WO2023246218A1 - Ionizable lipid for nucleic acid delivery and composition thereof - Google Patents

Ionizable lipid for nucleic acid delivery and composition thereof 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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PCT/CN2023/084750
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Chinese (zh)
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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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D295/00—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
    • 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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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • 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
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • 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

Provided are an ionizable lipid for nucleic acid delivery and a composition thereof, which belong to the field of pharmaceutical chemistry. Provided are a compound represented by formula I and use of the compound as a nucleic acid delivery carrier. When used as a delivery carrier of a nucleic acid drug, the compound shows stronger delivery efficiency and higher safety than traditional materials.

Description

用于核酸递送的可电离脂质及其组合物Ionizable lipids for nucleic acid delivery and compositions thereof 技术领域Technical field
本发明涉及用于核酸递送的可电离脂质及其组合物，属于药物化学领域。The present invention relates to ionizable lipids for nucleic acid delivery and compositions thereof, and belongs to the field of medicinal chemistry.
背景技术Background technique
核酸药物包括DNA、反义寡核苷酸(ASO)、小干扰RNA(siRNA)、微小RNA(miRNA)、miRNA mimics、antimiRs、核酶、mRNA、适配体、质粒、CRISPR RNA等。核酸药物的应用受其化学性质的不稳定限制，体外、体内易被核酸酶降解成单个核苷酸，而失去功效。Nucleic acid drugs include DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), miRNA mimics, antimiRs, ribozymes, mRNA, aptamers, plasmids, CRISPR RNA, etc. The application of nucleic acid drugs is limited by the instability of their chemical properties. They are easily degraded into single nucleotides by nucleases in vitro and in vivo, thus losing their efficacy.
核酸药物的应用往往需要特殊的递送载体，包括病毒载体和非病毒载体。常用病毒载体包括逆转录病毒、慢病毒、腺相关病毒等，病毒载体借助其天然的细胞感染活性能顺利侵入细胞，转运效率高，然而免疫原性、负载能力有限、生产工艺复杂等因素限制其临床应用。非病毒载体是目前研究较多、应用前景较好的一类基因递送载体，主要通过递送材料形成的阳离子与mRNA磷酸根离子的吸附作用负载mRNA，形成脂质体或纳米粒等结构，保护其免受核酸酶的降解并改变其入胞途径，优势在于载体相对易得、免疫原性低、安全性较高。The application of nucleic acid drugs often requires special delivery vectors, including viral vectors and non-viral vectors. Commonly used viral vectors include retroviruses, lentiviruses, adeno-associated viruses, etc. Viral vectors can successfully invade cells with their natural cell infection activity and have high transport efficiency. However, factors such as immunogenicity, limited loading capacity, and complex production processes limit their use. Clinical application. 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.
传统的非病毒核酸递送材料多为阳离子脂质或阳离子聚合物，因其强正电性，在体内容易被血浆蛋白吸附进而被网状内皮系统摄取，使所负载的核酸药物遭到破坏。非病毒载体中研究较多的为基于可电离脂质的脂质纳米粒，通过可电离脂质材料制备的纳米颗粒在体外酸性环境中显正电性，对核酸的静电吸附实现对核酸药物的负载，进入体内的中性环境后则显电中性，避免了血浆蛋白的吸附和网状内皮系统的捕获。基于此，可电离脂质纳米粒在核酸递送领域有着非常广阔的前景。Traditional 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. Among non-viral vectors, 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. Load, after entering the neutral environment in the body, it becomes electrically neutral, avoiding the adsorption of plasma proteins and capture by the reticuloendothelial system. Based on this, ionizable lipid nanoparticles have very broad prospects in the field of nucleic acid delivery.
然而，可电离脂质纳米粒的临床应用还相对较少，而核心难点在于安全有效的可电离脂质的开发。因此，开发具有更高递送效率、更安全的可电离脂质核酸递送材料，对于核酸药物基因治疗的广泛应用具有重要意义。However, 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.
发明内容Contents of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的目的在于提供用于核酸递送的可电离脂质。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.
