEPES sodium phosphate
  • sodium acetate sodium acetate
  • ammonium sulfate sodium bicarbonate or sodium citrate
  • the medium of the lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide
  • composition described in this patent also contains one or more of buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids, antioxidants, bacteriostatic agents, chelating agents, and adjuvants.
  • the acid described in the present invention includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzene
  • Base addition salts refer to salts prepared by adding an inorganic base or an organic base to a free base compound.
  • Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, etc.;
  • the 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, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin.
  • the organic bases include
  • the present invention provides a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof.
  • the therapeutic or preventive agent is encapsulated in or associated with a carrier.
  • the therapeutic or preventive agent includes one or more of nucleic acid molecules, genetic vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  • the nucleic acid includes any form of nucleic acid molecule, including but not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA ( aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of RNA molecules known in the art, or locked nucleic acids Nucleic acid mimics such as (LNA), peptide nucleic acid (PNA) and morpholino cyclic oligonucleotides.
  • LNA small interfering RNA
  • aiRNA asymmetric interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • the therapeutic or preventive agent comprises at least one mRNA encoding an antigen or a protein or a peptide or a fragment or epitope thereof.
  • the mRNA is monocistronic or polycistronic.
  • the antigen is a pathogenic antigen.
  • the mRNA contains one or more functional nucleotide analogs, including but not limited to pseudouridine, 1-methyl-pseudouridine and one or more of 5-methylcytosine.
  • the small molecule compounds include, but are not limited to, the active ingredients of therapeutic and/or preventive agents.
  • the therapeutic and/or preventive agents are currently known drugs, such as anti-tumor drugs, anti-infective drugs, and local anesthetics. , antidepressants, anticonvulsants, antibiotics/antimicrobials, antifungals, antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, anesthetics, or contrast media.
  • the lipids comprise three different lipid components, one of which is a cholic acid or derivative thereof based lipid.
  • the lipid further includes an auxiliary lipid with a neutral charge, a negative charge, or a bipolar charge.
  • the lipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and derivatives thereof.
  • the lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 11,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-(((2,3- Bis(oleoyloxy)propyl)dimethylammonium phosphate)ethylhydrogen (DOCP), sphingomyelin (SM), ceramide and its derivatives.
  • the lipids may be synthetic or derived from (isolated or modified) natural sources or compounds.
  • the carrier further includes a structurally modified lipid.
  • the structurally modified lipids mainly include disclosed or undisclosed lipid compounds, which can improve the stability of liposomes and reduce protein absorption of liposomes, such as polyethylene glycol, dextran, polyethylene glycol, and polyethylene glycol.
  • lipid compounds which can improve the stability of liposomes and reduce protein absorption of liposomes, such as polyethylene glycol, dextran, polyethylene glycol, and polyethylene glycol.
  • lactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, and dialkylglycerol are examples of lactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, and dialkylglycerol.
  • the structurally modified lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, Chol-PEG2000, 1-(monomethyl Oxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-bis( Tetradecanoyloxy)propyl-1-O-( ⁇ -methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyglycolated ceramide (PEG- cer), ⁇ -methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate, or 2,3-di(tetradecyloxy) methyl)propyl-
  • the mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 50:1, more preferably 5:1 to 35:1, and more preferably 10:1 to 30:1.
  • the carrier further includes, but is not limited to, lipids of cholic acid or its derivatives, charge-assisted lipids, and structurally modified lipids.
  • the molar ratio of the cholic acid lipid, the charge-assisted lipid, and the structurally modified lipid is (30-80): (5-50): (0.5-10).
  • lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution. In some embodiments, lipid nanoparticles are further purified by tangential flow filtration.
  • the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution.
  • the medium of the mRNA solution is HEPES, phosphate, acetate, ammonium sulfate, sodium bicarbonate or citrate.
  • the medium of lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide. 29.
  • the pharmaceutical composition is a nanoparticle preparation, and the average size of the nanoparticle preparation is 20 nm to 1000 nm, preferably 40 nm to 150 nm, further preferably 50 nm to 100 nm, and more preferably 70 nm to 100 nm.
  • the polydispersity index of the nanoparticle preparation is ⁇ 0.5, further preferably ⁇ 0.3, and more preferably ⁇ 0.25.
  • compositions of the present invention also typically include one or more buffers (eg neutral buffered saline or phosphate buffered water), carbohydrates (eg glucose, mannitol, sucrose, trehalose, dextrose or dextran). sugar), mannitol, proteins, peptides or amino acids (such as glycine and lysine), antioxidants (vitamin E and butylated hydroxytoluene), bacteriostatic agents, chelating agents (such as EDTA and glutathione), adjuvants (e.g. aluminum hydroxide), suspending agents/thickening agents/preservatives, etc. that make the formulation isotonic with the recipient's blood, or the composition of the invention can be formulated as a lyophilisate.
  • buffers eg neutral buffered saline or phosphate buffered water
  • carbohydrates eg glucose, mannitol, sucrose, trehalose, dextrose or dextran. sugar
  • the technical solution of the present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives.
  • the technical solution of the present invention is different from the existing patented technical solutions of foreign pharmaceutical companies. , enriches the types of lipid compounds, provides more options for the delivery of nucleic acid drugs, gene vaccines, peptides, proteins, antibodies and small molecule drugs, and can be significantly different from the technical routes of foreign companies such as Pfizer and Moderna. .
  • Figure 1 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 1 (compound 1);
  • Figure 2 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 2 (compound 2);
  • Figure 3 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 7 (compound 7);
  • Figure 4 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 9 (compound 9);
  • Figure 5 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 10 (compound 10);
  • Figure 6 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 13 (compound 13);
  • Figure 7 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 16 (compound 16);
  • Figure 8 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 17 (compound 17);
  • Figure 9 is a proton nuclear magnetic resonance spectrum of obeticholic acid derivative 18 (compound 18).
  • Figure 10 shows the experimental results of cells transfected with LNP-encapsulated cy3-siRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry;
  • Figure 11 shows the experimental results of cells transfected with LNP-encapsulated EGFP mRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry;
  • Figure 12 shows the fluorescence imaging results of LNP containing Luciferase mRNA 12h after intravenous injection in mice;
  • Figure 13 shows the fluorescence imaging results of LNP encapsulating Luciferase mRNA 12 hours after intramuscular injection in mice.
  • Cholic acid is a naturally ubiquitous steroid molecule in humans and mammals, synthesized in the liver from cholesterol. After eating, bile acid is actively secreted by liver cells, enters the gallbladder with bile, and then enters the intestine from the gallbladder to perform its digestive function. Cholic acid enters the small intestine in the form of sodium salt to help digest and absorb lipids, and then passes through the terminal ileum. It is returned to the liver through the portal vein through active absorption or passive transport, processed and transformed in liver cells, and then secreted into the small intestine together with newly synthesized bile acids.
  • This EHC (enterohepatic circulation) process of bile acids circulates 4 to 12 times a day, and about 95% of the bile acids are reabsorbed and utilized. If the EHC of bile acids is destroyed, it will not only affect the digestion and absorption of lipids in the body, but also cause the body to form cholesterol stones. Therefore, the biggest advantage of compound delivery carriers designed based on bile acid is that it has high enterohepatic circulation efficiency and participates in the enterohepatic circulation of bile acid, thereby improving drug absorption in the liver and gallbladder.
  • the structural formula of cholic acid is as follows.
  • the three six-membered rings and one five-membered ring on the steroid skeleton of the cholic acid molecule are on the same plane, and ring A and ring B are connected in reverse, making the molecule form a cave-like structure.
  • three methyl groups are distributed on one side of the plane where the steroid ring is located, forming the hydrophobic part of the molecule.
  • Three hydroxyl groups are distributed on the other side of the plane where the steroid ring is located, and together with the C24 carboxyl group, form the hydrophilic part of the molecule.
  • cholic acid The special structure of the cholic acid molecule determines that it is amphiphilic, acid-base, and easy to undergo chemical modification. Therefore, this patent uses cholic acid as a building block to prepare polymers or oligomers. These cholic acid-based products Functional molecules have good technical effects in drug delivery.
  • Cholic acid drugs have been on the market in China or the United States for many years, including ursodeoxycholic acid, obeticholic acid, chenodeoxycholic acid, and tauroursodeoxycholic acid.
  • ursodeoxycholic acid is used to treat cholesterol gallstones and bile reflux gastritis
  • obeticholic acid is used to treat primary biliary cirrhosis (PBC), and there is no adequate response to ursodeoxycholic acid or Patients who cannot tolerate it.
  • PBC primary biliary cirrhosis
  • Years of clinical application have shown that cholic acid compounds have good safety.
  • cholic acid compounds have hydrophilic and lipophilic amphiphilic properties, and can improve the bioavailability of small molecule chemical drugs through encapsulation or coupling.
  • Cholic acid compounds and cholesterol are both steroidal compounds with similar structures and more sites that can be chemically modified. Structural modification of cholic acid compounds to prepare new lipid components may potentially replace the ionizable lipids and cholesterol in the original four-component LNP to achieve similar or better mRNA delivery effects.
  • This patented study constructs lipid nanoparticle carriers based on bile acid analogs and explores their application in mRNA drug delivery.
  • the LNP of the present invention can simplify the production process and reduce costs; at the same time, it can avoid the patent blockade of four-component LNP, which is beneficial to promoting the localization of nucleic acid drugs.
  • cholic acid or its derivatives are composed of a rigid steroid ring and an aliphatic side chain
  • the steroid ring includes three six-membered rings and one five-membered ring.
  • the side chain structure of cholic acid, the conformation of the steroid ring, the number of hydroxyl groups and the orientation of the steroid ring will be different.
  • the common hydroxyl group of cholic acid or its derivatives and the carboxyl group of the fatty side chain are good chemical modification sites. Therefore, we believe that cholic acid or its derivatives can achieve the desired purpose through some common modifications.
  • Cholic acid or its derivatives in this patent is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3 ⁇ -hydroxy-D5-cholic acid, chenodeoxycholic acid, and lithocholic acid.
  • the lipid compound described in this patent is one or more compounds selected from the following structures;
  • Ursodeoxycholic acid derivative lipid 1 Ursodeoxycholic acid derivative lipid:
  • ursodeoxycholic acid (393mg, 1mmol) in DMF (8mL), add HBTU (569mg, 1.5mmol), DIEA (194mg, 1.5mmol), N,N-dimethylethylenediamine (132mg, 1.5 mmol), under nitrogen protection, stirred at room temperature overnight.
  • TLC detection after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product.
  • Silica gel column chromatography eluting with DCM/CH3OH (10:1), gave a white solid (333 mg, 72%).
  • Ursodeoxycholic acid (393 mg, 1 mmol) was dissolved in DMF (8 mL), and HBTU (569 mg, 1.5 mmol), DIEA (194 mg, 1.5 mmol), and 4-pyrrole-1-butylamine (213 mg, 1.5 mmol) were added in sequence. ), under nitrogen protection, the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid product X-4 (403 mg, 78%).
  • Ursodeoxycholic acid (393 mg, 1 mmol) was dissolved in acetonitrile (10 mL), K 2 CO 3 (415 mg, 3 mmol), BnBr (850 mg, 5 mmol) were added in sequence, protected by nitrogen, and the reaction was stirred at 80°C for 5 hours. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave compound 6 (390 mg, 81%) as a white solid product.
  • 4-Dimethylaminobutyric hydrochloride (336 mg, 2 mmol) was dissolved in anhydrous DCM (8 mL), oxalyl chloride (1 mL) was added, and stirred at room temperature for 4 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (3mL), and set aside. Dissolve compound 6 (241 mg, 0.5 mmol) and TEA (202 mg, 2 mmol) in anhydrous DCM (3 mL). Add the above standby product dropwise into the reaction solution and react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product.
  • Ursodeoxycholic acid (785 mg, 1 mmol) was dissolved in DMF (20 mL), K 2 CO 3 (829 mg, 6 mmol), CH 3 I (852 mg, 6 mmol) were added in sequence, protected by nitrogen, and the reaction was stirred at room temperature overnight.
  • TLC detection after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product.
  • Linoleic acid (476 mg, 1.7 mmol) was dissolved in anhydrous DCM (5 mL), oxalyl chloride (0.30 mL) was added, and the mixture was stirred at room temperature for 5 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (2mL), and set aside. Compound 2 (240 mg, 0.34 mmol) and TEA (69 mg, 0.68 mmol) were dissolved in anhydrous DCM (5 mL). The above standby product was dropped into the reaction solution and allowed to react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product.
  • nanoparticle compositions for delivering therapeutic and/or prophylactic agents to cells
  • a series of formulations were prepared and tested. Specifically, specific ingredients and their ratios in the lipid component of the nanoparticle composition are optimized.
  • Nanoparticles can be produced by mixing two fluid streams, one of which contains therapeutic and/or prophylactic agents and the other of which has a lipid component, by mixing methods such as microfluidization and T-junctions.
  • phospholipids such as DOPE or DSPC, available from Avanti Polar Lipids (Alabaster, AL)
  • PEG lipids such as 1,2-dimyristoyl -sn-Glycerylmethoxypolyethylene glycol, also known as PEG-DMG, available from Avanti Polar Lipids (Alabaster, AL)
  • structural lipids such as cholesterol, available from Sigma-Aldrich (Tauf Wegn, Germany)
  • lipid composition such as a concentration of about 50 mM.
  • a formulation containing a therapeutic agent is prepared by combining a lipid solution with a therapeutic agent and/or prophylactic agent in a lipid component to therapeutic agent and/or prophylactic agent wt:wt ratio of between about 5:1 and about 50:1.
