WO2023246218A1 - Lipide ionisable pour l'administration d'acides nucléiques et composition associée - Google Patents

Lipide ionisable pour l'administration d'acides nucléiques et composition associée Download PDF

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WO2023246218A1
WO2023246218A1 PCT/CN2023/084750 CN2023084750W WO2023246218A1 WO 2023246218 A1 WO2023246218 A1 WO 2023246218A1 CN 2023084750 W CN2023084750 W CN 2023084750W WO 2023246218 A1 WO2023246218 A1 WO 2023246218A1
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independently selected
mrna
compound
drug
lipid
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宋相容
魏霞蔚
魏于全
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成都威斯津生物医药科技有限公司
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    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
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    • A61P31/20—Antivirals for DNA viruses
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D245/00—Heterocyclic compounds containing rings of more than seven members having two nitrogen atoms as the only ring hetero atoms
    • C07D245/02—Heterocyclic compounds containing rings of more than seven members having two nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to ionizable lipids for nucleic acid delivery and compositions thereof, and belongs to the field of medicinal chemistry.
  • Nucleic acid drugs include DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), miRNA mimics, antimiRs, ribozymes, mRNA, aptamers, plasmids, CRISPR RNA, etc.
  • ASO antisense oligonucleotides
  • small interfering RNA siRNA
  • miRNA microRNA
  • miRNA mimics antimiRs
  • ribozymes ribozymes
  • mRNA aptamers
  • CRISPR RNA CRISPR RNA
  • Non-viral vectors are currently a type of gene delivery vector that has been much studied and has good application prospects. It mainly loads mRNA through the adsorption of cations formed by the delivery material and mRNA phosphate ions, forming structures such as liposomes or nanoparticles to protect them. It is protected from degradation by nucleases and changes its entry into cells. It has the advantage that the carrier is relatively easy to obtain, has low immunogenicity and is highly safe.
  • Non-viral nucleic acid delivery materials are mostly cationic lipids or cationic polymers. Due to their strong positive charge, they are easily adsorbed by plasma proteins in the body and then taken up by the reticuloendothelial system, causing the loaded nucleic acid drugs to be destroyed.
  • lipid nanoparticles based on ionizable lipids are the most studied. Nanoparticles prepared from ionizable lipid materials show positive electricity in an acidic environment in vitro, and electrostatic adsorption of nucleic acids enables the delivery of nucleic acid drugs.
  • ionizable lipid nanoparticles have very broad prospects in the field of nucleic acid delivery.
  • ionizable lipid nanoparticles there are relatively few clinical applications of ionizable lipid nanoparticles, and the core difficulty lies in the development of safe and effective ionizable lipids. Therefore, the development of ionizable lipid nucleic acid delivery materials with higher delivery efficiency and safer is of great significance for the widespread application of nucleic acid drugs and gene therapy.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide ionizable lipids for nucleic acid delivery.
  • the present invention provides compounds represented by formula I, or pharmaceutically acceptable salts, isomers, deuterated products or prodrugs thereof:
  • a 1, 2 or 3;
  • X 1 and X 2 are independently selected from N or C;
  • R 1 , R 2 , R 3 , R 4 are independently selected from C 1 -C 30 linear alkyl, C 1 -C 30 branched alkyl, C 2 -C 30 linear alkenyl, C 2 -C 30 Branched alkenyl, C 2 -C 30 linear alkynyl or C 2 -C 30 branched alkynyl;
  • R 5 and R 6 are independently selected from -H, substituted or unsubstituted alkyl
  • Q 1 and Q 2 are independently selected from O or S.
  • the lipid nanoparticles formed by the above-mentioned ionizable lipids are positively charged in the acidic environment in vitro, and the electrostatic adsorption of nucleic acids realizes the loading of nucleic acid drugs. After entering the neutral environment in the body, they become electrically neutral, avoiding plasma Protein adsorption and capture by the reticuloendothelial system.
  • the lipid delivery carrier formed by the above-mentioned ionizable lipid has stronger targeting ability to the liver and lungs.
  • the nucleic acid drugs it carries, such as mRNA are expressed more highly in receptor cells, and the antigen expressed by the antigen mRNA is more strongly presented in the body.
  • nucleic acid drugs such as mRNA to achieve up-regulation or down-regulation of corresponding genes, or to deliver antigen mRNA to express antigens in the body to achieve immunotherapy, or to deliver mRNA encoding antibodies to express antibodies in the body.
  • the inventor of the present application also unexpectedly discovered that after the lipid carrier formed by the above-mentioned ionizable lipid is loaded with drugs, when administered to mice via intramuscular injection, its local irritation is significantly lower than that of the positive control carrier, and the effect on mice is Weight gain had no significant effect, while positive Control vehicle had an inhibitory effect on weight gain in mice.
  • the writing order of the L1, L2, L3, and L4 connecting bonds defined above corresponds from left to right from the proximal nitrogen end to the far nitrogen end.
  • the writing order of the G1, G2, G3, and G4 connecting keys defined above is from left to right corresponding to the main chain direction of Formula I from left to right.
  • X 1 and X 2 are N at the same time.
  • the compound has the structure shown in Formula II, or is a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug of the structure shown in Formula II,
  • the k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • k is 1.
  • the R a is -H or unsubstituted C 1 to C 6 alkyl.