本发明提供了式Ⅰ所示的化合物，或其药学上可接受的盐、异构体、氘代物或前药：
The present invention provides compounds represented by formula I, or pharmaceutically acceptable salts, isomers, deuterated products or prodrugs thereof:
其中，a为1,2或3；Among them, a is 1, 2 or 3;
X1、X2分别独立地选自N或C；X 1 and X 2 are independently selected from N or C;
L1、L2、L3、L4独立地选自-ReCH(OH)-、-ReC(＝O)-、-ReC(＝O)O-、-ReOC(＝O)-、-ReC(＝O)S-、-ReSC(＝O)-、-ReC(＝O)NRa-、-ReNRaC(＝O)-、-ReNRaC(＝O)O-、-ReOC(＝O)NRa-、-ReO-、-Re-O-O-、-ReS-、-Re-S-S-、-Re-S-S-S-、-ReCH(OH)CH2O-、-ReCH(OH)CH2S-或不存在，Re为或不存在，k为1以上的整数，Ra为-H、取代或未取代的烷基；L 1 , L 2 , L 3 , L 4 are independently selected from -R e CH(OH)-, -R e C(=O)-, -R e C(=O)O-, -R e OC( =O)-, -R e C(=O)S-, -R e SC(=O)-, -R e C(=O)NR a -, -R e NR a C(=O)-, -R e NR a C(=O)O-, -R e OC(=O)NR a -, -R e O-, -R e -OO-, -R e S-, -R e -SS- , -R e -SSS-, -R e CH(OH)CH 2 O-, -R e CH(OH)CH 2 S- or absent, R e is or does not exist, k is an integer above 1, R a is -H, substituted or unsubstituted alkyl;
R1、R2、R3、R4独立地选自C1-C30直链烷基、C1-C30支化烷基、C2-C30直链烯基、C2-C30支化烯基、C2-C30直链炔基或C2-C30支化炔基；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;
G1、G2、G3、G4独立地选自-Rc-、-RcCH(OH)Rd-、-RcC(＝O)Rd-、-RcC(＝O)ORd-、-RcOC(＝O)Rd-、-RcC(＝O)SRd-、-RcSC(＝O)Rd-、-RcC(＝O)N(Rb)Rd-、-RcN(Rb)C(＝O)Rd-、-RcN(Rb)C(＝O)ORd-、-RcOC(＝O)N(Rb)Rd-、-RcORd-、-Rc-O-O-Rd-、-RcSRd-、-Rc-S-S-Rd-、-Rc-S-S-S-Rd-或不存在，Rb为-H、取代或未取代的烷基，Rc、Rd独立地选自或不存在，n为1以上的整数；G 1 , G 2 , G 3 , G 4 are independently selected from -R c -, -R c CH(OH)R d -, -R c C(=O)R d -, -R c C(=O )OR d -, -R c OC(＝O)R d -, -R c C(＝O)SR d -, -R c SC(＝O)R d -, -R c C(＝O)N (R b )R d -, -R c N(R b )C(=O)R d -, -R c N(R b )C(=O)OR d -, -R c OC(=O) N(R b )R d -, -R c OR d -, -R c -OOR d -, -R c SR d -, -R c -SSR d -, -R c -SSSR d - or not present, R b is -H, substituted or unsubstituted alkyl, R c and R d are independently selected from or does not exist, n is an integer above 1;
R5、R6独立地选自-H、取代或未取代的烷基；R 5 and R 6 are independently selected from -H, substituted or unsubstituted alkyl;
Q1、Q2独立地选自O或S。Q 1 and Q 2 are independently selected from O or S.
上述可电离脂质形成的脂质纳米粒，在体外酸性环境中显正电性，对核酸的静电吸附实现对核酸药物的负载，进入体内的中性环境后则显电中性，避免了血浆蛋白的吸附和网状内皮系统的捕获。上述可电离脂质形成的脂质递送载体，对肝脏和肺的靶向性更强。其所携带的核酸药物，如mRNA，在受体细胞的表达更高，抗原mRNA所表达的抗原在体内的递呈更强。可用于mRNA等核酸药物的体内递送，实现对相应基因的上调或下调，或递送抗原mRNA在体内表达抗原实现免疫治疗作用，或递送编码抗体的mRNA在体内表达抗体等目的。同时，本申请的发明人还意外地发现，上述可电离脂质形成的脂质载体负载药物后，对小鼠肌肉注射途径给药时，其局部刺激性显著低于阳性对照载体，对小鼠体重增长无显著影响，而阳性 对照载体对小鼠体重增长有抑制作用。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. It can be used for the in vivo delivery of 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. At the same time, 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.
其中，以上限定的L1、L2、L3、L4连接键的书写顺序，由左至右对应近氮端至远氮端。Among them, 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.
以上限定的G1、G2、G3、G4连接键的书写顺序，由左至右对应式Ⅰ主链方向由左至右。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.
根据本发明的实施例，所述X1和X2同时为N。发明人发现，当X1和X2同时为N时，上述化合物所形成的脂质纳米粒制剂学性质更优，对mRNA具有更好的包封效果，进一步地，在试验动物体内能更高效地表达mRNA。According to an embodiment of the present invention, X 1 and X 2 are N at the same time. The inventor found that when X 1 and express mRNA.
根据本发明的实施例，所述化合物具有式II所示结构，或为式II所示结构的药学上可接受的盐、立体异构体、氘代物或前药，
According to an embodiment of the present invention, 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,
根据本发明的实施例，所述k为1,2,3,4,5,6,7,8,9或10。According to an embodiment of the present invention, the k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
根据本发明的实施例，所述k为1。According to an embodiment of the present invention, k is 1.