  • Nanoparticle compositions of agents and/or prophylactic agents and lipid components are rapidly injected into the therapeutic agent and/or preventive agent solution at a flow rate between about 10 mL/min and about 18 mL/min to create a dispersion. , wherein the water to ethanol ratio is between about 2:1 and about 5:1.
  • Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, polydispersity index (PDI) and ⁇ potential of the nanoparticle composition.
  • the particle size is measured in 1 ⁇ PBS and ⁇ Potentials were measured in 15mM PBS.
  • RNA For nanoparticle compositions containing RNA, the QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA) assay can be used to evaluate the encapsulation of RNA by the nanoparticle composition. Samples were diluted in TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 7.5) to a concentration of approximately 5 ⁇ g/mL. Transfer 50 ⁇ L of the diluted sample to a polystyrene 96-well plate and add 50 ⁇ L of TE buffer or 50 ⁇ L of 2% Triton X-100 solution to each well. Incubate the plate at 37°C for 15 minutes.
  • TE buffer solution 10mM Tris-HCl, 1mM EDTA, pH 7.5
  • a fluorescent plate reader can be used ( Nivo TM Multimode Plate Readers, PerkinElmer, GER) measure fluorescence intensity at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm.
  • the fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence of the disrupted sample (caused by the addition of Triton X-100). value to determine the percentage of free RNA. See Table 1 for specific data.
  • ONE-GlO+TOX Luciferase Reporter and Cell Viability Assay can be used to evaluate its transfection effect and cytotoxicity. Calculate the volume of the required nanoparticle assembly based on the RNA concentration measured in the encapsulation efficiency determination, dilute the nanoparticle assembly to 20ng/ ⁇ L, and add it according to the cell density of 2 ⁇ 10 5 cells/well in a polystyrene 96-well plate. Add 5 ⁇ L of diluent to each well for transfection. A fluorescent plate reader can be used ( Nivo TM Multimode Plate Readers, PerkinElmer, GER) were used to measure chemiluminescence intensity. See Table 2 for specific data.
  • Example 11 Preparation and detection of lipid nanoparticles (to verify the ability to deliver siRNA in vitro)
  • Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 82 nm, the PDI (polydispersity index) was 0.13, and the encapsulation efficiency was 96.5%.
  • Example 12 Preparation and detection of lipid nanoparticles (to verify the ability to deliver mRNA in vitro)
  • the lipid nanoparticles are then filtered with a 0.22 ⁇ m sterile filter to obtain a preparation encapsulating EGFP mRNA.
  • Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit.
  • the particle size of the LNP preparation was measured to be 95nm, PDI 0.19, and the encapsulation rate was 93.1%.
  • Example 13 Preparation and detection of lipid nanoparticles (to verify the in vivo delivery effect of mRNA)
  • the weight ratio of total lipid to mRNA is Prepare liposomes at 20:1, remove ethanol using dialysis or tangential flow filtration, and replace with PBS solution.
  • the lipid nanoparticles are then filtered with a 0.22 ⁇ m sterile filter to obtain a preparation that encapsulates mRNA.
  • Lipid nanoparticle size was determined by dynamic light scattering using a Nanoparticle Size and Potentiometric Analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 98 nm, the PDI was 0.22, and the encapsulation rate was 88.9%.
  • Prepare liposomes use dialysis or tangential flow filtration to remove ethanol, and replace it with PBS solution.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 1249nm, the PDI was 0.67, and the encapsulation rate was 31%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 467 nm, the PDI was 0.40, and the encapsulation rate was 78%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 347nm, the PDI was 0.41, and the encapsulation rate was 81%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 94nm, the PDI was 0.50, and the encapsulation rate was 85%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 82nm, the PDI was 0.11, and the encapsulation rate was 96%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 145nm, the PDI was 0.23, and the encapsulation rate was 76%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
  • the lipid nanoparticles were then filtered with a 0.22 ⁇ m sterile filter to obtain an mRNA-encapsulated preparation.
  • the detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.

Abstract

Disclosed in the present invention are a lipid compound as represented by a general formula and a composition, and the preparation and the use thereof. The lipid compound of the present invention is a compound based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt of the compound. The lipid compound and the composition thereof of the present invention can provide a greater choice of vectors for the delivery of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies, small-molecule drugs, etc.

Description

脂质化合物及其组合物，制备和用途Lipid compounds and compositions thereof, preparation and uses 技术领域Technical field
本发明具体涉及一类脂类化合物及其组合物，制备和用途，尤其在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用。The present invention specifically relates to a type of lipid compound and its composition, preparation and use, especially its application in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
背景技术Background technique
核酸药物是指人为设计的具有疾病预防或治疗功能的DNA或RNA，其通过作用于致病靶基因或者靶mRNA，从根源上调控致病基因的表达，达到疾病预防或治疗的目的。核酸药物主要有反义核酸(antisense oligonucleotide,ASO)、小干扰RNA(small interference RNA，siRNA)、微小RNA(micro RNA,miRNA)、信使RNA(message,mRNA)等。截至2021年底，FDA已经批准了10多种核酸药物，多种候选药物处于临床试验或临床前试验阶段。Nucleic acid drugs refer to artificially designed DNA or RNA with disease prevention or treatment functions. They act on disease-causing target genes or target mRNA to fundamentally regulate the expression of disease-causing genes to achieve the purpose of disease prevention or treatment. Nucleic acid drugs mainly include antisense oligonucleotide (ASO), small interference RNA (siRNA), microRNA (miRNA), messenger RNA (message, mRNA), etc. As of the end of 2021, the FDA has approved more than 10 nucleic acid drugs, and multiple drug candidates are in clinical trials or preclinical trials.
FDA已经批准预防新冠病毒COVID-2019的RNA疫苗BNT162b2(辉瑞/BioNTech公司)和mRNA-1273(Moderna公司)上市，我国也有多个基于mRNA技术的新冠疫苗处于临床试验或临床前试验阶段。在新冠疫情战斗中，mRNA技术证明了它相比传统生物制药和疫苗技术的独特优势。治疗性核酸具有彻底改变疫苗接种、基因疗法、蛋白质替代疗法和其它遗传疾病疗法的潜力。The FDA has approved the RNA vaccines BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna) to prevent the new coronavirus COVID-2019. my country also has multiple new coronavirus vaccines based on mRNA technology in clinical trials or preclinical trials. In the fight against the COVID-19 epidemic, mRNA technology has proven its unique advantages over traditional biopharmaceutical and vaccine technologies. Therapeutic nucleic acids have the potential to revolutionize vaccinations, gene therapies, protein replacement therapies and the treatment of other genetic diseases.
RNA本身极容易降解的特性，决定了需要有一个优质的递送系统将其递送到体内。本发明侧重为RNA药物提供一种新的递送载体。The very easy degradation of RNA determines the need for a high-quality delivery system to deliver it into the body. The present invention focuses on providing a new delivery carrier for RNA drugs.
发明内容Contents of the invention
本发明提供了一类新的用于递送治疗或预防用药物的脂质化合物，即基于胆酸或其衍生物的脂质，丰富了脂质化合物种类，为核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物等的递送提供了更多选择。The present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives, which enriches the types of lipid compounds and can be used for nucleic acid drugs, gene vaccines, polypeptides, and proteins. The delivery of antibodies, small molecule drugs, etc. provides more options.
本发明提供一种如下通式1，通式2所示的脂质化合物，或其药物可用的盐，包括立体异构体、互变异构体、溶剂化物、螯合物、非共价化合物或前体药物，具体地，所述“其药物可用的盐”是指酸加成盐或碱加成盐。The present invention provides a lipid compound represented by the following general formula 1, general formula 2, or a pharmaceutically acceptable salt thereof, including stereoisomers, tautomers, solvates, chelates, and non-covalent compounds. Or prodrug, specifically, the "pharmaceutically acceptable salt thereof" refers to an acid addition salt or a base addition salt.
通式1：General formula 1:
Figure PCTCN2022107846-appb-000001
Figure PCTCN2022107846-appb-000001
或；通式2：Or; general formula 2:
Figure PCTCN2022107846-appb-000002
Figure PCTCN2022107846-appb-000002
优选的，所述脂质化合物母核为胆酸或其衍生物；其中所述连接键(Linker)包含酯基、酰胺基、氨基甲酸酯基、碳酸酯基或脲基的一种或多种。Preferably, the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes one or more of an ester group, an amide group, a carbamate group, a carbonate group or a urea group. kind.
优选的，所述的脂质化合物，其中胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠的一种或多种。Preferably, the lipid compound, wherein cholic acid or its derivatives is optionally selected from cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β-hydroxy-D5-cholenoic acid, Chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholic acid, glycinechenodeoxycholic acid, tauroursodeoxycholic acid Acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, hyodeoxycholic acid methyl ester, taurohodeoxycholic acid sodium, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate , sodium taurodeoxycholate, sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, tauroursodeoxycholic acid Sodium and one or more of sodium taurite cholate.
优选的，所述的脂质化合物，所述脂质化合物为选自熊去氧胆酸衍生物脂质化合物的一种或多种，或奥贝胆酸衍生物脂质化合物的一种或多种。Preferably, the lipid compound is one or more lipid compounds selected from ursodeoxycholic acid derivatives, or one or more obeticholic acid derivative lipid compounds. kind.
一种脂质化合物的组合物，所述组合物包括治疗或预防剂和用于递送所述治疗或预防剂的载体，所述载体包括前述的脂质化合物的一种或多种。A composition of lipid compounds, the composition includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, and the carrier includes one or more of the aforementioned lipid compounds.
优选的，所述的组合物，所述治疗或预防剂包括核酸分子、多肽、蛋白、抗体及小分子药物中的一种或多种。Preferably, the composition, therapeutic or preventive agent includes one or more of nucleic acid molecules, polypeptides, proteins, antibodies and small molecule drugs.
优选的，所述的组合物，所述载体与所述治疗或预防剂的质量比为1:1～100:1。Preferably, in the composition, the mass ratio of the carrier to the therapeutic or preventive agent is 1:1 to 100:1.
优选的，所述的组合物，所述组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为20nm～1000nm；所述纳米颗粒制剂的多分散系数≤0.5。Preferably, the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 20 nm to 1000 nm; the polydispersity coefficient of the nanoparticle preparation is ≤ 0.5.
优选的，所述的组合物，所述载体中包含三种不同的脂质组分，其中一种脂质是基于胆酸或其衍生物的脂质。Preferably, in the composition, the carrier contains three different lipid components, one of which is a lipid based on cholic acid or its derivatives.
优选的，所述的组合物，其特征在于，所述载体中还包括中性电荷、阴性电荷或双性电荷的电荷辅助脂质。Preferably, the composition is characterized in that the carrier further includes a charge-assisted lipid with neutral charge, negative charge or bipolar charge.
优选的，所述的组合物，其特征在于，所述电荷辅助脂质为如下的一种或多种：二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二油酰基磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰油酰基磷脂酰胆碱(POPC)、棕榈酰油酰基-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷脂酰乙醇胺(DMPE)、二硬脂酰基-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE,16-O-二甲基PE,18-1-反式PE,1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)或其混合物。Preferably, the composition is characterized in that the charge-auxiliary lipid is one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) , dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O- Dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or mixtures thereof.
优选的，所述的组合物，所述载体中还包括结构修饰脂质。Preferably, in the composition, the carrier further includes a structurally modified lipid.
优选的，所述的组合物，所述结构修饰脂质包括聚乙二醇、葡聚糖、聚乳酸或氨基酸修饰的磷脂酰乙醇胺、磷脂酸、神经酰胺、二烷基胺、二酰基甘油、二烷基甘油中的一种或多种。Preferably, in the composition, the structurally modified lipid includes polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, One or more dialkylglycerols.
优选的，所述的组合物，所述载体还包括但不限于胆酸或其衍生物的脂质、电荷辅助脂质以及结构修饰脂质，所述胆酸脂质、所述电荷辅助脂质以及所述结构修饰脂质的摩尔比为(30～80)：(5-50)：(0.5～10)。Preferably, the composition and the carrier also include but are not limited to lipids of cholic acid or its derivatives, charge-assisted lipids, and structurally modified lipids. The cholic acid lipids, the charge-assisted lipids And the molar ratio of the structurally modified lipid is (30-80): (5-50): (0.5-10).
优选的，所述的组合物，所述组合物还包括药物可用的赋形剂或稀释剂中的一种或多种。Preferably, the composition further includes one or more pharmaceutically acceptable excipients or diluents.
本发明所述脂质化合物或组合物可用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用。The lipid compound or composition of the present invention can be used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
本发明所述脂质化合物或组合物用于在制备核酸药物、基因疫苗、多肽、 蛋白、抗体及小分子药物中的应用中，其中所述脂质纳米颗粒具有20～1000nm粒径。The lipid compound or composition of the present invention is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticles have a particle size of 20 to 1000 nm.
用于制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物的组合物，其包含核酸和包载所述核酸的脂质纳米颗粒，其中每个单独的脂质纳米颗粒包含多种脂质组分，其中一种脂质组分是基于胆酸的脂质化合物，包括其化合物或其药学上可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价化合物或前体药物，并且其中所述脂质纳米颗粒具有至少70％的核酸包封比例。Compositions for preparing nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, which include nucleic acids and lipid nanoparticles encapsulating the nucleic acids, wherein each individual lipid nanoparticle contains a variety of lipids A lipid component, wherein one of the lipid components is a cholic acid-based lipid compound, including compounds thereof or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non- A covalent compound or prodrug, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
制备本专利所述组合物的方法，其中所述脂质纳米颗粒通过将mRNA溶液和本专利所述任一种脂质化合物的脂质溶液混合而形成，其中mRNA溶液的介质为HEPES、磷酸钠、乙酸钠、硫酸铵、碳酸氢钠或柠檬酸钠；脂质溶液的介质为乙醇、异丙醇或二甲亚砜；其中所述脂质纳米颗粒通过透析或超滤进一步纯化。Method for preparing the composition described in this patent, wherein the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution of any lipid compound described in this patent, wherein the medium of the mRNA solution is HEPES, sodium phosphate , sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate; the medium of the lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide; wherein the lipid nanoparticles are further purified by dialysis or ultrafiltration.