  • the R a is -H.
  • said L 1 and L 2 are selected from the same group and or L 3 and L 4 are selected from the same group.
  • said L 1 and L 3 are selected from the same group and or L 2 and L 4 are selected from the same group.
  • said L 1 , L 2 , L 3 and L 4 are selected from the same group.
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from C 1 -C 30 linear alkyl, C 2 -C 30 linear alkenyl, C 2 -C 30 linear Alkynyl.
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from unsubstituted C 1 to C 30 linear alkyl groups.
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from unsubstituted C 8 to C 18 linear alkyl groups.
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from unsubstituted C 10 to C 14 linear alkyl groups.
  • R 1 and R 2 are selected from the same group and or R 3 and R 4 are selected from the same group.
  • R 1 and R 3 are selected from the same group and or R 2 and R 4 are selected from the same group.
  • the R 1 , R 2 , R 3 and R 4 are selected from the same group.
  • the G 1 , G 2 , G 3 and G 4 are independently selected from or does not exist, n3 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the G 3 and G 4 do not exist.
  • the G 1 and G 2 are independently selected from -CH 2 - or -CH 2 CH 2 -.
  • the R 5 and R 6 are independently selected from -H, unsubstituted C 1 to C 6 alkyl or -OH substituted C 1 to C 6 alkyl.
  • the R 5 and R 6 are independently selected from -H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl or hydroxypropyl.
  • the R 5 and R 6 are selected from the same group.
  • the compound has one of the following structures:
  • the present invention provides the use of the aforementioned compounds, or pharmaceutically acceptable salts, stereoisomers, deuterated products or prodrugs thereof, as drug delivery carriers.
  • the active ingredient of the drug is optionally selected from at least one of nucleic acids, small molecule drugs, protein drugs, and polypeptides.
  • the active ingredient of the drug is selected from nucleic acids.
  • the drug has heart, liver, spleen, lung or kidney targeting.
  • the drug targets the lungs and spleen.
  • the present invention provides a drug delivery vehicle.
  • the drug delivery carrier includes a compound as described above, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof.
  • the present invention provides a drug complex.
  • the drug complex includes a carrier and an active ingredient, the carrier is connected to the active ingredient, the carrier includes a cationic lipid, and the cationic lipid includes the aforementioned compound, or its Pharmaceutically acceptable salts, stereoisomers, deuterates or prodrugs.
  • the active ingredient is optionally selected from at least one of nucleic acids, small molecule drugs, protein drugs, polypeptides, and antibodies.
  • the active ingredient of the drug is selected from nucleic acids
  • the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer.
  • the nucleic acid is mRNA
  • the delivery carrier is connected to the mRNA through ionic bonds.
  • the drug complex targets the lungs and spleen.
  • the drug complex exists in a form selected from lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, and polymer nanoparticles. .
  • the drug complex is formulated in the form of lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • the pharmaceutical composition further includes an excipient, and optionally, the excipient includes at least one selected from the group consisting of neutral phospholipids, steroids, and pegylated lipids.
  • the neutral phospholipid is selected from at least one of DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC, and POPC.
  • the neutral phospholipid is DOPE.
  • the molar ratio of the compound, or its pharmaceutically acceptable salt, isosteromer, deuterated product or prodrug to the neutral phospholipid is 1:10 to 10:1.
  • the steroid is selected from at least one selected from the group consisting of cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol, and fucosterol.
  • the steroid is cholesterol
  • the molar ratio of the compound, or its pharmaceutically acceptable salt, isosteromer, deuterated product or prodrug to the steroid is 1:10 to 10:1.
  • the PEGylated lipid is selected from at least one of DMG-PEG and DSPE-PEG;
  • the pegylated lipid is DMG-PEG2000.
  • the molar ratio of the compound, or its pharmaceutically acceptable salt, isosteromer, deuterated product or prodrug to the PEGylated lipid is 5:1 to 1000: 1.
  • the molar ratio of the compound, or its pharmaceutically acceptable salt, isosteromer, deuterated product or prodrug to the PEGylated lipid is 10:1 to 20: 1.
  • the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer.
  • the nucleic acid is mRNA.
  • the present invention proposes lipid nanoparticles.
  • the lipid nanoparticles include a lipid nanocapsid and a nucleic acid, the nucleic acid is wrapped in the lipid nanocapsid, the lipid nanocapsid includes a cationic lipid, and the cationic Lipids include compounds containing the aforementioned compounds, or pharmaceutically acceptable salts, stereoisomers, deuterated products or prodrugs thereof.
  • the average particle size of the lipid nanoparticles is 40 nm to 500 nm, Di (90) ⁇ 500 nm, and the polydispersity coefficient is ⁇ 30%.
  • the present invention provides a pharmaceutical composition. According to specific embodiments of the present invention, it includes the aforementioned drug complex or the aforementioned lipid nanoparticles.
  • pharmaceutically acceptable excipients are further included.
  • the composition is an injection.
  • the composition is suitable for intravenous or intramuscular injection.
  • the present invention proposes the use of the aforementioned drug complex or the aforementioned lipid nanoparticles or the aforementioned pharmaceutical composition in the preparation of medicines for treating or preventing diseases.
  • the disease is a heart, liver, spleen, lung or kidney related disease, preferably, the disease is a spleen or lung related disease.