根据本发明的实施例，所述L1、L2、L3、L4独立地选自-CH(OH)-、-C(＝O)-、-CH2C(＝O)O-、-C(＝O)O-、-OC(＝O)-、-C(＝O)S-、-SC(＝O)-、-CH2C(＝O)NRa-、-C(＝O)NRa-、-NRaC(＝O)-、-NRaC(＝O)O-、-OC(＝O)NRa-、-CH2O-、-O-、-CH2-O-O-、-CH2S-、-S-、-CH2-S-S-、-CH2-S-S-S-、-CH(OH)CH2O-、-CH(OH)CH2S-或不存在，Ra为-H、取代或未取代的烷基。According to an embodiment of the present invention, the L1, L2, L3, and L4 are independently selected from -CH(OH)-, -C(=O)-, -CH2C(=O)O-, -C(=O) O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -CH2C(=O)NRa-, -C(=O)NRa-, -NRaC(= O)-, -NRaC(=O)O-, -OC(=O)NRa-, -CH2O-, -O-, -CH2-O-O-, -CH2S-, -S-, -CH2-S-S-, -CH2-S-S-S-, -CH(OH)CH2O-, -CH(OH)CH2S- or absent, Ra is -H, substituted or unsubstituted alkyl.
根据本发明的实施例，所述L1、L2、L3、L4独立地选自-C(＝O)-、-C(＝O)NRa-、-CH2C(＝O)NRa-、-NRaC(＝O)-、-C(＝O)O-、-CH2C(＝O)O-、-OC(＝O)-、-CH2O-、-O-、-CH2S-、-S-、-CH(OH)-、-CH(OH)CH2O-、-CH(OH)CH2S-或不存在，Ra为-H或未取代的烷基。According to an embodiment of the present invention, the L 1 , L 2 , L 3 , and L 4 are independently selected from -C(=O)-, -C(=O)NR a -, -CH 2 C(=O) NR a -, -NR a C(=O)-, -C(=O)O-, -CH 2 C(=O)O-, -OC(=O)-, -CH 2 O-, -O -, -CH 2 S-, -S-, -CH(OH)-, -CH(OH)CH 2 O-, -CH(OH)CH 2 S- or absent, R a is -H or unsubstituted of alkyl.
根据本发明的实施例，所述Ra为-H或未取代的C1～C6烷基。According to an embodiment of the present invention, the R a is -H or unsubstituted C 1 to C 6 alkyl.
根据本发明的实施例，所述Ra为-H。According to an embodiment of the present invention, the R a is -H.
根据本发明的实施例，所述L1、L2、L3、L4独立地选自-C(＝O)-、-C(＝O)NH-、-CH2C(＝O)NH-、-C(＝O)O-、-CH2C(＝O)O-、-CH2O-、-CH2S-、-CH(OH)-、-CH(OH)CH2O-或不存在；优选地，L1、L2、L3、L4独立地选自-C(＝O)NH-、-C(＝O)O-、-CH(OH)-、-CH(OH)CH2O-或不存在。 According to an embodiment of the present invention, the L 1 , L 2 , L 3 , and L 4 are independently selected from -C(=O)-, -C(=O)NH-, -CH 2 C(=O)NH -, -C(=O)O-, -CH 2 C(=O)O-, -CH 2 O-, -CH 2 S-, -CH(OH)-, -CH(OH)CH 2 O- Or absent; preferably, L 1 , L 2 , L 3 , L 4 are independently selected from -C(=O)NH-, -C(=O)O-, -CH(OH)-, -CH( OH)CH 2 O- or absent.
根据本发明的实施例，所述L1和L2选自相同的基团和或L3和L4选自相同的基团。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述L1和L3选自相同的基团和或L2和L4选自相同的基团。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述L1、L2、L3和L4选自相同的基团。According to an embodiment of the present invention, said L 1 , L 2 , L 3 and L 4 are selected from the same group.
根据本发明的实施例，所述R1、R2、R3、R4独立地选自C1-C30直链烷基、C2-C30直链烯基、C2-C30直链炔基。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述R1、R2、R3、R4独立地选自未取代的C1～C30直链烷基。According to an embodiment of the present invention, the R 1 , R 2 , R 3 , and R 4 are independently selected from unsubstituted C 1 to C 30 linear alkyl groups.
根据本发明的实施例，所述R1、R2、R3、R4独立地选自未取代的C8～C18直链烷基。According to an embodiment of the present invention, the R 1 , R 2 , R 3 , and R 4 are independently selected from unsubstituted C 8 to C 18 linear alkyl groups.
根据本发明的实施例，所述R1、R2、R3、R4独立地选自未取代的C10～C14直链烷基。According to an embodiment of the present invention, the R 1 , R 2 , R 3 , and R 4 are independently selected from unsubstituted C 10 to C 14 linear alkyl groups.
根据本发明的实施例，所述R1和R2选自相同的基团和或R3和R4选自相同的基团。According to an embodiment of the present invention, R 1 and R 2 are selected from the same group and or R 3 and R 4 are selected from the same group.
根据本发明的实施例，所述R1和R3选自相同的基团和或R2和R4选自相同的基团。According to an embodiment of the present invention, R 1 and R 3 are selected from the same group and or R 2 and R 4 are selected from the same group.
根据本发明的实施例，所述R1、R2、R3和R4选自相同的基团。According to an embodiment of the present invention, the R 1 , R 2 , R 3 and R 4 are selected from the same group.