本专利所述的组合物，还包含缓冲剂、碳水化合物、甘露醇、蛋白质、多肽或者氨基酸、抗氧化剂、抑菌剂、螯合剂、佐剂中的一种或多种。The composition described in this patent also contains one or more of buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids, antioxidants, bacteriostatic agents, chelating agents, and adjuvants.
本发明所述酸包括但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、海藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯甲酸、4-乙酰氨基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸，柠檬酸、环酰胺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙磺酸、甲酸、富马酸、半乳糖酸、龙胆酸、葡庚酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲磺酸、、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、棕榈酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、酒石酸、对甲苯磺酸、三氟乙酸、以及十一碳烯酸。The acid described in the present invention includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzene Formic 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 sulfate, ethane-1, 2-Disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic acid, gentisic acid, gluconic acid, gluconic acid, glucuronic acid, glutamic acid, pentanoic acid Diacid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic 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, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid.
本发明所述的碱加成盐指通过将无机碱或有机碱加成至游离碱化合物而制备的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐，、等；所述有机碱包括但不限于氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、脱醇、2-二甲基氨基乙醇、2-二乙基氨基乙醇、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、肼苯胺、胆碱、甜菜碱、 苯那敏(benethamine)、、乙二胺、葡糖胺、甲基葡糖胺、可可碱、三乙醇胺、嘌呤、哌嗪、哌啶、N-乙基哌啶、以及聚胺树脂。优选地，有机碱是异丙胺、二乙胺、乙醇胺、三甲胺、二环己胺、胆碱和咖啡因。Base addition salts according to the present invention refer to salts prepared by adding an inorganic base or an organic base to a free base compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, etc.; the 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, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin. Preferably, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
本发明提供一种组合物，所述组合物包括治疗或预防剂和用于递送所述治疗或预防剂的载体，所述载体包括基于胆酸或其衍生物的脂质，或其药物可用的盐中的一种或多种。The present invention provides a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier comprising a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable agent thereof. One or more of the salts.
具体的，所述治疗或者预防剂被包封在载体内或与载体缔合。Specifically, the therapeutic or preventive agent is encapsulated in or associated with a carrier.
具体的，所述治疗或者预防剂包括核酸分子、基因疫苗、多肽、蛋白、抗体及小分子药物中的一种或多种。Specifically, the therapeutic or preventive agent includes one or more of nucleic acid molecules, genetic vaccines, polypeptides, proteins, antibodies and small molecule drugs.
具体的，所述核酸包括任何形式的核酸分子，包括但不限于单链DNA、双链DNA、短异构体、agomir、antagomir、反义分子、小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、microRNA(miRNA)、Dicer-substrate RNA(dsRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)和本领域已知的其他形式的RNA分子，或锁核酸(LNA)、肽核酸(PNA)和吗啉环寡聚核苷酸等核酸模拟物。Specifically, the nucleic acid includes any form of nucleic acid molecule, including but not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA ( aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of RNA molecules known in the art, or locked nucleic acids Nucleic acid mimics such as (LNA), peptide nucleic acid (PNA) and morpholino cyclic oligonucleotides.
根据一些具体地实施方式，所述治疗或预防剂包含至少一种编码抗原或蛋白或肽的mRNA或其片段或表位。According to some specific embodiments, the therapeutic or preventive agent comprises at least one mRNA encoding an antigen or a protein or a peptide or a fragment or epitope thereof.
更具体地，所述mRNA是单顺反子mRNA或多顺反子mRNA。More specifically, the mRNA is monocistronic or polycistronic.
更具体地，所述抗原是病原性抗原。More specifically, the antigen is a pathogenic antigen.
更具体地，所述mRNA包含一种或多种功能性核苷酸类似物，所述功能性核苷酸类似物包括但不限于假尿嘧啶核苷、1-甲基-假尿嘧啶核苷和5-甲基胞嘧啶中的一种或多种。More specifically, the mRNA contains one or more functional nucleotide analogs, including but not limited to pseudouridine, 1-methyl-pseudouridine and one or more of 5-methylcytosine.
具体地，所述小分子化合物包括但不限于治疗和/或预防剂的有效成分，所述治疗和/或预防剂为现有已知的药物，例如抗肿瘤药、抗感染药、局部麻醉药、抗抑郁药、抗惊厥药、抗生素/抗菌剂、抗真菌药、抗寄生虫药、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼剂、麻醉剂、或造影剂。Specifically, the small molecule compounds include, but are not limited to, the active ingredients of therapeutic and/or preventive agents. The therapeutic and/or preventive agents are currently known drugs, such as anti-tumor drugs, anti-infective drugs, and local anesthetics. , antidepressants, anticonvulsants, antibiotics/antimicrobials, antifungals, antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, anesthetics, or contrast media.
优选地，所述脂质包含三种不同的脂质组分，其中一种脂质是基于胆酸或其衍生物的脂质。优选地，所述脂质还包括带带中性电荷、阴性电荷或双性电荷的辅助脂质。Preferably, the lipids comprise three different lipid components, one of which is a cholic acid or derivative thereof based lipid. Preferably, the lipid further includes an auxiliary lipid with a neutral charge, a negative charge, or a bipolar charge.
更具体地，所述脂质包括磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂、神经酰胺、甾醇及其衍生物中的一种或多种。More specifically, the lipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and derivatives thereof.
更具体地，所述脂质包括但不限于1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱(DMPC)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱(POPC)、11,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)、1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)、2-(((2,3-双(油酰氧基)丙基))磷酸二甲基铵)乙基氢(DOCP)、鞘磷脂(SM)、神经酰胺及其衍生物。所述的脂质为可为合成的或者衍生自天然来源或化合物(自其分离或改性)。More specifically, the lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine base (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 11,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-(((2,3- Bis(oleoyloxy)propyl)dimethylammonium phosphate)ethylhydrogen (DOCP), sphingomyelin (SM), ceramide and its derivatives. The lipids may be synthetic or derived from (isolated or modified) natural sources or compounds.
根据一些具体实施方式，所述载体还包括结构修饰脂质。According to some embodiments, the carrier further includes a structurally modified lipid.
具体地，所述结构修饰脂质主要包括已公开的或未公开的脂质化合物，可以改善脂质体的稳定性并减少脂质体的蛋白质吸收，例如聚乙二醇、葡聚糖、聚乳酸或氨基酸修饰的磷脂酰乙醇胺、磷脂酸、神经酰胺、二烷基胺、二酰基甘油、二烷基甘油中的一种或多种。Specifically, the structurally modified lipids mainly include disclosed or undisclosed lipid compounds, which can improve the stability of liposomes and reduce protein absorption of liposomes, such as polyethylene glycol, dextran, polyethylene glycol, and polyethylene glycol. One or more of lactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine, diacylglycerol, and dialkylglycerol.
更具体地，所述结构修饰脂质可以是PEG-c-DOMG、PEG-DMG、PEG-DLPE、PEGDMPE、PEG-DPPC、PEG-DSPE、神经酰胺-PEG2000、Chol-PEG2000、1-(单甲氧基-聚乙二醇)-2,3-二肉豆蔻基甘油(PEG-DMG)、聚乙二醇化磷脂酰乙醇胺(PEG-PE)、4-O-(2'，3'-二(十四烷酰氧基)丙基-1-O-(ω-甲氧基(聚乙氧基)乙基)丁二酸酯(PEG-S-DMG)、聚乙二醇化神经酰胺(PEG-cer)、ω-甲氧基(聚乙氧基)乙基-N-(2,3-二(十四烷氧基)丙基)氨基甲酸酯、或2,3-二(四癸氧基)丙基-N-(ω-甲氧基)(聚乙氧基)乙基)氨基甲酸酯。More specifically, the structurally modified lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, Chol-PEG2000, 1-(monomethyl Oxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-bis( Tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyglycolated ceramide (PEG- cer), ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate, or 2,3-di(tetradecyloxy) methyl)propyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate.
优选地，所述载体与所述有治疗或预防剂的质量比为5:1～50:1，进一步优选为5:1～35:1，更优选为10:1～30:1。Preferably, the mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 50:1, more preferably 5:1 to 35:1, and more preferably 10:1 to 30:1.
根据前述的组合物，其特征在于，所述载体还包括但不限于胆酸或其衍生物的脂质、电荷辅助脂质以及结构修饰脂质。所述胆酸脂质、所述电荷辅助脂质，以及所述结构修饰脂质的摩尔比为(30～80)：(5～50)：(0.5～10)。According to the aforementioned composition, the carrier further includes, but is not limited to, lipids of cholic acid or its derivatives, charge-assisted lipids, and structurally modified lipids. The molar ratio of the cholic acid lipid, the charge-assisted lipid, and the structurally modified lipid is (30-80): (5-50): (0.5-10).
在一些实施例中，脂质纳米颗粒通过将mRNA溶液和脂质溶液混合而形成。在一些实施例中，脂质纳米颗粒通过切向流过滤进一步纯化。In some embodiments, lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution. In some embodiments, lipid nanoparticles are further purified by tangential flow filtration.
根据前述制备组合物的方法，其中所述脂质纳米颗粒通过将mRNA溶液和脂质溶液混合而形成。其中mRNA溶液的介质为HEPES、磷酸盐、乙酸盐、硫酸铵、碳酸氢钠或柠檬酸盐。脂质溶液的介质为乙醇、异丙醇或二甲亚砜。29.优选地，所述药物组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为20nm～1000nm，优选为40nm～150nm，进一步优选为50nm～100nm，更优选为70nm～100nm。According to the aforementioned method of preparing the composition, the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution. The medium of the mRNA solution is HEPES, phosphate, acetate, ammonium sulfate, sodium bicarbonate or citrate. The medium of lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide. 29. Preferably, the pharmaceutical composition is a nanoparticle preparation, and the average size of the nanoparticle preparation is 20 nm to 1000 nm, preferably 40 nm to 150 nm, further preferably 50 nm to 100 nm, and more preferably 70 nm to 100 nm.
进一步优选地，所述纳米颗粒制剂的多分散指数≤0.5，进一步优选≤0.3，更优选≤0.25。Further preferably, the polydispersity index of the nanoparticle preparation is ≤0.5, further preferably ≤0.3, and more preferably ≤0.25.
本发明的药物组合物通常还包含一种或多种缓冲剂(例如中性缓冲盐水或磷酸盐缓冲液水)、碳水化合物(例如葡萄糖、甘露醇、蔗糖、海藻糖、右旋糖或葡聚糖)、甘露醇、蛋白质、多肽或者氨基酸(例如甘氨酸和赖氨酸)、抗氧化剂(维生素E和丁基羟基甲苯)、抑菌剂、螯合剂(例如EDTA和谷胱甘肽)、佐剂(例如氢氧化铝)、使制剂与接受者的血液等渗的助悬剂/增稠剂/防腐剂等，或者可以将本发明的组合物配制成冻干物。Pharmaceutical compositions of the present invention also typically include one or more buffers (eg neutral buffered saline or phosphate buffered water), carbohydrates (eg glucose, mannitol, sucrose, trehalose, dextrose or dextran). sugar), mannitol, proteins, peptides or amino acids (such as glycine and lysine), antioxidants (vitamin E and butylated hydroxytoluene), bacteriostatic agents, chelating agents (such as EDTA and glutathione), adjuvants (e.g. aluminum hydroxide), suspending agents/thickening agents/preservatives, etc. that make the formulation isotonic with the recipient's blood, or the composition of the invention can be formulated as a lyophilisate.
本发明技术方案提供了一类全新的用于递送治疗或预防用药物的脂质化合物，即基于胆酸或其衍生物的脂质，本发明技术方案不同于国外医药公司的现有专利技术方案，丰富了脂质化合物种类，为核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物等的递送提供了更多选择，并且能够与国外辉瑞公司、莫德纳公司的技术路线具有明显区别。The technical solution of the present invention provides a new class of lipid compounds for delivering therapeutic or preventive drugs, namely lipids based on cholic acid or its derivatives. The technical solution of the present invention is different from the existing patented technical solutions of foreign pharmaceutical companies. , enriches the types of lipid compounds, provides more options for the delivery of nucleic acid drugs, gene vaccines, peptides, proteins, antibodies and small molecule drugs, and can be significantly different from the technical routes of foreign companies such as Pfizer and Moderna. .