  • the diseases are infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases and metabolic diseases.
  • the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or a variety of herpes.
  • the cancer is a solid tumor.
  • the cancer is liver cancer or lung cancer.
  • the drug treats or prevents diseases by presenting antigens and/or activating immune responses.
  • the invention provides a new type of ionizable lipid, the hydrophilic center of which is composed of four tertiary amine available nitrogens, and the hydrophobic tail is composed of four saturated or unsaturated fatty chains.
  • the novel ionizable lipid provided by the present invention is positively charged in an acidic environment and almost uncharged in a neutral environment. This property can be used to load nucleic acid drugs in an acidic buffer system. After the nucleic acid drugs are loaded, they enter the neutral liquid in the body. Environment, lipid nanoparticles are electrically neutral, which can effectively prevent nucleic acid drug complexes from being adsorbed by plasma proteins, thereby achieving higher delivery efficiency of nucleic acid drugs and significantly improving safety.
  • Figure 1 Particle size, PDI and potential of LNPs@mRNA prepared in Example 4;
  • FIG. 1 Encapsulation efficiency of LNPs@mRNA prepared in Example 4.
  • Figure 3 LNPs@mRNA cell level expression of reporter gene: (A) LNPs@EGFP mRNA transfected HEK293T cells; (B) LNPs@EGFP mRNA transfected DC2.4 cells; (C) LNPs@FLuc mRNA transfected DC2.4 cell;
  • Figure 4 MTT method to measure 4N4T-LNPs@FLuc mRNA cytotoxicity
  • alkyl is a linear or branched saturated hydrocarbon radical.
  • Examples of C1 to C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4) , sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2- Butyl (C5), tert-pentyl (C5) and n-hexyl (C6).
  • alkenyl refers to a straight or branched chain hydrocarbon radical containing at least one double bond.
  • alkenyl groups include, but are not limited to, vinyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, pent-3-enyl, Hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl.
  • alkynyl refers to a straight or branched chain hydrocarbon group containing at least one triple bond.
  • alkynyl groups include, but are not limited to, ethynyl, propargyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, Hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl.
  • pharmaceutically acceptable refers to a carrier, excipient, salt, etc. that is generally chemically or physically compatible with the other ingredients constituting a pharmaceutical dosage form, and is physiologically compatible with the receptor.
  • the compound or complex is chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or with the human or mammal in which it is used to prevent or treat a disease or condition.
  • subject or “patient” as used herein includes humans and mammals.
  • treatment refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of such disease for the purpose of curing, alleviating, alleviating, ameliorating or affecting the disease or Symptoms of said disease.
  • treatment may also include prevention unless specifically stated to the contrary.
  • salts refers to acidic and/or basic salts of the compounds of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, For example, alkylammonium salts.
  • These salts can be obtained directly from the final isolation and purification of the compounds. It can also be obtained by appropriately mixing the above compound with a certain amount of acid or base (for example, equivalent amounts). These salts may form a precipitate in the solution and be collected by filtration, or may be recovered after evaporation of the solvent, or may be obtained by reacting in an aqueous medium and then freeze-drying.
  • the salt mentioned in the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, butylene salt of the compound. salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
  • a compound of Formula I, or a pharmaceutically acceptable salt thereof may be isolated as a solvate and that any such solvate is therefore included within the scope of the present invention.
  • a compound of Formula I, or a pharmaceutically acceptable salt thereof may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • Stereoisomers include geometric isomers, diastereomers and enantiomers. Accordingly, compounds claimed in this disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. It will be appreciated by those skilled in the art that one stereoisomer may have better efficacy and/or fewer side effects than other stereoisomers.
  • Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. The racemate can be resolved by chromatography Or chemical resolution method for chiral resolution.
  • chiral acid resolving reagents such as chiral tartaric acid and chiral malic acid can be added to form a salt with the compound of the present disclosure, and the physical and chemical properties of the product, such as differences in solubility, can be utilized for separation.
  • the present invention also includes all suitable isotopic variations of the compounds of the present disclosure.
  • Isotopic variants are defined as compounds in which at least one atom is replaced by an atom with the same atomic number but whose atomic mass differs from the atomic mass commonly found or predominantly found in nature.
  • isotopes that may be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as 2H (deuterium), 3H (tritium), 11C, 13C, 14C, 15N, 17O, and 18O, respectively.
  • a “therapeutically effective amount” is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or symptoms.
  • a “prophylactically effective amount” is an amount of a prophylactic agent that prevents disease or symptoms when administered to a subject.
  • the amount of therapeutic agent that constitutes a “therapeutically effective amount” or the amount of prophylactic agent that constitutes a “prophylactically effective amount” varies with the therapeutic agent/prophylactic agent, the disease state and its severity, the age of the patient/subject to be treated/prevented, Changes in weight, etc.
  • a therapeutically effective amount and a prophylactically effective amount can be routinely determined by one of ordinary skill in the art based on their knowledge and this disclosure.
  • cationic lipids refers to lipids that are positively charged at a selected pH value.
  • LNP lipid nanoparticles
  • the invention provides a new type of ionizable lipid, which has a structure represented by formula (I) or formula (II). Its hydrophilic center is composed of four tertiary amine available nitrogens, and its hydrophobic tail is composed of four saturated or unsaturated composed of fat chains.