根据本发明的实施例，所述G1、G2、G3、G4独立地选自或不存在，n3为1,2,3,4,5,6,7,8,9或10。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述G3、G4不存在。According to an embodiment of the present invention, the G 3 and G 4 do not exist.
根据本发明的实施例，所述G1、G2独立地选自-CH2-或-CH2CH2-。According to an embodiment of the present invention, the G 1 and G 2 are independently selected from -CH 2 - or -CH 2 CH 2 -.
根据本发明的实施例，所述R5、R6独立地选自-H、未取代的C1～C6烷基或-OH取代的C1～C6烷基。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述R5、R6独立地选自-H、甲基、乙基、丙基、羟甲基、羟乙基或羟丙基。According to an embodiment of the present invention, the R 5 and R 6 are independently selected from -H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl or hydroxypropyl.
根据本发明的实施例，所述R5、R6选自相同的基团。According to an embodiment of the present invention, the R 5 and R 6 are selected from the same group.
根据本发明的具体实施例，所述化合物具有以下结构中的一种：According to specific embodiments of the present invention, the compound has one of the following structures:
其药学上可接受的盐、立体异构体、氘代物或前药。 Its pharmaceutically acceptable salts, stereoisomers, deuterated products or prodrugs.
另一方面，本发明提供前面所述的化合物，或其药学上可接受的盐、立体异构体、氘代物或前药作为药物递送载体的用途。On the other hand, the present invention provides the use of the aforementioned compounds, or pharmaceutically acceptable salts, stereoisomers, deuterated products or prodrugs thereof, as drug delivery carriers.
进一步地，所述药物的活性成分任选自核酸、小分子化药、蛋白药物、多肽的至少一种。Further, the active ingredient of the drug is optionally selected from at least one of nucleic acids, small molecule drugs, protein drugs, and polypeptides.
具体地，所述药物的活性成分选自核酸。Specifically, the active ingredient of the drug is selected from nucleic acids.
具体地，所述药物具有心、肝、脾、肺或肾靶向性。Specifically, the drug has heart, liver, spleen, lung or kidney targeting.
较优地，所述药物靶向肺、脾脏。Preferably, the drug targets the lungs and spleen.
再一方面，本发明提出一种药物递送载体。根据本发明的实施例，所述药物递送载体包括含有前面所述的化合物，或其药学上可接受的盐、立体异构体、氘代物或前药。In yet another aspect, the present invention provides a drug delivery vehicle. According to an embodiment of the present invention, the drug delivery carrier includes a compound as described above, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof.
再一方面，本发明提出了一种药物复合物。根据本发明的实施例，所述药物复合物包括载体以及活性成分，所述载体与所述活性成分相连，所述载体包括阳离子脂质，所述阳离子脂质包括前面所述的化合物，或其药学上可接受的盐、立体异构体、氘代物或前药。In yet another aspect, the present invention provides a drug complex. According to an embodiment of the present invention, 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.
根据本发明的具体实施例，所述活性成分任选自核酸、小分子化药、蛋白药物、多肽、抗体中的至少一种。According to specific embodiments of the present invention, the active ingredient is optionally selected from at least one of nucleic acids, small molecule drugs, protein drugs, polypeptides, and antibodies.
根据本发明的具体实施例，所述药物的活性成分选自核酸；According to a specific embodiment of the present invention, the active ingredient of the drug is selected from nucleic acids;
根据本发明的具体实施例，所述核酸选自DNA、ASO、siRNA、miRNA、mRNA、适配体中至少一种。According to a specific embodiment of the present invention, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer.
根据本发明的具体实施例，所述核酸为mRNA；According to specific embodiments of the present invention, the nucleic acid is mRNA;
根据本发明的具体实施例，所述递送载体与所述mRNA通过离子键相连。According to a specific embodiment of the present invention, the delivery carrier is connected to the mRNA through ionic bonds.
根据本发明的具体实施例，所述药物复合物靶向肺、脾脏。 According to specific embodiments of the invention, the drug complex targets the lungs and spleen.
根据本发明的具体实施例，所述药物复合物以任选自脂质纳米颗粒LNP、PLGA纳米粒、胶束、脂质体、核-壳纳米粒、聚合物纳米颗粒中的一种形式存在。According to specific embodiments of the present invention, the drug complex exists in a form selected from lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, and polymer nanoparticles. .
根据本发明的具体实施例，所述药物复合物的制剂形式以脂质纳米颗粒LNP形式存在。According to a specific embodiment of the present invention, the drug complex is formulated in the form of lipid nanoparticles (LNP).
根据本发明的具体实施例，所述药物组合物进一步包含赋形剂，任选地，所述赋形剂包括选自中性磷脂、类固醇、聚乙二醇化脂质中的至少一种。According to a specific embodiment of the present invention, 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.
根据本发明的具体实施例，所述中性磷脂选自DOPE、DSPC、DOPC、DSPE、DMPC、DMPE、DPPC、DPPE、DEPC、HSPC、POPC中至少一种。According to a specific embodiment of the present invention, the neutral phospholipid is selected from at least one of DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC, and POPC.