附图说明Description of the drawings
图1为熊去氧胆酸衍生物1(化合物1)核磁共振氢谱图；Figure 1 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 1 (compound 1);
图2为熊去氧胆酸衍生物2(化合物2)核磁共振氢谱图；Figure 2 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 2 (compound 2);
图3为熊去氧胆酸衍生物7(化合物7)核磁共振氢谱图；Figure 3 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 7 (compound 7);
图4为熊去氧胆酸衍生物9(化合物9)核磁共振氢谱图；Figure 4 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 9 (compound 9);
图5为熊去氧胆酸衍生物10(化合物10)核磁共振氢谱图；Figure 5 is a proton nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 10 (compound 10);
图6为熊去氧胆酸衍生物13(化合物13)核磁共振氢谱图；Figure 6 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 13 (compound 13);
图7为熊去氧胆酸衍生物16(化合物16)核磁共振氢谱图；Figure 7 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 16 (compound 16);
图8为熊去氧胆酸衍生物17(化合物17)核磁共振氢谱图；Figure 8 is a hydrogen nuclear magnetic resonance spectrum of ursodeoxycholic acid derivative 17 (compound 17);
图9为奥贝胆酸衍生物18(化合物18)核磁共振氢谱图；Figure 9 is a proton nuclear magnetic resonance spectrum of obeticholic acid derivative 18 (compound 18);
图10为LNP包载cy3-siRNA转染细胞实验结果，其中，a)荧光显微镜明场,b)荧光显微镜暗场，c)细胞流式图；Figure 10 shows the experimental results of cells transfected with LNP-encapsulated cy3-siRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry;
图11为LNP包载EGFP mRNA转染细胞实验结果，其中，a)荧光显微镜明场,b)荧光显微镜暗场，c)细胞流式图；Figure 11 shows the experimental results of cells transfected with LNP-encapsulated EGFP mRNA, including a) bright field of fluorescence microscope, b) dark field of fluorescence microscope, c) cell flow cytometry;
图12为包载Luciferase mRNA的LNP,小鼠静脉注射后12h荧光成像结果；Figure 12 shows the fluorescence imaging results of LNP containing Luciferase mRNA 12h after intravenous injection in mice;
图13为包载Luciferase mRNA的LNP,小鼠肌肉注射后12h荧光成像结果。Figure 13 shows the fluorescence imaging results of LNP encapsulating Luciferase mRNA 12 hours after intramuscular injection in mice.
具体实施方式Detailed ways
胆酸是人和哺乳动物体内天然普遍存在的甾类分子，由胆固醇在肝脏中合成。进食后，胆酸通过肝细胞的主动分泌，随胆汁进入胆囊，再由胆囊进入肠道发挥它的消化功能，胆酸以钠盐形式进入小肠，帮助脂类的消化吸收，随后在回肠末端通过主动吸收或被动运输的方式经门静脉回到肝脏，在肝细胞内进行加工转化，然后同新合成的胆汁酸一起又被分泌入小肠。胆汁酸的这种EHC(肝肠循环)过程每天会循环4～12次，大约有95％的胆汁酸被回吸利用。如果胆汁酸的EHC遭到破坏，不仅会影响体内脂类的消化吸收，也会使机体形成胆固醇结石。因此基于胆酸设计的化合物递送载体最大的优势就是肝肠循环效率高，参与胆酸的肝肠循环，从而提高药物在肝脏和胆囊中的吸收。胆酸结构式如下。Cholic acid is a naturally ubiquitous steroid molecule in humans and mammals, synthesized in the liver from cholesterol. After eating, bile acid is actively secreted by liver cells, enters the gallbladder with bile, and then enters the intestine from the gallbladder to perform its digestive function. Cholic acid enters the small intestine in the form of sodium salt to help digest and absorb lipids, and then passes through the terminal ileum. It is returned to the liver through the portal vein through active absorption or passive transport, processed and transformed in liver cells, and then secreted into the small intestine together with newly synthesized bile acids. This EHC (enterohepatic circulation) process of bile acids circulates 4 to 12 times a day, and about 95% of the bile acids are reabsorbed and utilized. If the EHC of bile acids is destroyed, it will not only affect the digestion and absorption of lipids in the body, but also cause the body to form cholesterol stones. Therefore, the biggest advantage of compound delivery carriers designed based on bile acid is that it has high enterohepatic circulation efficiency and participates in the enterohepatic circulation of bile acid, thereby improving drug absorption in the liver and gallbladder. The structural formula of cholic acid is as follows.
Figure PCTCN2022107846-appb-000003
Figure PCTCN2022107846-appb-000003
胆酸分子中甾体骨架上的3个六元环和1个五元环在同一平面上,其中环A和环B反向连接,使分子形成1个穴状结构。分子中3个甲基分布在甾环所在平面的一边,形成分子憎水部分,3个羟基分布在甾环所在平面的另一边,同C24位羧基一起形成分子的亲水部分。The three six-membered rings and one five-membered ring on the steroid skeleton of the cholic acid molecule are on the same plane, and ring A and ring B are connected in reverse, making the molecule form a cave-like structure. In the molecule, three methyl groups are distributed on one side of the plane where the steroid ring is located, forming the hydrophobic part of the molecule. Three hydroxyl groups are distributed on the other side of the plane where the steroid ring is located, and together with the C24 carboxyl group, form the hydrophilic part of the molecule.
胆酸分子的特殊结构,决定了它具有双亲性、酸碱性,并容易进行化学修饰，因此，本专利利用胆酸作为构筑基元用来制备高分子或者低聚物，这些基于胆酸的功能性分子在药物传输具有良好的技术效果。The special structure of the cholic acid molecule determines that it is amphiphilic, acid-base, and easy to undergo chemical modification. Therefore, this patent uses cholic acid as a building block to prepare polymers or oligomers. These cholic acid-based products Functional molecules have good technical effects in drug delivery.
胆酸类药物已经在中国或美国市场上市多年，包括熊去氧胆酸、奥贝胆酸、鹅去氧胆酸、牛磺熊去氧胆酸。如熊去氧胆酸，用于治疗胆固醇型胆结石及胆汁反流性胃炎；奥贝胆酸，用于治疗原发性胆汁性肝硬化(PBC)，针对熊去氧 胆酸没有充分应答或不能耐受的患者。多年的临床应用表明，胆酸类化合物具有较好的安全性。另外，胆酸类化合物具有亲水亲油的两亲性质，通过包裹或偶联的方式可以提高小分子化学药物的生物利用度。Cholic acid drugs have been on the market in China or the United States for many years, including ursodeoxycholic acid, obeticholic acid, chenodeoxycholic acid, and tauroursodeoxycholic acid. For example, ursodeoxycholic acid is used to treat cholesterol gallstones and bile reflux gastritis; obeticholic acid is used to treat primary biliary cirrhosis (PBC), and there is no adequate response to ursodeoxycholic acid or Patients who cannot tolerate it. Years of clinical application have shown that cholic acid compounds have good safety. In addition, cholic acid compounds have hydrophilic and lipophilic amphiphilic properties, and can improve the bioavailability of small molecule chemical drugs through encapsulation or coupling.
胆酸类化合物与胆固醇同属于甾体类化合物，结构相似，并且可进行化学修饰的位点更多。将胆酸类化合物进行结构修饰制备新的脂质成分，有可能替代原四组分LNP中的可离子化脂和胆固醇，达到相似或更优的递送mRNA效果。本专利研究基于胆酸类似物构建脂质纳米颗粒载体，并探索其在mRNA药物递送中的应用。本发明的LNP可以简化生产流程，降低成本；同时可以避开四组分LNP的专利封锁，有利于推动核酸药物的国产化。Cholic acid compounds and cholesterol are both steroidal compounds with similar structures and more sites that can be chemically modified. Structural modification of cholic acid compounds to prepare new lipid components may potentially replace the ionizable lipids and cholesterol in the original four-component LNP to achieve similar or better mRNA delivery effects. This patented study constructs lipid nanoparticle carriers based on bile acid analogs and explores their application in mRNA drug delivery. The LNP of the present invention can simplify the production process and reduce costs; at the same time, it can avoid the patent blockade of four-component LNP, which is beneficial to promoting the localization of nucleic acid drugs.
因为胆酸或其衍生物均由一个刚性甾环和一个脂肪侧链组成，其中甾环包括三个六元环和一个五元环。只是根据来源的不同，胆酸的侧链结构、甾环构象、羟基的数目及在甾环上的朝向会有所不同。胆酸或其衍生物的共有的羟基和脂肪侧链的羧基，均是很好的化学修饰位点。因此我们认为胆酸或其衍生物均可以通过一些共同的修饰，达到预期目的。Because cholic acid or its derivatives are composed of a rigid steroid ring and an aliphatic side chain, the steroid ring includes three six-membered rings and one five-membered ring. However, depending on the source, the side chain structure of cholic acid, the conformation of the steroid ring, the number of hydroxyl groups and the orientation of the steroid ring will be different. The common hydroxyl group of cholic acid or its derivatives and the carboxyl group of the fatty side chain are good chemical modification sites. Therefore, we believe that cholic acid or its derivatives can achieve the desired purpose through some common modifications.
本专利中的胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠，具体结构如下。Cholic acid or its derivatives in this patent is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, 3β-hydroxy-D5-cholic acid, chenodeoxycholic acid, and lithocholic acid. Cholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholancholic acid, glycochenodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholic acid Cholic acid, glycocholic acid, hyodeoxycholic acid, methyl hyodeoxycholate, sodium taurodeoxycholate, sodium dehydrocholate, sodium cholate, sodium deoxyglycholate, sodium taurodeoxycholate , sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, sodium tauroursodeoxycholate and sodium taurolithocholate , the specific structure is as follows.
Figure PCTCN2022107846-appb-000004
Figure PCTCN2022107846-appb-000004
Figure PCTCN2022107846-appb-000005
Figure PCTCN2022107846-appb-000005
Figure PCTCN2022107846-appb-000006
Figure PCTCN2022107846-appb-000006
本专利所述的脂质化合物为选自如下结构所示化合物的一种或多种；The lipid compound described in this patent is one or more compounds selected from the following structures;
①熊去氧胆酸衍生物脂质：① Ursodeoxycholic acid derivative lipid:
Figure PCTCN2022107846-appb-000007
Figure PCTCN2022107846-appb-000007
Figure PCTCN2022107846-appb-000008
Figure PCTCN2022107846-appb-000008
Figure PCTCN2022107846-appb-000009
Figure PCTCN2022107846-appb-000009
Figure PCTCN2022107846-appb-000010
Figure PCTCN2022107846-appb-000010
Figure PCTCN2022107846-appb-000011
Figure PCTCN2022107846-appb-000011
Figure PCTCN2022107846-appb-000012
Figure PCTCN2022107846-appb-000012
Figure PCTCN2022107846-appb-000013
Figure PCTCN2022107846-appb-000013
Figure PCTCN2022107846-appb-000014
Figure PCTCN2022107846-appb-000014
Figure PCTCN2022107846-appb-000015
Figure PCTCN2022107846-appb-000015
Figure PCTCN2022107846-appb-000016
Figure PCTCN2022107846-appb-000016
②奥贝胆酸衍生物脂质：② Obeticholic acid derivative lipids:
Figure PCTCN2022107846-appb-000017
Figure PCTCN2022107846-appb-000017
Figure PCTCN2022107846-appb-000018
Figure PCTCN2022107846-appb-000018
Figure PCTCN2022107846-appb-000019
Figure PCTCN2022107846-appb-000019
Figure PCTCN2022107846-appb-000020
Figure PCTCN2022107846-appb-000020
Figure PCTCN2022107846-appb-000021
Figure PCTCN2022107846-appb-000021
Figure PCTCN2022107846-appb-000022
Figure PCTCN2022107846-appb-000022
Figure PCTCN2022107846-appb-000023
Figure PCTCN2022107846-appb-000023
Figure PCTCN2022107846-appb-000024
Figure PCTCN2022107846-appb-000024
Figure PCTCN2022107846-appb-000025
Figure PCTCN2022107846-appb-000025
Figure PCTCN2022107846-appb-000026
Figure PCTCN2022107846-appb-000026
下面结合实施例对本发明作进一步描述，但本发明并不限于以下实施例。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未做注明的实施条件为本行业的常规条件。本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互结合。The present invention will be further described below with reference to examples, but the present invention is not limited to the following examples. The implementation conditions used in the embodiments can be further adjusted according to different requirements for specific use. Implementation conditions that are not noted are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.
实施例1：熊去氧胆酸衍生物1(化合物1)的合成Example 1: Synthesis of Ursodeoxycholic Acid Derivative 1 (Compound 1)
Figure PCTCN2022107846-appb-000027
Figure PCTCN2022107846-appb-000027
将熊去氧胆酸(393mg,1mmol)溶解于DMF(8mL)中,依次加入HBTU(569mg,1.5mmol),DIEA(194mg,1.5mmol),N,N-二甲基乙二胺(132mg,1.5mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH3OH(10:1)洗脱，得白色固体(333mg,72％)。 1HNMR(400MHz,CD 3OD,)δ3.29-3.44(m,3H),2.89(t,J＝8.0Hz,2H),2.64(s,6H,CH 3×2),0.95-0.97(m,6H,CH 3×2),0.70(s,3H,CH 3).ESI-MS m/z Calc.C 28H 51N 2O 3[M+H] +463.72,Found 464.15.化合物1的核磁共振氢谱图见说明书附图中的图1。 Dissolve ursodeoxycholic acid (393mg, 1mmol) in DMF (8mL), add HBTU (569mg, 1.5mmol), DIEA (194mg, 1.5mmol), N,N-dimethylethylenediamine (132mg, 1.5 mmol), under nitrogen protection, stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH3OH (10:1), gave a white solid (333 mg, 72%). 1 HNMR (400MHz, CD 3 OD,) δ3.29-3.44 (m, 3H), 2.89 (t, J = 8.0Hz, 2H), 2.64 (s, 6H, CH 3 × 2), 0.95-0.97 (m ,6H,CH 3 ×2),0.70(s,3H,CH 3 ).ESI-MS m/z Calc.C 28 H 51 N 2 O 3 [M+H] + 463.72, Found 464.15. NMR of compound 1 The resonance hydrogen spectrum is shown in Figure 1 in the accompanying drawings of the specification.