  • the novel ionizable lipid provided by the present invention is positively charged in an acidic environment and almost uncharged in a neutral environment. This property can be used to load nucleic acid drugs in an acidic buffer system. After the nucleic acid drugs are loaded, they enter the neutral liquid in the body. Environment, lipid nanoparticles are positively charged, which can effectively prevent nucleic acid drug complexes from being adsorbed by plasma proteins, thereby achieving higher delivery efficiency of nucleic acid drugs and significantly improving safety.
  • a pharmaceutical complex which includes a carrier, the carrier includes a cationic lipid, and the cationic lipid includes the above-mentioned compound of formula (I) formula (II) or a pharmaceutically acceptable salt thereof or stereoisomers.
  • the composite is a nanoparticle preparation, and the average size of the nanoparticle preparation is 40 nm to 500 nm, preferably 100 nm to 205 nm; the polydispersity coefficient of the nanoparticle preparation is ⁇ 50%, preferably ⁇ 30%, more preferably ⁇ 25%.
  • the cationic lipid is selected from the above-mentioned formula (I) or formula (II) One or more deuterated compounds of the compound or its pharmaceutically acceptable salts or stereoisomers. In one embodiment, the cationic lipid is a compound of formula (I) selected from the above.
  • the cationic lipid includes: (a) a compound of formula (I) selected from the above or a deuterated product, a pharmaceutically acceptable salt or a stereoisomer thereof; one or more in the body; (b) one or more other ionizable lipid compounds different from (a). (b)
  • the cationic lipid compound may be a commercially available cationic lipid, or a cationic lipid compound reported in the literature.
  • the molar ratio of the cationic lipid to the carrier is 30% to 70%, such as 35%, 45%, 50%, 55%, 60%, 65%.
  • the carrier can be used to deliver active ingredients such as therapeutic or prophylactic agents.
  • the active ingredient can be enclosed within or associated with a carrier.
  • the therapeutic or preventive agent includes one or more of nucleic acid molecules, small molecule compounds, macromolecular compounds, polypeptides, antibodies or proteins.
  • the nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA.
  • Suitable RNAs include, but are not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and its mixture.
  • the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 10:1, such as about 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1. In a preferred embodiment, the molar ratio of said cationic lipid to said neutral lipid is about 5:1.
  • the carrier component containing the cationic lipid complex may include one or more neutral lipid phospholipids, such as one or more (poly)unsaturated lipids.
  • Phospholipids can assemble into one or more lipid bilayers.
  • phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
  • the neutral lipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.
  • a phospholipid may be functionalized with or cross-linked with one or more alkynes (eg, an alkenyl group in which one or more double bonds are replaced by a triple bond).
  • alkynyl groups may undergo copper-catalyzed cycloaddition reactions when exposed to azides. These reactions can be used to functionalize the lipid bilayer of the complex to facilitate membrane permeability or cell recognition, or to couple the complex with useful components such as targeting or imaging moieties (e.g., dyes).
  • Neutral lipids that may be used in these complexes may be selected from the non-limiting group consisting of: 1,2 dilinoleyl sn glyceryl phosphocholine (DLPC), 1,2 dimyristoyl sn glycerophosphocholine base (DMPC), 1,2 dioleoyl sn glyceryl 3 phosphocholine (DOPC), 1,2 dipalmitoyl sn glyceryl 3 phosphocholine (DPPC), 1,2 distearoyl sn glyceryl 3 phosphocholine (DSPC), 1,2 disundecanoyl sn glycerophosphocholine (DUPC), 1 palmitoyl 2 oleoyl sn glycerol 3 phosphocholine (POPC), 1,2 diOoctadecenyl sn glycerol 3 Phosphocholine (18:0 Diether PC), 1 oleoyl 2 cholesteryl hemi-s
  • the neutral lipid includes DSPC. In certain embodiments, the neutral lipid includes DOPE.
  • neutral lipids include both DSPC and DOPE.
  • the carrier of the cationic lipid-containing complex may also include one or more structural lipids, such as steroids.
  • Structural lipids in this disclosure refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.
  • the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1 , 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.
  • the carrier for the cationic lipid-containing complex may also include one or more polymeric conjugated lipids, such as pegylated lipids.
  • Polymer conjugated lipids mainly refer to lipids modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, regulates nanoparticle size by limiting lipid fusion, and increases nanoparticle half-life by reducing nonspecific interactions with macrophages.
  • the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dioxane amine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.
  • the molecular weight of PEG-modified PEG is usually 350-5000Da.
  • the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE PEG2000), dimyristoylglycerol-3methoxypolyethylene glycol Alcohol 2000 (DMG PEG2000) and methoxy polyethylene glycol distetradecyl acetamide (ALC 0159).
  • DSPE PEG2000 distearoylphosphatidylethanolamine polyethylene glycol 2000
  • DMG PEG2000 dimyristoylglycerol-3methoxypolyethylene glycol Alcohol 2000
  • ALC 0159 methoxy polyethylene glycol distetradecyl acetamide
  • the polymeric conjugated lipid is DMG PEG2000.
  • the carrier includes neutral lipids, structural lipids, and polymeric conjugated lipids, the ionizable lipid, the neutral lipid, the structural lipid
  • the molar ratio of lipids to the polymer-conjugated lipids is 50:10:38.5:1.