根据本发明的具体实施例，所述中性磷脂为DOPE。According to a specific embodiment of the present invention, the neutral phospholipid is DOPE.
根据本发明的具体实施例，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述中性磷脂的摩尔比为1:10～10:1。According to specific embodiments of the present invention, 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.
根据本发明的具体实施例，所述类固醇选自胆固醇、谷甾醇、豆固醇、羊毛固醇、麦角固醇、岩藻甾醇中至少一种。According to a specific embodiment of the present invention, the steroid is selected from at least one selected from the group consisting of cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol, and fucosterol.
根据本发明的具体实施例，所述类固醇为胆固醇。According to a specific embodiment of the invention, the steroid is cholesterol.
根据本发明的具体实施例，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述类固醇的摩尔比为1:10～10:1。According to specific embodiments of the present invention, 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.
根据本发明的具体实施例，所述聚乙二醇化脂质选自DMG-PEG、DSPE-PEG中至少一种；According to a specific embodiment of the present invention, the PEGylated lipid is selected from at least one of DMG-PEG and DSPE-PEG;
优选地，所述聚乙二醇化脂质为DMG-PEG2000。Preferably, the pegylated lipid is DMG-PEG2000.
根据本发明的具体实施例，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述聚乙二醇化脂质的摩尔比为5:1～1000:1。According to specific embodiments of the present invention, 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.
根据本发明的具体实施例，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述聚乙二醇化脂质的摩尔比为10:1～20:1。According to specific embodiments of the present invention, 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.
根据本发明的具体实施例，所述核酸选自DNA、ASO、siRNA、miRNA、mRNA、适配体中至少一种。According to a specific embodiment of the present invention, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer.
根据本发明的具体实施例，所述核酸为mRNA。According to a specific embodiment of the invention, the nucleic acid is mRNA.
另一方面，本发明提出了一种脂质纳米颗粒。根据本发明的实例，所述脂质纳米颗粒包括脂质纳米衣壳和核酸，所述核酸包裹于所述脂质纳米衣壳内，所述脂质纳米衣壳包括阳离子脂质，所述阳离子脂质包括含前述的化合物，或其药学上可接受的盐、立体异构体、氘代物或前药。 On the other hand, the present invention proposes lipid nanoparticles. According to an example of the present invention, 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.
根据本发明的具体实施例，所述脂质纳米颗粒的平均粒径为40nm～500nm，Di(90)≤500nm，多分散系数≤30％。According to specific embodiments of the present invention, the average particle size of the lipid nanoparticles is 40 nm to 500 nm, Di (90) ≤ 500 nm, and the polydispersity coefficient is ≤ 30%.
另一方面，本发明提出了一种药物组合物。根据本发明的具体实施例，包括前述的药物复合物或前述的脂质纳米颗粒。In another aspect, 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.
根据本发明的具体实施例，进一步包括药学上可接受的辅料。According to specific embodiments of the present invention, pharmaceutically acceptable excipients are further included.
根据本发明的具体实施例，所述组合物为注射剂。According to a specific embodiment of the invention, the composition is an injection.
根据本发明的具体实施例，所述组合物适于静脉或肌肉注射。According to specific embodiments of the invention, the composition is suitable for intravenous or intramuscular injection.
另一方面，本发明提出了前述药物复合物或前述的脂质纳米颗粒或前述的药物组合物在制备药物中的用途，所述药物用于治疗或预防疾病。On the other hand, 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.
根据本发明的具体实施例，所述疾病为心、肝、脾、肺或肾相关疾病，优选地，所述疾病为脾或肺相关疾病。According to a specific embodiment of the present invention, the disease is a heart, liver, spleen, lung or kidney related disease, preferably, the disease is a spleen or lung related disease.
根据本发明的具体实施例，所述疾病为感染性疾病、癌症和增生性疾病、遗传性疾病、自体免疫性疾病、糖尿病、神经退化性疾病、心血管和肾血管疾病以及代谢性疾病。According to specific embodiments of the invention, the diseases are infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases and metabolic diseases.
根据本发明的具体实施例，所述感染性疾病选自：由冠状病毒、流感病毒或HIV病毒引起的疾病，小儿肺炎，裂谷热，黄热病，狂犬病，或多种疱疹。According to a specific embodiment of the present invention, 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.
根据本发明的具体实施例，所述癌症为实体肿瘤，优选地，所述癌症为肝癌或肺癌。According to a specific embodiment of the present invention, the cancer is a solid tumor. Preferably, the cancer is liver cancer or lung cancer.
根据本发明的具体实施例，所述药物通过递呈抗原和/或激活免疫反应治疗或预防疾病。According to specific embodiments of the present invention, the drug treats or prevents diseases by presenting antigens and/or activating immune responses.