实施例2：熊去氧胆酸衍生物2(化合物2)的合成Example 2: Synthesis of Ursodeoxycholic Acid Derivative 2 (Compound 2)
Figure PCTCN2022107846-appb-000028
Figure PCTCN2022107846-appb-000028
将熊去氧胆酸(393mg,1mmol)溶解于DMF(8mL)中,依次加入HBTU(569mg,1.5mmol),DIEA(194mg,1.5mmol),4-吡咯-1-丁胺(213mg,1.5mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩 溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体产品X-4(403mg,78％)。 1HNMR(400MHz,CD 3OD)δ5.52(s,1H,CONH),3.50-3.54(m,2H),3.22-3.26(m,4H),2.64(s,6H,CH 3×2),1.00-1.02(m,6H,CH 3×2),0.75(s,3H CH3).ESI-MS m/z Calc.C 33H 57N 2O 5[M+HCOO] -561.43,Found 561.35。化合物2的核磁共振氢谱图见说明书附图中的图2. Ursodeoxycholic acid (393 mg, 1 mmol) was dissolved in DMF (8 mL), and HBTU (569 mg, 1.5 mmol), DIEA (194 mg, 1.5 mmol), and 4-pyrrole-1-butylamine (213 mg, 1.5 mmol) were added in sequence. ), under nitrogen protection, the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave white solid product X-4 (403 mg, 78%). 1 HNMR (400MHz, CD 3 OD) δ5.52 (s, 1H, CONH), 3.50-3.54 (m, 2H), 3.22-3.26 (m, 4H), 2.64 (s, 6H, CH 3 × 2), 1.00-1.02(m,6H,CH 3 ×2),0.75(s,3H CH3).ESI-MS m/z Calc.C 33 H 57 N 2 O 5 [M+HCOO] - 561.43, Found 561.35. The proton nuclear magnetic resonance spectrum of compound 2 is shown in Figure 2 in the accompanying drawings of the description.
实施例3：熊去氧胆酸衍生物7(化合物7)的合成Example 3: Synthesis of Ursodeoxycholic Acid Derivative 7 (Compound 7)
Figure PCTCN2022107846-appb-000029
Figure PCTCN2022107846-appb-000029
将熊去氧胆酸(393mg,1mmol)溶解于乙腈(10mL)中,依次加入K 2CO 3(415mg,3mmol),BnBr(850mg,5mmol),氮气保护，80℃搅拌反应5h。TLC检测，反应结束后，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(1:1)洗脱，得白色固体产品化合物6(390mg,81％)。 1HNMR(400MHz,CDCl 3)δ7.32-7.36(m,5H),5.08-5.15(m,2H),3.55-3.62(m,2H),0.94(s,3H,CH 3),0.91(d,J＝4Hz,3H,CH 3),0.65(s,3H,CH 3). Ursodeoxycholic acid (393 mg, 1 mmol) was dissolved in acetonitrile (10 mL), K 2 CO 3 (415 mg, 3 mmol), BnBr (850 mg, 5 mmol) were added in sequence, protected by nitrogen, and the reaction was stirred at 80°C for 5 hours. TLC detection, after the reaction is completed, filter and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave compound 6 (390 mg, 81%) as a white solid product. 1 HNMR (400MHz, CDCl 3 ) δ7.32-7.36(m,5H),5.08-5.15(m,2H),3.55-3.62(m,2H),0.94(s,3H,CH 3 ),0.91(d ,J＝4Hz,3H,CH 3 ),0.65(s,3H,CH 3 ).
4-二甲基氨基丁酸盐酸盐(336mg,2mmol)溶解于无水DCM(8mL)中，加入草酰氯(1mL)，室温搅拌4h。真空浓缩至无溶剂,用无水DCM(3mL)溶解,备用。把化合物6(241mg,0.5mmol)，TEA(202mg,2mmol)溶解于无水DCM(3mL)中，将上述备用产物滴入反应液中，室温反应过夜。加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体产品化合物7(213mg,60％)。 1HNMR(400MHz,d 6-DMSO)δ7.33-7.37(m,5H),5.04-5.11(nm,2H),4.59-4.68 (m,2H),2.92-2.98(m,4H),2.66(s,6H),2.65(s,6H),0.93(s,3H,CH 3),0.87(d,J＝8Hz,3H,CH 3),0.60(s,3H,CH 3)；ESI-MS m/z Calc.C 43H 69N 2O 6[M+H] +709.52,Found 709.85.化合物7的核磁公正氢谱图见说明书附图中的图3。 4-Dimethylaminobutyric hydrochloride (336 mg, 2 mmol) was dissolved in anhydrous DCM (8 mL), oxalyl chloride (1 mL) was added, and stirred at room temperature for 4 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (3mL), and set aside. Dissolve compound 6 (241 mg, 0.5 mmol) and TEA (202 mg, 2 mmol) in anhydrous DCM (3 mL). Add the above standby product dropwise into the reaction solution and react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 7 as a white solid product (213 mg, 60%). 1 HNMR (400MHz, d 6 -DMSO) δ7.33-7.37(m,5H),5.04-5.11(nm,2H),4.59-4.68 (m,2H),2.92-2.98(m,4H),2.66( s,6H),2.65(s,6H),0.93(s,3H,CH 3 ),0.87(d,J＝8Hz,3H,CH 3 ),0.60(s,3H,CH 3 ); ESI-MS m /z Calc.C 43 H 69 N 2 O 6 [M+H] + 709.52, Found 709.85. The nuclear magnetic spectrum of compound 7 is shown in Figure 3 in the appendix of the description.
实施例4：熊去氧胆酸衍生物9(化合物9)的合成Example 4: Synthesis of Ursodeoxycholic Acid Derivative 9 (Compound 9)
Figure PCTCN2022107846-appb-000030
Figure PCTCN2022107846-appb-000030
将熊去氧胆酸(785mg,1mmol)溶解于DMF(20mL)中,依次加入K 2CO 3(829mg,6mmol)，CH 3I(852mg,6mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(1:1)洗脱，得白色固体产品化合物8(700mg,86％)。 Ursodeoxycholic acid (785 mg, 1 mmol) was dissolved in DMF (20 mL), K 2 CO 3 (829 mg, 6 mmol), CH 3 I (852 mg, 6 mmol) were added in sequence, protected by nitrogen, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE/EA (1:1), gave compound 8 (700 mg, 86%) as a white solid product.
4-二甲基氨基丁酸盐酸盐(336mg,2mmol)溶解于无水DCM(8mL)中，加入草酰氯(0.5mL)，室温搅拌4h。真空浓缩至无溶剂,用无水DCM(3mL)溶解,备用。把化合物8(203mg,0.5mmol)，TEA(202mg,2mmol)溶解于无水DCM(3mL)中，将上述备用产物滴入反应液中，室温反应过夜。加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体产品化合物9(221mg,70％)。 1HNMR(400MHz,d 6-DMSO)δ4.58-4.70(m,2H),3.57(s,3H),2.63(s,6H),2.61(s,6H),0.93(s,3H,CH 3),0.88(d,J＝4Hz,3H,CH 3),0.63(s,3H,CH 3)；ESI-MS m/z  Calc.C 37H 65N 2O 6[M+H] +633.48,Found 634.15.化合物-9的核磁共振氢谱图见说明书附图中的图4。 4-Dimethylaminobutyric hydrochloride (336 mg, 2 mmol) was dissolved in anhydrous DCM (8 mL), oxalyl chloride (0.5 mL) was added, and stirred at room temperature for 4 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (3mL), and set aside. Compound 8 (203 mg, 0.5 mmol) and TEA (202 mg, 2 mmol) were dissolved in anhydrous DCM (3 mL). The above standby product was dropped into the reaction solution and allowed to react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 9 as a white solid product (221 mg, 70%). 1 HNMR (400MHz, d 6 -DMSO) δ4.58-4.70 (m, 2H), 3.57 (s, 3H), 2.63 (s, 6H), 2.61 (s, 6H), 0.93 (s, 3H, CH 3 ),0.88(d,J＝4Hz,3H,CH 3 ),0.63(s,3H,CH 3 ); ESI-MS m/z Calc.C 37 H 65 N 2 O 6 [M+H] + 633.48, Found 634.15. The hydrogen nuclear magnetic resonance spectrum of compound-9 is shown in Figure 4 in the accompanying drawings of the description.
实施例5：熊去氧胆酸衍生物10(化合物10)的合成Example 5: Synthesis of Ursodeoxycholic Acid Derivative 10 (Compound 10)
Figure PCTCN2022107846-appb-000031
Figure PCTCN2022107846-appb-000031
亚油酸(476mg,1.7mmol)溶解于无水DCM(5mL)中，加入草酰氯(0.30mL)，室温搅拌5h。真空浓缩至无溶剂,用无水DCM(2mL)溶解,备用。把化合物2(240mg,0.34mmol)，TEA(69mg,0.68mmol)溶解于无水DCM(5mL)中，将上述备用产物滴入反应液中，室温反应过夜。加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得淡黄色半固体产品化合物10(212mg,60％)。 1HNMR(400MHz,CD 3OD)δ5.34-5.36(m,8H),1.01(s,3H,CH 3),0.98(d,J＝4Hz,3H,CH 3),0.72(s,3H,CH 3)；ESI-MS m/z Calc.C 68H 119N 2O 6[M+H 2O+H] +1059.91,Found 1060.50.化合物10的核磁共振氢谱图见说明书附图中的图5。 Linoleic acid (476 mg, 1.7 mmol) was dissolved in anhydrous DCM (5 mL), oxalyl chloride (0.30 mL) was added, and the mixture was stirred at room temperature for 5 h. Concentrate under vacuum to no solvent, dissolve with anhydrous DCM (2mL), and set aside. Compound 2 (240 mg, 0.34 mmol) and TEA (69 mg, 0.68 mmol) were dissolved in anhydrous DCM (5 mL). The above standby product was dropped into the reaction solution and allowed to react at room temperature overnight. Add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 10, a light yellow semi-solid product (212 mg, 60%). 1 HNMR (400MHz, CD 3 OD) δ5.34-5.36 (m, 8H), 1.01 (s, 3H, CH 3 ), 0.98 (d, J = 4Hz, 3H, CH 3 ), 0.72 (s, 3H, CH 3 ); ESI-MS m/z Calc.C 68 H 119 N 2 O 6 [M+H 2 O+H] + 1059.91, Found 1060.50. The hydrogen nuclear magnetic resonance spectrum of compound 10 is shown in the figure in the appendix of the description. 5.
实施例6：熊去氧胆酸衍生物13(化合物13)的合成Example 6: Synthesis of Ursodeoxycholic Acid Derivative 13 (Compound 13)
Figure PCTCN2022107846-appb-000032
Figure PCTCN2022107846-appb-000032
中间体化合物11的合成：将化合物6(3.0g,6.2mmol)溶解于干燥的吡啶(20mL)中,依次加入DMAP(159mg,1.3mmol),乙酸酐(3.2g,31mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(2:1)洗脱，得白色固体产品化合物11(3.0g,86％)。 1HNMR(400MHz,CDCl 3)δ7.33-7.37(m,5H),5.08-5.15(m,2H),4.74-4.79(m,1H),4.64-4.70(m,1H)，2.03(s,3H),1.90(s,3H),0.97(s,3H),0.91(d,J＝8.0Hz,3H),0.75(s,3H). Synthesis of intermediate compound 11: Dissolve compound 6 (3.0g, 6.2mmol) in dry pyridine (20mL), add DMAP (159mg, 1.3mmol), acetic anhydride (3.2g, 31mmol) in sequence, under nitrogen protection, at room temperature The reaction was stirred overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE/EA (2:1), gave compound 11 (3.0 g, 86%) as a white solid product. 1 HNMR (400MHz, CDCl 3 ) δ7.33-7.37(m,5H),5.08-5.15(m,2H),4.74-4.79(m,1H),4.64-4.70(m,1H), 2.03(s, 3H),1.90(s,3H),0.97(s,3H),0.91(d,J=8.0Hz,3H),0.75(s,3H).
中间体化合物12的合成：将化合物11(1.7g,3.0mmol)溶解于干燥的甲醇(40mL)中,加入K 2CO 3(0.83g,6.0mmol),室温搅拌反应2h。TLC检测，反应结束后，过滤，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，PE/EA(2:1)洗脱，得白色固体产品化合物12(1.1g,70％)。 1HNMR(400MHz,CDCl 3)δ4.74-4.81(m,1H),3.66(s,3H),3.54-3.62(m,1H),1.98(s,3H)，0.96(s,3H),0.91(d,J＝4.0Hz,3H),0.68(s,3H). Synthesis of intermediate compound 12: Dissolve compound 11 (1.7g, 3.0mmol) in dry methanol (40mL), add K 2 CO 3 (0.83g, 6.0mmol), and stir for 2 hours at room temperature. TLC detection, after the reaction is completed, filter, add an appropriate amount of water, extract three times with ethyl acetate, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with PE/EA (2:1), gave compound 12 (1.1 g, 70%) as a white solid product. 1 HNMR (400MHz, CDCl 3 ) δ4.74-4.81(m,1H),3.66(s,3H),3.54-3.62(m,1H),1.98(s,3H),0.96(s,3H),0.91 (d,J＝4.0Hz,3H),0.68(s,3H).