  • compositions and alternatively "active agents” may include one or more therapeutic and/or prophylactic agents.
  • the mass ratio of the ionizable lipid to the therapeutic or preventive agent is 0.1:1-1000:1.
  • the therapeutic or preventive agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins.
  • the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
  • the vectors of the present disclosure can deliver therapeutic and/or prophylactic agents to mammalian cells or organs, and thus the present disclosure also provides methods of treating a disease or condition in a mammal in need thereof, the methods comprising administering to the mammal a therapeutic agent and/or a prophylactic agent. or a complex, pharmaceutical composition of a prophylactic agent and/or contacting a mammalian cell with the complex or pharmaceutical composition.
  • Therapeutic and/or prophylactic agents may be substances that, upon delivery to a cell or organ, cause a desired change in the cell or organ, or in other body tissues or systems. Such species may be used to treat one or more diseases, disorders or conditions. exist In some embodiments, therapeutic and/or prophylactic agents are small molecule drugs useful in treating a particular disease, disorder or condition.
  • drugs examples include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin (cisplatin), bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (such as actinomycin D, vinifera Neosine, vinblastine, cytosine arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purines and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, chlorpromazine) (timolol and labetalol), antihypertensive agents (such as clonidine and
  • the therapeutic and/or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and/or another therapeutic and/or prophylactic agent.
  • Cytotoxic or cytotoxic agents include any agent that is harmful to cells.
  • Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin (mitomycin), etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene Dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine lidocaine, propranolol, puromycin, maytansinoid such as maytansinol, rachelmycin (CC 1065), and analogs or homologs thereof.
  • Radioactive ions include, but are not limited to, iodine (eg, iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium.
  • iodine eg, iodine-125 or iodine-131
  • strontium-89 phosphorus
  • palladium cesium
  • iridium phosphate
  • cobalt yttrium-90
  • samarium-153 praseodymium
  • Vaccines include compounds capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis and preparations and may include mRNA encoding infectious disease-derived antigens and/or epitopes.
  • Vaccines may also include compounds and agents that direct an immune response against cancer cells and may include mRNA encoding tumor cell-derived antigens, epitopes, and/or neo-epitopes.
  • Compounds that elicit immune responses may include vaccines, corticosteroids (eg, dexamethasone), and other species.
  • vaccines and/or compounds capable of eliciting an immune response are administered intramuscularly by comprising a complex according to Formula (I).
  • Other therapeutic and/or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine
  • alkylating agents such as mechlorethamine, thiotepa, chlorambucil, laxithromycin (CC 1065), melphalan (melphalan), carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP, cisplatin), anthracyclines (such as daunorubicin (formerly daunomycin) and doxorubicin), antibiotics ( Examples include dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)) as well as antimitotic agents (eg, vincristine, vinblastine bases, taxols and maytansinoids).
  • alkylating agents such as mechlorethamine, thiotepa, chlorambucil
  • the therapeutic and/or prophylactic agent is a protein.
  • Therapeutic proteins that may be used in nanoparticles in the present disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G CSF), Granulocyte macrophage colony-stimulating factor (GM CSF), factor VIR, luteinizing hormone releasing hormone (LHRH) analogues, interferon, heparin, hepatitis B surface antigen, typhoid vaccine and cholera vaccine.
  • EPO erythropoietin
  • G CSF granulocyte colony-stimulating factor
  • GM CSF Granulocyte macrophage colony-stimulating factor
  • LHRH luteinizing hormone releasing hormone
  • the therapeutic agent is a polynucleotide or nucleic acid (eg, ribonucleic acid or deoxyribonucleic acid).
  • polynucleotide in its broadest sense includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger RNA (mRNA), hybrids thereof; RNAi-inducing factors; RNAi Factors; siRNA; shRNA; miRNA; antisense RNA; ribozyme; catalytic DNA; RNA that induces triple helix formation; aptamers, etc.
  • the therapeutic and/or prophylactic agent is RNA.
  • RNA useful in the complexes and methods described herein may be selected from the group consisting of, but not limited to: shortmer, antagomir, antisense RNA, ribozyme, 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 mixtures thereof.
  • the RNA is mRNA.
  • the therapeutic and/or prophylactic agent is mRNA.
  • the mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide.
  • Polypeptides encoded by mRNA can be of any size and can have any secondary structure or activity.
  • the polypeptide encoded by the mRNA when in the cell It can have therapeutic effects when expressed.
  • the therapeutic and/or prophylactic agent is siRNA.
  • siRNA can selectively reduce the expression of a gene of interest or downregulate the expression of the gene.
  • the siRNA can be selected such that a gene associated with a particular disease, disorder or condition is silenced upon administration of a complex including the siRNA to a subject in need thereof.
  • siRNA can comprise a sequence complementary to an mRNA sequence encoding a gene or protein of interest.
  • the siRNA can be an immunomodulatory siRNA.
  • the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA.
  • sgRNA and/or cas9 mRNA can be used as gene editing tools.
  • the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.
  • the therapeutic and/or prophylactic agent is shRNA or a vector or plasmid encoding it.
  • shRNA can be produced inside the target cell after delivering the appropriate construct into the nucleus.