本发明提供了一种新型可电离脂质，其亲水中心由4个叔胺有效氮构成，疏水尾部由4条饱和或不饱和的脂肪链构成。本发明所提供的新型可电离脂质，在酸性环境中带正电，中性环境下几乎不带电，利用这一属性可在酸性缓冲系统中装载核酸药物，核酸药物装载后进入体内的中性环境，脂质纳米粒显电中性，可有效避免核酸药物复合物被血浆蛋白的吸附，从而实现更高的对核酸药物的递送效率，安全性也显著提高。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.
附图说明Description of the drawings
图1实施例4制得的LNPs@mRNA的粒径、PDI及电位；Figure 1 Particle size, PDI and potential of LNPs@mRNA prepared in Example 4;
图2实施例4制得的LNPs@mRNA的包封率；Figure 2 Encapsulation efficiency of LNPs@mRNA prepared in Example 4;
图3报告基因的LNPs@mRNA细胞水平表达：(A)LNPs@EGFP mRNA转染HEK293T细胞；(B)LNPs@EGFP mRNA转染DC2.4细胞；(C)LNPs@FLuc mRNA转染DC2.4细胞；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;
图4 MTT法测4N4T-LNPs@FLuc mRNA细胞毒性； Figure 4 MTT method to measure 4N4T-LNPs@FLuc mRNA cytotoxicity;
图5 LNPs@FLuc mRNA表达及分布的生物发光图像；Figure 5 Bioluminescence image of LNPs@FLuc mRNA expression and distribution;
图6静脉给药LNPs@FLuc mRNA表达及分布的统计；Figure 6 Statistics of mRNA expression and distribution of intravenously administered LNPs@FLuc;
图7 LNPs@OVA mRNA疫苗对BMDC的激活作用：Figure 7 Activation effect of LNPs@OVA mRNA vaccine on BMDC:
图8 LNPs@OVA mRNA疫苗处理BMDC的TNF-α细胞因子分泌。Figure 8 TNF-α cytokine secretion from BMDC treated with LNPs@OVA mRNA vaccine.
具体实施方式Detailed ways
本发明可在不偏离本发明基本属性的情况下以其它具体形式来实施。应该理解的是，在不冲突的前提下，本发明的任一和所有实施方案都可与任一其它实施方案或多个其它实施方案中的技术特征进行组合以得到另外的实施方案。本发明包括这样的组合得到的另外的实施方案。The invention may be embodied in other specific forms without departing from the essential attributes of the invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with technical features of any other embodiment or multiple other embodiments to obtain additional embodiments. The invention includes additional embodiments resulting from such combinations.
本公开中提及的所有出版物和专利在此通过引用以它们的全部内容纳入本公开。如果通过引用纳入的任何出版物和专利中使用的用途或术语与本公开中使用的用途或术语冲突，那么以本公开的用途和术语为准。All publications and patents mentioned in this disclosure are hereby incorporated by reference into the disclosure in their entirety. If the usage or terminology used in any publications and patents incorporated by reference conflicts with the usage or terminology used in this disclosure, the usage or terminology used in this disclosure shall control.
本文所用的章节标题仅用于组织文章的目的，而不应被解释为对所述主题的限制。The section headings used in this article are for the purpose of organizing the article only and should not be construed as limitations on the subject matter described.
除非另有规定，本文使用的所有技术术语和科学术语具有要求保护主题所属领域的通常含义。倘若对于某术语存在多个定义，则以本文定义为准。Unless otherwise defined, all technical and scientific terms used herein have their ordinary meaning in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.
除了在工作实施例中或另外指出之外，在说明书和权利要求中陈述的定量性质例如剂量的所有数字应理解为在所有情况中被术语“约”修饰。还应理解的是，本申请列举的任何数字范围意在包括该范围内的所有的子范围和该范围或子范围的各个端点的任何组合。Except in the working examples or where otherwise indicated, all numbers reciting quantitative properties, such as dosages, in the specification and claims are to be understood as modified in all instances by the term "about". It should also be understood that any numerical range recited herein is intended to include all subranges within that range and any combination of the individual endpoints of such ranges or subranges.
本公开中使用的“包括”、“含有”或者“包含”等类似的词语意指出现该词前面的要素涵盖出现在该词后面列举的要素及其等同，而不排除未记载的要素。本文所用的术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…组成”、或“由…组成”。The use of similar words such as "includes", "contains" or "includes" in this disclosure means that the elements appearing before the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The term "contains" or "includes" as used herein can be open, semi-closed and closed. In other words, the term also includes "consisting essentially of," or "consisting of."
本发明中提供的化合物和衍生物可以根据IUPAC(国际纯粹与应用化学联合会)或CAS(化学文摘服务社，Columbus，OH)命名系统命名。Compounds and derivatives provided in the present invention may be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature systems.
术语“烷基”是直链或支链的饱和烃基的基团。C1～C6烷基的实例包括但不限于甲基(C1)、乙基(C2)、正丙基(C3)、异丙基(C3)、正丁基(C4)、叔丁基(C4)、仲丁基(C4)、异丁基(C4)、正戊基(C5)、3-戊基(C5)、戊基(C5)、新戊基(C5)、3-甲基-2-丁基(C5)、叔戊基(C5)和正己基(C6)。 The term "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).