将化合物12(1.0g,2.2mmol)溶解于DMF(8mL)中,依次加入HBTU(1.15g,3.0mmol),DIEA(786mg,6.1mmol),4-二甲基氨基丁酸盐酸盐(509mg,3.0mmol)氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体产品化合物13(900mg,73％)。 1HNMR(400MHz,d 6-DMSO)δ4.56-4.68(m,2H),3.57(s,3H,OCH 3),3.02-3.06(m,2H),2.77(s,6H),2.37(t,J＝8.0Hz,2H),1.94(s,3H),0.93(s,3H,CH 3),0.87(d,J＝4Hz,3H,CH 3),0.63(s,3H,CH 3)；ESI-MS m/z Calc.C 33H 56N 2O 6[M+H] +562.41,Found 562.95.化合物13的核磁共振氢谱图见说明书附图中的图6。 Compound 12 (1.0g, 2.2mmol) was dissolved in DMF (8mL), and HBTU (1.15g, 3.0mmol), DIEA (786mg, 6.1mmol), and 4-dimethylaminobutyric hydrochloride (509mg) were added in sequence. , 3.0 mmol) under nitrogen protection, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave compound 13 (900 mg, 73%) as a white solid product. 1 HNMR (400MHz, d 6 -DMSO) δ4.56-4.68(m,2H),3.57(s,3H,OCH 3 ),3.02-3.06(m,2H),2.77(s,6H),2.37(t ,J＝8.0Hz,2H),1.94(s,3H),0.93(s,3H,CH 3 ),0.87(d,J＝4Hz,3H,CH 3 ),0.63(s,3H,CH 3 ); ESI-MS m/z Calc.C 33 H 56 N 2 O 6 [M+H] + 562.41, Found 562.95. The hydrogen nuclear magnetic resonance spectrum of compound 13 is shown in Figure 6 in the appendix of the description.
实施例7：熊去氧胆酸衍生物16(化合物16)的合成Example 7: Synthesis of Ursodeoxycholic Acid Derivative 16 (Compound 16)
Figure PCTCN2022107846-appb-000033
Figure PCTCN2022107846-appb-000033
将化合物6(965mg,2.0mmol)溶解于DMF(30mL)中,依次加入HBTU(1.15g,3.0mmol),DIEA(517mg,4.0mmol),4-二甲基氨基丁酸盐酸盐(335mg,2.0mmol)氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体产品化合物13(400mg,33％). 1HNMR(400MHz,d 6-DMSO)δ7.33-7.38(m,5H),5.05-5.12(m,2H),4.55-4.63(m,1H),3.92(d,J＝8.0Hz,1H),2.98-3.02(m,2H),2.74(s,6H),2.23-2.43(m,4H),0.91(s,3H,CH 3),0.87(d,J＝4Hz,3H,CH 3),0.59(s,3H,CH 3)；ESI-MS m/z Calc.C 37H 58NO 5[M+H] +596.4,Found 597.3.化合物16的核磁共振氢谱图见说明书附图中的图7。 Compound 6 (965mg, 2.0mmol) was dissolved in DMF (30mL), and HBTU (1.15g, 3.0mmol), DIEA (517mg, 4.0mmol), 4-dimethylaminobutyric hydrochloride (335mg, 2.0 mmol) under nitrogen protection, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave the white solid product compound 13 (400 mg, 33%). 1 HNMR (400MHz, d 6 -DMSO) δ7.33-7.38 (m, 5H) ),5.05-5.12(m,2H),4.55-4.63(m,1H),3.92(d,J＝8.0Hz,1H),2.98-3.02(m,2H),2.74(s,6H),2.23- 2.43(m,4H),0.91(s,3H,CH 3 ),0.87(d,J＝4Hz,3H,CH 3 ),0.59(s,3H,CH 3 ); ESI-MS m/z Calc.C 37 H 58 NO 5 [M+H] + 596.4, Found 597.3. The hydrogen nuclear magnetic resonance spectrum of compound 16 is shown in Figure 7 in the appendix of the description.
实施例8：熊去氧胆酸衍生物17(化合物17)的合成Example 8: Synthesis of Ursodeoxycholic Acid Derivative 17 (Compound 17)
Figure PCTCN2022107846-appb-000034
Figure PCTCN2022107846-appb-000034
将化合物4(200mg,0.39mmol)溶解于干燥的吡啶(4mL)中,依次加入DMAP(19mg,0.16mmol),乙酸酐(0.5mL)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用DCM萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体产品化合物17(100mg,44％)。 1HNMR(400MHz,d 6-DMSO)δ4.52-4.60(m,1H),4.63-4.68(m,2H),3.08-3.12(m,2H),3.02-3.07(m,2H),1.98(s,3H),1.94(s,3H),0.93(s,3H),0.89(d,J＝8.0Hz,3H),0.63(s,3H).化合物17的核磁共振氢谱图见说明书附图中的图8。 Compound 4 (200 mg, 0.39 mmol) was dissolved in dry pyridine (4 mL), DMAP (19 mg, 0.16 mmol) and acetic anhydride (0.5 mL) were added in sequence, under nitrogen protection, and the reaction was stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with DCM three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluted with DCM/CH 3 OH (10:1), gave compound 17 (100 mg, 44%) as a white solid product. 1 HNMR (400MHz, d 6 -DMSO) δ4.52-4.60(m,1H),4.63-4.68(m,2H),3.08-3.12(m,2H),3.02-3.07(m,2H),1.98( s, 3H), 1.94 (s, 3H), 0.93 (s, 3H), 0.89 (d, J = 8.0Hz, 3H), 0.63 (s, 3H). The hydrogen nuclear magnetic resonance spectrum of compound 17 is shown in the attached figure of the description. Figure 8 in.
实施例9：奥贝胆酸衍生物(化合物18)的合成Example 9: Synthesis of Obeticholic Acid Derivative (Compound 18)
Figure PCTCN2022107846-appb-000035
Figure PCTCN2022107846-appb-000035
将奥贝胆酸(421mg,1mmol)溶解于DMF(8mL)中,依次加入HBTU(569mg,1.5mmol),DIEA(194mg,1.5mmol),N,N-二甲基乙二胺(132mg,1.5mmol)，氮气保护，室温搅拌反应过夜。TLC检测，反应结束后，加入适量水，用乙酸乙酯萃取3次，合并有机层，无水硫酸钠干燥，滤过，浓缩溶剂得粗品。硅胶柱层析，DCM/CH 3OH(10:1)洗脱，得白色固体(343mg,70％)。 1HNMR(400MHz,d 6-DMSO)δ7.83(brs,1H),4.31(brs,1H),4.05(d,J＝4.0Hz,1H),3.49(s,2H),2.36(s 2H),2.36(s,6H),0.88(d,J＝4.0Hz,3H),0.81-0.83(m,6H),0.60(s,3H).ESI-MS m/z Calc.C 30H 56N 2O 3[M+H] +491.42,Found 492.20.化合物18的核磁共振氢谱图见说明书附图中的图9。 Dissolve obeticholic acid (421mg, 1mmol) in DMF (8mL), then add HBTU (569mg, 1.5mmol), DIEA (194mg, 1.5mmol), N,N-dimethylethylenediamine (132mg, 1.5 mmol), under nitrogen protection, and stirred at room temperature overnight. TLC detection, after the reaction is completed, add an appropriate amount of water, extract with ethyl acetate three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, and concentrate the solvent to obtain a crude product. Silica gel column chromatography, eluting with DCM/CH 3 OH (10:1), gave a white solid (343 mg, 70%). 1 HNMR (400MHz, d 6 -DMSO) δ7.83 (brs, 1H), 4.31 (brs, 1H), 4.05 (d, J = 4.0Hz, 1H), 3.49 (s, 2H), 2.36 (s 2H) ,2.36(s,6H),0.88(d,J＝4.0Hz,3H),0.81-0.83(m,6H),0.60(s,3H).ESI-MS m/z Calc.C 30 H 56 N 2 O 3 [M+H] + 491.42, Found 492.20. The hydrogen nuclear magnetic resonance spectrum of compound 18 is shown in Figure 9 in the accompanying drawings of the description.
实施例10：纳米颗粒组合体的制备与测试Example 10: Preparation and testing of nanoparticle assemblies
1)纳米粒子组合物的制备1) Preparation of nanoparticle composition
为了研究用于将治疗剂和/或预防剂递送至细胞的安全且有效的纳米粒子组合物，制备并测试一系列配制物。确切地说，对纳米粒子组合物的脂质组分中的特定成分和其比率进行优化。To investigate safe and effective nanoparticle compositions for delivering therapeutic and/or prophylactic agents to cells, a series of formulations were prepared and tested. Specifically, specific ingredients and their ratios in the lipid component of the nanoparticle composition are optimized.
纳米粒子可以通过混合方法如微流体化和T型接头混合两种流体流制得，所述两种流体流之一含有治疗剂和/或预防剂且另一种具有脂质组分。通过在乙醇中合并根据实施例1-9中合成的脂质、磷脂(如DOPE或DSPC，可从Avanti Polar Lipids(Alabaster,AL)获得)、PEG脂质(如1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇，又称为PEG-DMG，可从Avanti Polar Lipids(Alabaster,AL)获得)和结构性脂质(如胆固醇，可从Sigma-Aldrich(Taufkirchen,Germany)获得；或皮质类固醇(如泼尼松龙、地塞米松、泼尼龙和氢化可的松)；或其组合)，制备出浓度为约50mM的脂质组合物。溶液应在例如-20℃下冷藏储存。合并脂质得到所希望的摩尔比并用水和乙醇稀释至在约5.5mM与约25mM之间的最终脂质浓度。Nanoparticles can be produced by mixing two fluid streams, one of which contains therapeutic and/or prophylactic agents and the other of which has a lipid component, by mixing methods such as microfluidization and T-junctions. By combining in ethanol the lipids synthesized according to Examples 1-9, phospholipids (such as DOPE or DSPC, available from Avanti Polar Lipids (Alabaster, AL)), PEG lipids (such as 1,2-dimyristoyl -sn-Glycerylmethoxypolyethylene glycol, also known as PEG-DMG, available from Avanti Polar Lipids (Alabaster, AL)) and structural lipids such as cholesterol, available from Sigma-Aldrich (Taufkirchen, Germany) Obtain; or a corticosteroid (such as prednisolone, dexamethasone, prednisolone, and hydrocortisone; or a combination thereof), and prepare a lipid composition at a concentration of about 50 mM. The solution should be stored refrigerated, e.g. -20°C. The lipids are combined to achieve the desired molar ratio and diluted with water and ethanol to a final lipid concentration between about 5.5mM and about 25mM.
通过将脂质溶液与包括治疗剂和/或预防剂以在约5:1与约50:1之间的脂质组分比治疗剂和/或预防剂wt:wt比率组合，制备出包含治疗剂和/或预防剂和脂质组分的纳米粒子组合物。使用迈安纳-S微流体纳米粒子制备系统，将脂质溶液以在约10mL/min～约18mL/min之间的流动速率迅速地注入治疗剂和/或预防剂溶液中，制造出分散液，其中水比乙醇比率在约2:1与约5:1之间。A formulation containing a therapeutic agent is prepared by combining a lipid solution with a therapeutic agent and/or prophylactic agent in a lipid component to therapeutic agent and/or prophylactic agent wt:wt ratio of between about 5:1 and about 50:1. Nanoparticle compositions of agents and/or prophylactic agents and lipid components. Using the Maianna-S microfluidic nanoparticle preparation system, the lipid solution is rapidly injected into the therapeutic agent and/or preventive agent solution at a flow rate between about 10 mL/min and about 18 mL/min to create a dispersion. , wherein the water to ethanol ratio is between about 2:1 and about 5:1.
2)纳米颗粒组合体的表征2) Characterization of nanoparticle assemblies
纳米颗粒理化性状的测定：可以使用Zetasizer NanoZS(Malvern Instruments Ltd,Malvern,Worcestershire,UK)测定纳米粒子组合物的粒度、多分散指数(PDI)和ζ电势，粒度是在1×PBS中测定且ζ电势是在15mM的PBS中测定的。Determination of the physical and chemical properties of nanoparticles: Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to determine the particle size, polydispersity index (PDI) and ζ potential of the nanoparticle composition. The particle size is measured in 1×PBS and ζ Potentials were measured in 15mM PBS.
纳米颗粒包封率的测定：对于包含RNA的纳米粒子组合物，可以使用QUANT-ITTM RNA(Invitrogen Corporation Carlsbad,CA)测定评价纳米粒子组合物对RNA的包封情况。在TE缓冲溶液(10mM Tris-HCl、1mM EDTA，pH 7.5)中将样品稀释至约5μg/mL的浓度。将50μL稀释过的样品转移至聚苯乙烯96孔板上并向各孔中添加50μL TE缓冲液或50μL 2％Triton X-100溶液。在 37℃温度下孵育板15分钟。将试剂以1:100稀释于TE缓冲液中，并向各孔中添加100μL该溶液。可以使用荧光板读取器(
Figure PCTCN2022107846-appb-000036
Nivo TM Multimode Plate Readers,PerkinElmer，GER)在例如约480nm激发波长和例如约520nm发射波长下测量荧光强度。从各样品的荧光度值中减去试剂空白的荧光度值并通过用完全样品(未添加Triton X-100)的荧光强度除以被破坏的样品(由添加Triton X-100引起)的荧光度值来测定游离RNA的百分比。具体数据见表1。 Determination of nanoparticle encapsulation efficiency: For nanoparticle compositions containing RNA, the QUANT-ITTM RNA (Invitrogen Corporation Carlsbad, CA) assay can be used to evaluate the encapsulation of RNA by the nanoparticle composition. Samples were diluted in TE buffer solution (10mM Tris-HCl, 1mM EDTA, pH 7.5) to a concentration of approximately 5 μg/mL. Transfer 50 μL of the diluted sample to a polystyrene 96-well plate and add 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution to each well. Incubate the plate at 37°C for 15 minutes. Dilute the reagent 1:100 in TE buffer and add 100 μL of this solution to each well. A fluorescent plate reader can be used (
Figure PCTCN2022107846-appb-000036
Nivo ™ Multimode Plate Readers, PerkinElmer, GER) measure fluorescence intensity at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and determined by dividing the fluorescence intensity of the intact sample (without the addition of Triton X-100) by the fluorescence of the disrupted sample (caused by the addition of Triton X-100). value to determine the percentage of free RNA. See Table 1 for specific data.