  • the constructs and mechanisms associated with shRNA are well known in the relevant fields.
  • the complexes/carriers of the present disclosure can deliver active ingredients, including therapeutic or prophylactic agents, to a subject or patient.
  • the therapeutic or preventive agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins. Therefore, the complex of the present disclosure can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of therapeutic or preventive agents described above, the complexes of the present disclosure can be used to treat or prevent a variety of diseases or conditions.
  • the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
  • the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases .
  • the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various types of herpes.
  • the complex may include one or more components in addition to those described in the preceding sections.
  • the complex may include one or more small hydrophobic molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.
  • the complex may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface altering agents, or other components.
  • Permeability enhancing molecules may be, for example, those described in US Patent Application Publication No. 2005/0222064.
  • Carbohydrates may include simple sugars (such as glucose) and polysaccharides (such as glycogen and derivatives and analogs thereof).
  • Surface altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (such as cyclodextrin), nucleic acids, polymers (such as heparin, polyethylene glycol, and poloxamer), mucolytic agents (such as acetylcysteine, mugwort, bromelain, papain, daqing ( clerodendrum), bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostine, stironin stepronin, tiopronin, gelsolin, thymosin beta4, streptococcal DNase alpha, neltenexine and erdosteine) and DNase (e.g. rh
  • the complex may also contain one or more functionalized lipids.
  • lipids can be functionalized with alkynyl groups that may undergo cycloaddition reactions when exposed to azide under appropriate reaction conditions.
  • lipid bilayers can be functionalized in this way with one or more groups that are effective in promoting membrane permeability, cell recognition, or imaging.
  • the surface of the complex may also be coupled to one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane permeation are well known in the art.
  • the complex can further be formed into pharmaceutical compositions.
  • the pharmaceutical composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, Granule aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavorings agents, colorants, etc.
  • Excipients such as starch, lactose or dextrin.
  • Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington’s The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).
  • diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, Sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and/or combinations thereof.
  • compositions including one or more lipids described herein may also include one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxy Ribonucleotides (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.
  • GLA glucopyranosyl lipid adjuvant
  • CpG oligodeoxy Ribonucleotides e.g., class A or B
  • poly(I:C) poly(I:C)
  • aluminum hydroxide e.g., aluminum hydroxide
  • Pam3CSK4 glucopyranosyl lipid adjuvant
  • the pharmaceutical composition of the present disclosure may be formulated in the form of solid, semi-solid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols.
  • the pharmaceutical compositions of the present disclosure can be prepared by methods well known in the pharmaceutical art.
  • sterile injectable solutions can be prepared by incorporating the required amount of the therapeutic or prophylactic agent with various other ingredients as required above in a suitable solvent, such as sterile distilled water, followed by filtered sterilization.
  • Surfactants may also be added to promote the formation of a uniform solution or suspension.
  • the complexes and pharmaceutical compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or via Administer by inhalation.
  • the pharmaceutical composition is administered intravenously.
  • compositions of the present disclosure are administered in therapeutically effective amounts, which amounts may vary not only with the particular agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be at the discretion of the attending physician or clinician.
  • the present invention will be further described below with reference to examples, but the present invention is not limited to the following examples. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
  • reaction solvent is removed by rotary evaporation, and the product is separated and purified by silica gel column chromatography.
  • the eluent is spin-dried. A light yellow semi-solid was obtained, which was the final product I-1, with a yield of 35%.
  • reaction solvent is removed by rotary evaporation, and the product is separated and purified by silica gel column chromatography.
  • the eluent is spin-dried. A light yellow semi-solid, the final product II-1, was obtained with a yield of 34%.
  • C12-200 and MC3 are classic mRNA delivery lipids
  • ionizable lipids SM-102 is the ionizable lipid used in Moderna’s marketed mRNA vaccine products
  • ALC-0315 is the ionizable lipid used in CureVac and BioNTeach’s marketed mRNA vaccine products. Ionized lipids.
  • the synthesis route is based on literature reports, and can be synthesized by a third-party company or obtained commercially.
  • the overall structure of the compound of the present invention can be divided into a hydrophilic center composed of four tertiary amines (N) and a hydrophobic tail composed of four saturated aliphatic chains (T).
  • Each ionizable lipid molecule has The electropositive and hydrophilic and hydrophobic properties are equivalent, ensuring that its loading capacity for mRNA is equivalent.
  • the difference in fine structure constitutes a difference in specific compounds.
  • the synthesis of the compound of the present invention is mainly divided into two steps: first, constructing a 4N positive center through amide-forming reaction and Michael addition (Michael addition); second, through epoxy ring-opening reaction, alkylation reaction or Michael addition
  • Michael addition Michael addition
  • the reaction ligates the 4T hydrophobic tail.
  • Other exemplary compounds disclosed herein were prepared by similar routes described in Example 1, using different starting materials.
  • the synthesis route of the compound of the present invention is simple and easy to implement , the structure is clearly characterized and the compound yield is sufficient.
  • the inventor constructed the mRNA delivery system LNPs@mRNA based on the ionizable lipid prepared in Example 1, and examined its pharmaceutical properties such as particle size, potential, encapsulation rate, and TEM to evaluate its pharmaceutical properties. Formulated.