术语“烯基”是指含有至少一个双键的直链或支链烃基。烯基的例子包括但不限于乙烯基、烯丙基、丁-1-烯基、丁-2-烯基、戊-1-烯基、戊-2-烯基、戊-3-烯基、己-1-烯基、己-2-烯基、己-3-烯基、己-4-烯基。The term "alkenyl" refers to a straight or branched chain hydrocarbon radical containing at least one double bond. Examples of 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.
术语“炔基”是指含有至少一个叁键的直链或支链烃基。炔基的例子包括但不限于乙炔基、炔丙基、丁-1-炔基、丁-2-炔基、戊-1-炔基、戊-2-炔基、戊-3-炔基、己-1-炔基、己-2-炔基、己-3-炔基、己-4-炔基。The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one triple bond. Examples of 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.
术语“药学上可接受的”是指某载体、赋形剂、盐等通常在化学上或物理上与构成某药物剂型的其它成分相兼容，并在生理上与受体相兼容，具体地，化合物或复合物在化学上和/或在毒理学上与构成制剂的其它成分和/或与用其预防或治疗疾病或病症的人类或哺乳动物相容。The term "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. Specifically, 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.
术语“受试者”或“患者”在本申请中包括人类和哺乳动物。The term "subject" or "patient" as used herein includes humans and mammals.
本文所用的术语“治疗”是指给患有疾病或者具有所述疾病的症状的患者或受试者施用一种或多种药物物质，用以治愈、缓解、减轻、改善或影响所述疾病或者所述疾病的症状。在本申请的上下文中，除非作出相反的具体说明，术语“治疗”也可包括预防。The term "treatment" as used herein 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. In the context of this application, the term "treatment" may also include prevention unless specifically stated to the contrary.
术语“药学上可接受的盐”是指本发明化合物与无机和/或有机酸和碱形成的酸式和/或碱式盐，也包括两性离子盐(内盐)，还包括季铵盐，例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将上述化合物与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集，或在溶剂蒸发后回收而得到，或在水介质中反应后冷冻干燥制得。本发明中所述盐可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。The term "pharmaceutically acceptable 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.
应进一步理解，式I化合物或其药学上可接受的盐可以溶剂合物形式分离，并且因此任何所述溶剂合物皆包括于本发明的范围内。例如，式I化合物或其药学上可接受的盐可以未溶剂化形式以及与药学上可接受的溶剂(诸如，水、乙醇等)形成的溶剂化形式存在。It is further understood that 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. For example, 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.
本公开的某些化合物可以以一种或多种立体异构体的形式存在。立体异构体包括几何异构体、非对映异构体和对映异构体。因此，本公开要求保护的化合物还包括外消旋混合物、单一的立体异构体和具有光学活性的混合物。本领域技术人员应该理解的是，一种立体异构体可能比其它立体异构体具有更好的功效和/或更低的副作用。单一的立体异构体和具有光学活性的混合物可手性源合成法、手性催化、手性拆分等方法得到。消旋体可通过色谱拆分法 或者化学拆分法进行手性拆分。例如，可通过加入手性酒石酸、手性苹果酸等手性酸类拆分试剂与本公开的化合物成盐，利用产物的物理化学性质例如溶解度不同进行分离。Certain compounds of the present disclosure may exist as one or more stereoisomers. 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. For example, 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.
本发明还包括本公开化合物所有适合的同位素变体。同位素变体定义为这样的化合物，其中至少一个原子被具有相同原子序数但其原子质量不同于自然界中常见的或主要存在的原子质量的原子替代。可以引入到本公开化合物中的同位素的实例包括氢、碳、氮和氧的同位素，分别例如2H(氘)、3H(氚)、11C、13C、14C、15N、17O和18O。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. Examples of 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.
“治疗有效量”为当给予患者时能改善疾病或症状的治疗剂的量。“预防有效量”为当给予受试者时能预防疾病或症状的预防剂的量。构成“治疗有效量”的治疗剂的量或“预防有效量”的预防剂的量随着治疗剂/预防剂、疾病状态及其严重性、待治疗/预防的患者/受试者的年龄、体重等而变化。本领域普通技术人员可根据其知识以及本公开常规地确定治疗有效量和预防有效量。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.
在本申请中，当化合物的名称与结构式不一致时，以结构式为准。In this application, when the name of a compound is inconsistent with its structural formula, the structural formula shall prevail.
应理解，本申请所用的术语“本公开化合物”根据语境可包括：式I化合物、其溶剂合物、其药学上可接受的盐、其立体异构体、以及它们的混合物。It should be understood that the term "compounds of the present disclosure" as used in this application may include, depending on the context: compounds of Formula I, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.
本文所用术语阳离子脂质是指在选定的pH值带正电荷的脂质。The term cationic lipids as used herein refers to lipids that are positively charged at a selected pH value.
阳离子脂质体易于与带负电荷的核酸结合，即通过静电力与核酸中存在的带负电的磷酸基团相互作用，形成脂质纳米颗粒(LNP)。LNP是目前主流的递送载体之一。Cationic liposomes easily bind to negatively charged nucleic acids by interacting with the negatively charged phosphate groups present in the nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNP is one of the current mainstream delivery vehicles.