表1：不同化合物制备的纳米颗粒组合体的特征Table 1: Characteristics of nanoparticle assemblies prepared from different compounds
Figure PCTCN2022107846-appb-000037
Figure PCTCN2022107846-appb-000037
3)纳米颗粒组合体的体外性能研究3) Study on the in vitro properties of nanoparticle assemblies
对于包含RNA的纳米颗粒组合体，可以采用ONE-GlO+TOX Luciferase Reporter and Cell Viability Assay(Promega Corporation，US)评价其转染效果和细胞毒性。根据包封率测定中测得的RNA浓度计算所需纳米颗粒组合体的体积，将纳米颗粒组合体稀释到20ng/μL，按照聚苯乙烯96孔板2×10 5细胞/孔的细胞密度向每孔中添加5μL稀释液进行转染。可以使用荧光板读取器(
Figure PCTCN2022107846-appb-000038
Nivo TM Multimode Plate Readers,PerkinElmer，GER)测量化学发光强度。具体数据见表2。 For nanoparticle combinations containing RNA, ONE-GlO+TOX Luciferase Reporter and Cell Viability Assay (Promega Corporation, US) can be used to evaluate its transfection effect and cytotoxicity. Calculate the volume of the required nanoparticle assembly based on the RNA concentration measured in the encapsulation efficiency determination, dilute the nanoparticle assembly to 20ng/μL, and add it according to the cell density of 2×10 5 cells/well in a polystyrene 96-well plate. Add 5 μL of diluent to each well for transfection. A fluorescent plate reader can be used (
Figure PCTCN2022107846-appb-000038
Nivo ™ Multimode Plate Readers, PerkinElmer, GER) were used to measure chemiluminescence intensity. See Table 2 for specific data.
表2：不同化合物制备的纳米组合体的体外性能研究Table 2: In vitro performance study of nanoassemblies prepared with different compounds
Figure PCTCN2022107846-appb-000039
Figure PCTCN2022107846-appb-000039
Figure PCTCN2022107846-appb-000040
Figure PCTCN2022107846-appb-000040
实施例11：脂质纳米颗粒的制备和检测(验证体外递送siRNA能力)Example 11: Preparation and detection of lipid nanoparticles (to verify the ability to deliver siRNA in vitro)
1)将实施例1合成的化合物-1，与DOPE(1、2-二油酰-SN-甘油-3-磷酰乙醇胺)和DMG-PEG2000(1、2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇)以65:30:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将cy3标记的siCont(Sense 5’to 3’:CUUACGCUGAGUACUUCGAdTdT-Cy3；Antisense 5’to 3’:UCGAAGUACUCAGCGUAAGdTdT)在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到siRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和siRNA溶液，以总脂质与siRNA的重量比为约20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液(磷酸缓冲盐溶液)代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封cy3-siCont的制剂。1) Compound-1 synthesized in Example 1, and DOPE (1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine) and DMG-PEG2000 (1, 2-dimyristoyl-rac-glycerol -3-methoxypolyethylene glycol) was dissolved in absolute ethanol at a molar ratio of 65:30:5 to prepare a lipid ethanol solution, and cy3-labeled siCont (Sense 5'to 3':CUUACGCUGAGUACUUCGAdTdT-Cy3; Antisense 5'to 3':UCGAAGUACUCAGCGUAAGdTdT) was diluted in 50mM citrate buffer (pH=4) to obtain a siRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the siRNA solution in a volume of 1:3. Prepare liposomes at a weight ratio of about 20:1 to siRNA. Use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution (phosphate buffered saline). The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain a formulation encapsulating cy3-siCont.
通过动态光散射，使用纳米粒度及电位分析仪(NS-90Z)测定脂质纳米颗粒大小。使用Quant-it Ribogreen RNA定量分析试剂盒测定脂质纳米颗粒的包封效率。测得脂质纳米颗粒的粒径为82nm，PDI(多分散指数)是0.13，包封率是96.5％。Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 82 nm, the PDI (polydispersity index) was 0.13, and the encapsulation efficiency was 96.5%.
2)将上述制备的包载siRNA的脂质纳米颗粒转染293T细胞。将293T细胞以1*10 5个细胞/孔的密度铺到24孔板上，加入含有10％小牛血清的DMEM培养液中培养24小时至细胞密度长到70％后，移除24孔板中的培养液。将制备的LNP/cy3-siRNA复合物用DMEM培养基稀释至1ml，加入24孔板中。37℃，5％CO 2条件下培养箱继续孵育24h。 2) Transfect 293T cells with the siRNA-encapsulated lipid nanoparticles prepared above. Plate 293T cells on a 24-well plate at a density of 1* 105 cells/well, add DMEM culture medium containing 10% calf serum, and culture for 24 hours until the cell density reaches 70%, then remove the 24-well plate. culture medium in. Dilute the prepared LNP/cy3-siRNA complex with DMEM medium to 1 ml and add it to a 24-well plate. Continue incubation for 24h in the incubator at 37°C and 5% CO2 .
然后采用荧光显微镜拍照记录cy3-siRNA的转染情况。细胞转染效果见说明书附图中的图10。荧光显微镜结果表明，制备的包载siRNA的脂质纳米颗粒具有较好的细胞转染效果。流式细胞仪测试结果表明68.90％的细胞被转染。Then a fluorescence microscope was used to take pictures to record the transfection status of cy3-siRNA. The cell transfection effect is shown in Figure 10 in the accompanying drawings of the instructions. Fluorescence microscopy results showed that the prepared lipid nanoparticles encapsulating siRNA had good cell transfection effect. Flow cytometry test results showed that 68.90% of cells were transfected.
实施例12：脂质纳米颗粒的制备和检测(验证体外递送mRNA能力)Example 12: Preparation and detection of lipid nanoparticles (to verify the ability to deliver mRNA in vitro)
1)将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以65:30:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将EGFP mRNA(编码绿色荧光蛋白mRNA)在20-100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为约20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒同0.22μm无菌过滤器过滤，得到包封EGFP mRNA的制剂。1) Dissolve the lipid compound-1 synthesized in Example 1 with DOPE and DMG-PEG2000 in absolute ethanol at a molar ratio of 65:30:5 to prepare a lipid ethanol solution, and add EGFP mRNA (encoding green fluorescent protein mRNA ) was diluted in 20-100mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, with a weight ratio of total lipid to mRNA. To prepare liposomes at approximately 20:1, use dialysis or tangential flow filtration to remove ethanol and replace it with PBS solution. The lipid nanoparticles are then filtered with a 0.22μm sterile filter to obtain a preparation encapsulating EGFP mRNA.
通过动态光散射，使用纳米粒度及电位分析仪(NS-90Z)测定脂质纳米颗粒大小。使用Quant-it Ribogreen RNA定量分析试剂盒测定脂质纳米颗粒的包封效率。测得LNP制剂粒径为95nm，PDI 0.19，包封率是93.1％。Lipid nanoparticle size was determined by dynamic light scattering using a nanoparticle size and potential analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the LNP preparation was measured to be 95nm, PDI 0.19, and the encapsulation rate was 93.1%.
2)将上述制备的的包载mRNA的脂质纳米颗粒转染293T细胞。将293T细胞以1*10 5个细胞/孔的密度铺到24孔板上，加入含有10％小牛血清的DMEM培养液中培养24小时至细胞密度长到70％后，移除24孔板中的培养液。将制备的LNP/EGFP复合物用DMEM培养基稀释至1ml，加入24孔板中。37℃，5％CO 2条件下培养箱孵育24h。然后采用荧光显微镜拍照记录EGFP的转染情况。细胞转染效果见说明书附图中的图11。荧光显微镜结果表明，制备的包载mRNA的脂质纳米颗粒具有较好的细胞转染效果。流式细胞仪结果表明77.79％的细胞被转染。 2) Transfect 293T cells with the mRNA-encapsulated lipid nanoparticles prepared above. Plate 293T cells on a 24-well plate at a density of 1* 105 cells/well, add DMEM culture medium containing 10% calf serum, and culture for 24 hours until the cell density reaches 70%, then remove the 24-well plate. culture medium in. Dilute the prepared LNP/EGFP complex with DMEM medium to 1 ml and add it to a 24-well plate. Incubate for 24h in an incubator at 37°C and 5% CO2 . Then a fluorescence microscope was used to take pictures to record the transfection status of EGFP. The cell transfection effect is shown in Figure 11 in the accompanying drawings of the instructions. Fluorescence microscopy results showed that the prepared lipid nanoparticles encapsulating mRNA had good cell transfection effect. Flow cytometry results showed that 77.79% of cells were transfected.
实施例13：脂质纳米颗粒的制备和检测(验证mRNA体内递送效果)Example 13: Preparation and detection of lipid nanoparticles (to verify the in vivo delivery effect of mRNA)
1)将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以65:30:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒同0.22μm无菌过滤器过滤，得到包封mRNA的制剂。1) Dissolve the lipid compound-1 synthesized in Example 1 with DOPE and DMG-PEG2000 in absolute ethanol at a molar ratio of 65:30:5 to prepare a lipid ethanol solution, and add Luciferase mRNA (encoding luciferase mRNA ) were diluted in 50mM citrate buffer (pH=4) to obtain an mRNA solution. Use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3. The weight ratio of total lipid to mRNA is Prepare liposomes at 20:1, remove ethanol using dialysis or tangential flow filtration, and replace with PBS solution. The lipid nanoparticles are then filtered with a 0.22 μm sterile filter to obtain a preparation that encapsulates mRNA.
通过动态光散射，使用纳米粒度及电位分析仪(NS-90Z)测定脂质纳米颗 粒大小。使用Quant-it Ribogreen RNA定量分析试剂盒测定脂质纳米颗粒的包封效率。测得脂质纳米颗粒的粒径为98nm，PDI为0.22，包封率为88.9％。Lipid nanoparticle size was determined by dynamic light scattering using a Nanoparticle Size and Potentiometric Analyzer (NS-90Z). Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantitative Assay Kit. The particle size of the lipid nanoparticles was measured to be 98 nm, the PDI was 0.22, and the encapsulation rate was 88.9%.
2)将上述制备的LNP分别通过尾静脉或肌肉注入6周龄SPF级别BALB/c小鼠体内，剂量为每只小鼠100ul(含10μg mRNA)，12h小时候后用活体成像仪测试小鼠体内的荧光强度，活体成像仪的曝光时间设置为30s。包载Luciferase mRNA的脂质纳米粒，在小鼠活体成像结果见说明书附图中的图12和图13。结果表明，当制备的LNP通过静脉注射进小鼠体内时，LNP在体内的递送部位主要是腹部。而当LNP通过肌肉注射进小鼠体内时，LNP在体内的递送部位主要是腹部和肝脏。上述结果均表明，LNP具有较好的体内递送效果。2) Inject the LNPs prepared above into 6-week-old SPF grade BALB/c mice through the tail vein or muscle respectively. The dose is 100ul per mouse (containing 10μg mRNA). After 12 hours, the mice are tested with an in vivo imager. of fluorescence intensity, and the exposure time of the in vivo imager was set to 30 s. The results of in vivo mouse imaging of lipid nanoparticles encapsulating Luciferase mRNA are shown in Figure 12 and Figure 13 in the appendix of the instruction manual. The results showed that when the prepared LNP was injected intravenously into mice, the delivery site of LNP in the body was mainly the abdomen. When LNP was injected intramuscularly into mice, the main delivery sites of LNP in the body were the abdomen and liver. The above results all show that LNP has good in vivo delivery effect.
实施例14：将合成的脂质化合物-1，与DOPE和DMG-PEG2000以89.9:10:0.1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Example 14: Dissolve the synthesized lipid compound-1, DOPE and DMG-PEG2000 in absolute ethanol at a molar ratio of 89.9:10:0.1 to prepare a lipid ethanol solution, and add Luciferase mRNA (encoding luciferase mRNA) Dilute the ethanol lipid solution and the mRNA solution in 50mM citrate buffer (pH=4) respectively, and use a microfluidic device to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, so that the weight ratio of total lipid to mRNA is 20 :1 Prepare liposomes, use dialysis or tangential flow filtration to remove ethanol, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为1249nm，PDI为0.67，包封率为31％。The detected particle size was 1249nm, the PDI was 0.67, and the encapsulation rate was 31%.
实施例15：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以89.5:10:0.5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在50mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Embodiment 15: The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 89.5:10:0.5 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 50mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3. Prepare liposomes at a ratio of 20:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为467nm，PDI为0.40，包封率为78％。The detected particle size was 467 nm, the PDI was 0.40, and the encapsulation rate was 78%.
实施例16：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以89:10:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Example 16: The lipid compound-1 synthesized in Example 1, DOPE and DMG-PEG2000 were dissolved in absolute ethanol at a molar ratio of 89:10:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 100mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, and the weight of the total lipid and mRNA was calculated. Prepare liposomes at a ratio of 20:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为347nm，PDI为0.41，包封率为81％。The detected particle size was 347nm, the PDI was 0.41, and the encapsulation rate was 81%.