  • Solution preparation Dissolve ionizable lipids, DOPE (or DSPC), Chol, and DMG-PEG2000 in absolute ethanol to make the concentration of ionizable lipids 10 mg/mL.
  • the molar ratio of the ionizable lipid prepared in Example 1 of the present invention and C12-200, MC3, SM-102 and ALC-0315 is 50:10: 38.5:1.5.
  • the mRNA was diluted to an appropriate concentration with PBS buffer (prepared with RNase-free water) and set aside for use.
  • PBS buffer prepared with RNase-free water
  • LNP preparation Mix the ionizable lipid solution obtained in step (1) and the mRNA solution.
  • Microfluidic process parameters the volume ratio of ethanol phase and water phase is 1:3, and the flow rate is 9mL/min.
  • Ultrafiltration Dilute the LNP initial preparation 25 times with PBS buffer, and ultrafiltrate to the initial volume through the ultrafiltration cup to obtain the final LNP preparation. During the ultrafiltration process, the ethanol in the initial preparation is removed. Ultrafiltration process parameters: filter membrane 100kDa, air pressure 0.2MPa, rotation speed 100-200rpm.
  • Particle size potential measurement Take an appropriate amount of LNPs@mRNA preparation and dilute it 10 times with purified water, and use a Malvern nanoparticle size potentiometer to detect its particle size, particle size distribution, ⁇ potential and other pharmaceutical properties.
  • Transmission electron microscopy characterization dilute LNPs@mRNA with purified water to make the nanoparticle mass concentration 2 mg/mL, and drop carefully Put it on the TEM special copper grid, let it stand for 2 minutes, use filter paper to absorb the excess liquid, add 2% phosphotungstic acid for negative staining for 3 minutes, absorb the excess dyeing liquid, blow dry with an ear cleaning ball, load the sample for detection and take pictures.
  • Encapsulation efficiency detection solution preparation: dilute LNPs@mRNA with RNase-free water to an mRNA concentration of 0.1 ⁇ g/ ⁇ L. Under light-proof conditions, take an appropriate amount of RiboGreen dye and dilute it 400 times with 1 ⁇ TE buffer, and store it in light-proof conditions for later use.
  • Membrane rupture Add 25 ⁇ L of diluted mRNA preparation and 75 ⁇ L of 1 ⁇ TE buffer to the sample well, and add 25 ⁇ L of diluted mRNA preparation, 25 ⁇ L of 1 ⁇ TE buffer and 50 ⁇ L of 1% Triton to the total mRNA well. Incubate the above 96-well plate at 37°C in the dark for 10 minutes.
  • encapsulation rate (%) (1-sample well fluorescence value/total mRNA fluorescence value) ⁇ 100%.
  • LNPs prepared from different ionizable lipids prepared in Example 1 of the present invention have similar particle sizes, with average particle sizes around 100nm, Di (90) within 500nm, and PDI below 0.3, indicating that LNP particle size distribution is uniform.
  • the preparation potential is around 5mV.
  • the LNPs@mRNA prepared from different ionizable lipids prepared in Example 1 of the present invention were solid spherical, with a particle size of about 100 nm under TEM, and a fingerprint-like structure was found, which is a representative feature of LNP nanostructures.
  • the encapsulation rate test results are shown in Figure 2. Compared with the positive controls MC3, ALC-0315, and SM-102, the encapsulation rate of LNPs@mRNA prepared from the ionizable lipid in Example 1 of the present invention is significantly higher than that of the positive control. .
  • the foregoing examples verified the pharmaceutical properties of the ionizable lipid nanoparticles provided by the present invention. Furthermore, the inventor used EGFP mRNA and FLuc mRNA as reporter genes and conducted transfection experiments and toxicity experiments on HEK293T and DC2.4. Evaluate the in vitro effect and safety of LNPs@mRNA provided by the present invention.
  • LNPs@EGFP mRNA transfection experiment HEK293T and DC2.4 cells were plated. Then prepare LNPs@EGFP mRNA, positive control preparations MC3-LNPs@EGFP mRNA, ALC-0315-LNPs@EGFP mRNA, SM-102-LNPs@EGFP mRNA according to the method described in Example 4; control the mRNA concentration of the dosage preparation to be 0.02 ⁇ g/ ⁇ L, 50 ⁇ L was administered to each well of the 24-well cell plate, that is, 1 ⁇ g/well, and continued to be cultured in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, flow cytometry was used to detect the GFP positivity rate and mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • LNPs@FLuc mRNA transfection experiment HEK293T and DC2.4 cells were plated.
  • positive control preparations MC3-LNPs@FLuc mRNA, ALC-0315-LNPs@FLuc mRNA, SM-102-LNPs@FLuc mRNA were prepared by the method; the mRNA concentration of the dosing preparation was controlled to 0.02 ⁇ g/ ⁇ L, and 24-well cells were used.
  • substrate fluorescein fluorescein potassium salt, 15 mg/mL
  • Cytotoxicity experiment LNPs@FLuc mRNA and positive control preparation C12-200-LNPs@FLuc mRNA were selected to examine the MTT toxicity of DC2.4 cells.
  • the key experimental parameters are as follows: 10 ⁇ L per well in a 96-well plate; culture for 24 hours in an incubator at 37°C and 5% CO2; 5 mg/mL MTT solution, add 40 ⁇ L of the above MTT solution to each well, 37°C, 5% CO2 Incubate in the incubator for 4 hours.
  • substrate solution 15 mg/mL, fluorescein potassium salt
  • the exposure time is 60 seconds. After imaging, the total flux of each organ was counted.
  • LNP@Fluc mRNA positive control preparations MC3-LNPs@Fluc mRNA, ALC-0315-LNPs@Fluc mRNA, SM-102-LNPs@Fluc mRNA were prepared according to the method described in Example 4, using Adjust the mRNA concentration of the preparation to 0.1 mg/mL with a PBS solution (prepared in RNase-free water), and adjust the osmotic pressure of the preparation to isotonicity. Inject 200 ⁇ L into the hind leg muscle of each BALB/c mouse, that is, 20 ⁇ g FLuc mRNA/mouse. , 3 animals in each group, with PBS as negative control.
  • mice After administration, the mice were kept on a normal diet. 8 hours after administration, 200 ⁇ L of substrate solution (15 mg/mL, fluorescein potassium salt) was injected intraperitoneally, that is, 3 mg/animal. Start timing after injecting the substrate. After 10 minutes, put the mouse into the gas anesthesia device. After complete anesthesia, detect the bioluminescence intensity of the whole body in the IVIS instrument and expose it. The time is 60s. Eat normally after recovery. The above-mentioned anesthesia and testing were repeated 24 hours and 48 hours after administration. Calculate the total flux of the whole body and draw the luminous intensity-time curve.
  • substrate solution 15 mg/mL, fluorescein potassium salt
  • the in vivo expression results of the intravenous injection route show that, unexpectedly, compared with the control C12-200, the LNPs@mRNA of the present invention was expressed in the lungs via the intravenous injection route.
  • High expression which will be beneficial to nucleic acid drugs targeting the lungs; at the same time, it also has a certain expression level in the spleen, suggesting that the LNPs@mRNA of the present invention can also be used for nucleic acid drugs via intravenous administration.
  • LNPs of the present invention can be further used for the preparation of nucleic acid drugs targeting lung organs.
  • the E.G7-OVA (Ovalbumin ovalbumin) tumor model was further constructed, and OVA-mRNA was selected as the model antigen to evaluate the effectiveness of LNPs@mRNA as an mRNA tumor vaccine delivery system, and then screened out the high-efficiency tumor inhibitory effect of the present invention.
  • mRNA tumor vaccine delivery system, and the potential impact of the ionizable lipid chemical structure of the present invention on the effectiveness of LNPs@mRNA tumor vaccine was investigated.
  • the method for establishing the E.G7-OVA tumor model is as follows: when the E.G7-OVA cells are cultured to the logarithmic growth phase, collect the cells, wash the cells with sterile PBS, centrifuge and discard the supernatant, and reuse with sterile PBS. Suspend the cells and adjust the cell concentration to 10 7 /mL.
  • Each male C57BL/6 mouse was subcutaneously inoculated with 100 ⁇ L E.G7-OVA cells on the right flank, that is, 10 6 /mouse. Observe the growth status of the mice and the size of the subcutaneous tumors. After 5 days of inoculation, small visible tumors will form, and the immunotherapy experiment can be carried out.
  • E.G7-OVA cells (mouse T lymphoma cells) were purchased from ATCC and cultured in 1640 complete medium (containing 10% FBS and 1% P/S) in an incubator at 37°C and 5% CO2 at a cell density of Passage when reaching 80-90%.
  • the experimental plan is as follows: LNPs@OVA mRNA was prepared through microfluidic control agent technology, and its activation effect on BMDC in vitro and its effect on cytokine secretion were examined.
  • BMDC maturation and activation examination method cell plating.
  • LNPs@OVA mRNA and positive control preparation C12-200-LNPs@OVA mRNA were prepared according to the method described in Example 4, and the mRNA concentration of the dosage preparation was controlled to be 0.05 ⁇ g/ ⁇ L.
  • the cells were washed twice by centrifugation at 1500 rpm for 5 min with 1 mL of pre-cooled PBS, then 300 ⁇ L of PBS was added and mixed, and the mature activation status of BMDC was detected by flow cytometry.
  • Cytokine detection Detect the LNPs@OVA mRNA vaccine to stimulate BMDC maturation and activation. At the same time, after the incubation, collect the cell supernatant and dilute it 5 times with the sample diluent in the ELISA kit for testing. Dilute the standard and enzyme-labeled antibodies according to the kit instructions, establish a standard working curve for TNF- ⁇ , and detect the level of TNF- ⁇ secreted by BMDC in the cell supernatant using the double-antibody sandwich method.

Abstract

L'invention concerne un lipide ionisable pour l'administration d'acide nucléique et une composition de celui-ci, se rapportant au domaine de la chimie pharmaceutique. L'invention concerne un composé représenté par la formule I et l'utilisation du composé en tant que véhicule d'administration d'acide nucléique. Le composé, lorsqu'il est utilisé en tant que véhicule d'administration d'un médicament à base d'acide nucléique, a une efficacité d'administration plus forte et une sécurité plus élevée que les matériaux classiques.
PCT/CN2023/084750 2022-06-20 2023-06-15 Lipide ionisable pour l'administration d'acides nucléiques et composition associée WO2023246218A1 (fr)

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