实施例17：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以85:10:5的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Embodiment 17: The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 85:10:5 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 100mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, and the weight of the total lipid and mRNA was calculated. Prepare liposomes at a ratio of 20:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为94nm，PDI为0.50，包封率为85％。The detected particle size was 94nm, the PDI was 0.50, and the encapsulation rate was 85%.
实施例18：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以64:35:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为20:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Example 18: The lipid compound-1 synthesized in Example 1, DOPE and DMG-PEG2000 were dissolved in absolute ethanol at a molar ratio of 64:35:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 100mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, and the weight of the total lipid and mRNA was calculated. Prepare liposomes at a ratio of 20:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为82nm，PDI为0.11，包封率为96％。The detected particle size was 82nm, the PDI was 0.11, and the encapsulation rate was 96%.
实施例19：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以39:60:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA (编码荧光素酶mRNA)分别在100mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为40:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Embodiment 19: The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 39:60:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 100mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3, and the weight of the total lipid and mRNA was calculated. Prepare liposomes at a ratio of 40:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为145nm，PDI为0.23，包封率为76％。The detected particle size was 145nm, the PDI was 0.23, and the encapsulation rate was 76%.
实施例20：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以39:60:1的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在20mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为40:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Embodiment 20: The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 39:60:1 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 20mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3. Prepare liposomes at a ratio of 40:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为302nm，PDI为0.38，包封率为73％。The detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
实施例21：将实施例1合成的脂质化合物-1，与DOPE和DMG-PEG2000以60:39.2:0.8的摩尔比溶于无水乙醇制备脂质乙醇溶液，并将Luciferase mRNA(编码荧光素酶mRNA)分别在20mM柠檬酸盐缓冲液(pH＝4)中稀释得到mRNA溶液，使用微流控装置以1:3的体积混合乙醇脂质溶液和mRNA溶液，以总脂质与mRNA的重量比为40:1制备脂质体，采用透析或切向流过滤除去乙醇，并用PBS溶液代替。之后将脂质纳米颗粒用0.22μm无菌过滤器过滤，得到包封mRNA的制剂。Embodiment 21: The lipid compound-1 synthesized in Example 1 was dissolved in absolute ethanol with DOPE and DMG-PEG2000 at a molar ratio of 60:39.2:0.8 to prepare a lipid ethanol solution, and Luciferase mRNA (encoding fluorescein Enzyme mRNA) were diluted in 20mM citrate buffer (pH=4) to obtain an mRNA solution, and a microfluidic device was used to mix the ethanol lipid solution and the mRNA solution in a volume of 1:3. Prepare liposomes at a ratio of 40:1, remove ethanol by dialysis or tangential flow filtration, and replace it with PBS solution. The lipid nanoparticles were then filtered with a 0.22 μm sterile filter to obtain an mRNA-encapsulated preparation.
检测粒径为302nm，PDI为0.38，包封率为73％。The detected particle size was 302nm, the PDI was 0.38, and the encapsulation rate was 73%.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (20)

  1. 一种脂质化合物，所述脂质化合物为基于胆酸或其衍生物的脂质，或所述脂质化合物为基于胆酸或其衍生物的脂质的药物可用的盐、前药或立体异构体，所述脂质化合物具有如下通式1或通式2的结构：A lipid compound, which is a lipid based on cholic acid or a derivative thereof, or a pharmaceutically acceptable salt, prodrug or steric compound of a lipid based on cholic acid or a derivative thereof. Isomers, the lipid compound has the structure of the following general formula 1 or general formula 2:
    通式1：General formula 1:
    Figure PCTCN2022107846-appb-100001
    Figure PCTCN2022107846-appb-100001
    通式2：General formula 2:
    Figure PCTCN2022107846-appb-100002
    Figure PCTCN2022107846-appb-100002
  2. 根据权利要求1所述的脂质化合物，所述脂质化合物母核为胆酸或其衍生物；其中所述连接键(Linker)包含酯基、酰胺基、氨基甲酸酯基、碳酸酯基或脲基的一种或多种。The lipid compound according to claim 1, wherein the core of the lipid compound is cholic acid or a derivative thereof; wherein the linker includes an ester group, an amide group, a carbamate group, and a carbonate group. Or one or more urea groups.
  3. 根据权利要求1所述的脂质化合物，其中胆酸或其衍生物是任选自胆酸、奥贝胆酸、熊去氧胆酸、熊果胆酸、3β-羟基-D5-胆烯酸、鹅去氧胆酸、石胆酸、脱氧胆酸、牛磺胆酸、5β-胆酸、去氢胆酸、猪胆酸、络胆酸、甘氨鹅脱氧胆酸、牛磺熊去氧胆酸、牛磺鹅去氧胆酸、甘氨胆酸、猪脱氧胆酸、猪去氧胆酸甲酯、牛磺猪去氧胆酸钠、去氢胆酸钠、胆酸钠、脱氧甘胆酸钠、牛磺脱氧胆酸钠、牛磺胆酸钠、牛磺鹅去氧胆酸钠、甘氨胆酸钠盐、牛磺胆酸-3-硫酸酯二钠盐、牛磺熊去氧胆酸钠和牛磺石胆酸钠的一种或多种。The lipid compound according to claim 1, wherein cholic acid or its derivative is optionally selected from the group consisting of cholic acid, obeticholic acid, ursodeoxycholic acid, ursolic acid, and 3β-hydroxy-D5-cholenoic acid. , chenodeoxycholic acid, lithocholic acid, deoxycholic acid, taurocholic acid, 5β-cholic acid, dehydrocholic acid, hyocholic acid, cholic acid, glycinechenodeoxycholic acid, tauroursodeoxy Cholic acid, taurochenodeoxycholic acid, glycocholic acid, hyodeoxycholic acid, hyodeoxycholic acid methyl ester, sodium taurohodeoxycholate, sodium dehydrocholic acid, sodium cholate, deoxyglycholic acid Sodium, sodium taurodeoxycholate, sodium taurocholate, sodium taurochenodeoxycholate, glycocholic acid sodium salt, taurocholic acid-3-sulfate disodium salt, tauroursodeoxycholate One or more of sodium bisulfate and sodium taurocholate.
  4. 根据权利要求1所述的脂质化合物，所述脂质化合物为选自熊去氧胆酸衍生物脂质化合物的一种或多种，或奥贝胆酸衍生物脂质化合物的一种或多种。The lipid compound according to claim 1, which is one or more selected from the group consisting of ursodeoxycholic acid derivative lipid compounds, or one or more obeticholic acid derivative lipid compounds. Various.
  5. 一种脂质化合物的组合物，其特征在于所述组合物包括治疗或预防剂和用于递送所述治疗或预防剂的载体，所述载体包括前述权利要求1-4中所述的 脂质化合物的一种或多种。A composition of lipid compounds, characterized in that the composition includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, the carrier including the lipid described in the preceding claims 1-4 One or more compounds.
  6. 根据权利要求5所述的组合物，其特征在于，所述治疗或预防剂包括核酸分子、多肽、蛋白、抗体及小分子药物中的一种或多种。The composition according to claim 5, wherein the therapeutic or preventive agent includes one or more of nucleic acid molecules, polypeptides, proteins, antibodies and small molecule drugs.
  7. 根据权利要求5所述的组合物，其特征在于所述载体与所述治疗或预防剂的质量比为1:1～100:1。The composition according to claim 5, characterized in that the mass ratio of the carrier to the therapeutic or preventive agent is 1:1 to 100:1.
  8. 根据权利要求5所述的组合物，其特征在于，所述组合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为20nm～1000nm；所述纳米颗粒制剂的多分散系数≤0.5。The composition according to claim 5, characterized in that the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 20 nm to 1000 nm; the polydispersity coefficient of the nanoparticle preparation is ≤0.5.
  9. 根据权利要求5所述的组合物，其特征在于，所述载体中包含三种不同的脂质组分，其中一种脂质是基于胆酸或其衍生物的脂质。The composition of claim 5, wherein the carrier contains three different lipid components, one of which is a lipid based on cholic acid or a derivative thereof.
  10. 根据权利要求5所述的组合物，其特征在于，所述载体中还包括中性电荷、阴性电荷或双性电荷的电荷辅助脂质。The composition according to claim 5, characterized in that the carrier further includes a charge-assisted lipid with neutral charge, negative charge or amphiphilic charge.
  11. 根据权利要求10所述的组合物，其特征在于，所述电荷辅助脂质为如下的一种或多种：二硬脂酰基磷脂酰胆碱(DSPC)、二油酰基磷脂酰胆碱(DOPC)、二棕榈酰基磷脂酰胆碱(DPPC)、二油酰基磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰基磷脂酰乙醇胺(DOPE)、棕榈酰油酰基磷脂酰胆碱(POPC)、棕榈酰油酰基-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺基甲基)-环己烷-1-甲酸酯(DOPE-mal)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷脂酰乙醇胺(DMPE)、二硬脂酰基-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE,16-O-二甲基PE,18-1-反式PE,1-硬脂酰基-2-油酰基-磷脂酰乙醇胺(SOPE)或其混合物。The composition according to claim 10, wherein the charge-auxiliary lipid is one or more of the following: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) ), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine Base (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE) -mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O -DimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or mixtures thereof.
  12. 根据权利要求5所述的组合物，其特征在于，所述载体中还包括结构修饰脂质。The composition according to claim 5, wherein the carrier further includes a structurally modified lipid.
  13. 根据权利要求12所述的组合物，其特征在于，所述结构修饰脂质包括聚乙二醇、葡聚糖、聚乳酸或氨基酸修饰的磷脂酰乙醇胺、磷脂酸、神经酰胺、二烷基胺、二酰基甘油、二烷基甘油中的一种或多种。The composition according to claim 12, wherein the structurally modified lipids include polyethylene glycol, dextran, polylactic acid or amino acid modified phosphatidylethanolamine, phosphatidic acid, ceramide, dialkylamine , one or more of diacylglycerol and dialkylglycerol.
  14. 根据权利要求5所述的组合物，其特征在于，所述载体还包括但不限于胆酸或其衍生物的脂质、电荷辅助脂质以及结构修饰脂质，所述胆酸脂质、所述电荷辅助脂质以及所述结构修饰脂质的摩尔比为(30～80)：(5-50)： (0.5～10)。The composition according to claim 5, wherein the carrier further includes but is not limited to lipids of cholic acid or its derivatives, charge-assisted lipids and structurally modified lipids, and the cholic acid lipid, the The molar ratio of the charge-assisted lipid and the structurally modified lipid is (30-80): (5-50): (0.5-10).
  15. 根据权利要求5所述的组合物，其特征在于，所述组合物还包括药物可用的赋形剂或稀释剂中的一种或多种。The composition of claim 5, further comprising one or more pharmaceutically acceptable excipients or diluents.
  16. 根据权利要求1-15中任一项脂质化合物或组合物用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用。The lipid compound or composition according to any one of claims 1 to 15 is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs.
  17. 根据权利要求1-15中任一项脂质化合物或组合物用于在制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物中的应用中，其中所述脂质纳米颗粒具有20～1000nm粒径。The lipid compound or composition according to any one of claims 1 to 15 is used in the preparation of nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, wherein the lipid nanoparticles have a particle size of 20 to 1000nm particle size.
  18. 用于制备核酸药物、基因疫苗、多肽、蛋白、抗体及小分子药物的组合物，其包含核酸和包载所述核酸的脂质纳米颗粒，其中每个单独的脂质纳米颗粒包含多种脂质组分，其中一种脂质组分是基于胆酸的脂质化合物，包括其化合物或其药学上可用的盐、立体异构体、互变异构体、溶剂化物、螯合物、非共价化合物或前体药物，并且其中所述脂质纳米颗粒具有至少70％的核酸包封比例。Compositions for preparing nucleic acid drugs, gene vaccines, polypeptides, proteins, antibodies and small molecule drugs, which include nucleic acids and lipid nanoparticles encapsulating the nucleic acids, wherein each individual lipid nanoparticle contains a variety of lipids A lipid component, wherein one of the lipid components is a cholic acid-based lipid compound, including compounds thereof or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non- A covalent compound or prodrug, and wherein the lipid nanoparticle has a nucleic acid encapsulation ratio of at least 70%.
  19. 制备权利要求18所述组合物的方法，其中所述脂质纳米颗粒通过将mRNA溶液和包括权利要求1-4中任一种脂质化合物的脂质溶液混合而形成，其中mRNA溶液的介质为HEPES、磷酸钠、乙酸钠、硫酸铵、碳酸氢钠或柠檬酸钠；脂质溶液的介质为乙醇、异丙醇或二甲亚砜；其中所述脂质纳米颗粒通过透析或超滤进一步纯化。A method for preparing the composition of claim 18, wherein the lipid nanoparticles are formed by mixing an mRNA solution and a lipid solution including any one of the lipid compounds of claims 1-4, wherein the medium of the mRNA solution is HEPES, sodium phosphate, sodium acetate, ammonium sulfate, sodium bicarbonate or sodium citrate; the medium of the lipid solution is ethanol, isopropyl alcohol or dimethyl sulfoxide; wherein the lipid nanoparticles are further purified by dialysis or ultrafiltration .
  20. 根据权利要求18所述的组合物，还包含缓冲剂、碳水化合物、甘露醇、蛋白质、多肽或者氨基酸、抗氧化剂、抑菌剂、螯合剂、佐剂中的一种或多种。The composition according to claim 18, further comprising one or more of a buffer, carbohydrate, mannitol, protein, polypeptide or amino acid, antioxidant, bacteriostatic agent, chelating agent, and adjuvant.
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