本发明提供了一种新型可电离脂质，其具有式(I)或式(II)所示的结构，其亲水中心由4个叔胺有效氮构成，疏水尾部由4条饱和或不饱和的脂肪链构成。本发明所提供的新型可电离脂质，在酸性环境中带正电，中性环境下几乎不带电，利用这一属性可在酸性缓冲系统中装载核酸药物，核酸药物装载后进入体内的中性环境，脂质纳米粒显正电性，可有效避免核酸药物复合物被血浆蛋白的吸附，从而实现更高的对核酸药物的递送效率，安全性也显著提高。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.
本公开的又一方面提供一种药物复合物，其包含载体，所述载体包括阳离子脂质，所述阳离子脂质包括上述的式(I)式(II)化合物或其药学上可接受的盐或立体异构体。Another aspect of the present disclosure provides 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.
在一种实施方案中，所述复合物为纳米颗粒制剂，所述纳米颗粒制剂的平均尺寸为40nm～500nm，优选为100nm～205nm；所述纳米颗粒制剂的多分散系数≤50％，优选≤30％，更优选≤25％。In one embodiment, 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%.
在本公开的复合物/载体的一种实施方式中，所述阳离子脂质为选自上述的式(I)或式(II) 化合物或其药学上可接受的盐或立体异构体中、氘代物的一种或多种。在一种实施方案中，所述阳离子脂质为选自上述的式(I)化合物。In one embodiment of the complex/carrier of the present disclosure, 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.
在本公开的复合物/载体的另一种实施方式中，所述阳离子脂质包括：(a)选自上述的式(I)化合物或其氘代物、药学上可接受的盐或立体异构体中的一种或多种；(b)一种或多种与(a)不同的其它可电离的脂质化合物。(b)阳离子脂质化合物可以是商购的阳离子脂质，或者文献中报道的阳离子脂质化合物。In another embodiment of the complex/carrier of the present disclosure, 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.
在一种实施方案中，所述阳离子脂质占载体的摩尔比为30％～70％，例如35％、45％、50％、55％、60％、65％。In one embodiment, the molar ratio of the cationic lipid to the carrier is 30% to 70%, such as 35%, 45%, 50%, 55%, 60%, 65%.
该载体可用于递送活性成分例如治疗剂或预防剂。活性成分可包封在载体内或者与载体结合。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.
例如，所述治疗剂或预防剂包括核酸分子、小分子化合物、大分子化合物、多肽、抗体或蛋白质中的一种或多种。所述核酸包括但不限于单链DNA、双链DNA和RNA。适宜的RNA包括但不限于小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、微RNA(miRNA)、Dicer底物RNA(dsRNA)、小发夹RNA(shRNA)、信使RNA(mRNA)以及其混合物。For example, 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.
载体可包含中性脂质，如中性磷脂。中性脂质在本公开中是指在选定的pH值不带电荷或者以两性离子形式存在的起辅助作用的脂质。该中性脂质可能通过促进脂质相变来调节纳米颗粒流动性成脂质双层结构并提高效率，同时还可能影响靶器官的特异性。The carrier may comprise neutral lipids, such as neutral phospholipids. Neutral lipids are used in this disclosure to refer to supporting lipids that are uncharged or exist in zwitterionic form at a selected pH value. This neutral lipid may regulate nanoparticle fluidity into a lipid bilayer structure and improve efficiency by promoting lipid phase transition, and may also affect target organ specificity.
在一种实施方案中，所述阳离子脂质与所述中性脂质的摩尔比为约1:1～10:1，例约9:1、8:1、7:1、6:1、5:1、4:1、3:1。在一种优选的实施方案中，所述阳离子脂质与所述中性脂质的摩尔比为约5:1。In one embodiment, 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.
例如，中性脂质可包括磷脂酰胆碱、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。For example, the neutral lipid may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.
包含阳离子脂质复合物的载体组分可以包括一种或多种中性脂质磷脂，如一种或多种(多)不饱和脂质。磷脂可以组装成一个或多个脂质双层。一般来说，磷脂可以包括磷脂部分和一个或多个脂肪酸部分。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. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
中性脂质部分可以选自由以下组成的非限制性组：磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰甘油、磷脂酰丝氨酸、磷脂酸、2溶血磷脂酰胆碱和鞘磷脂。脂肪酸部分可以选自由以下组成的非限制性组：月桂酸、肉豆蔻酸、肉豆蔻烯酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、α亚麻酸、芥酸、植烷酸、花生酸、花生四烯酸、二十碳五烯酸、山萮酸、二十二碳 五烯酸和二十二碳六烯酸。还涵盖包括带有修饰和取代的天然物种的非天然物种，所述修饰和取代包括分支、氧化、环化和炔烃。例如，磷脂可以用一种或多种炔烃(例如一个或多个双键被三键置换的烯基)官能化或与该一种或多种炔烃交联。在适当反应条件下，炔基在暴露于叠氮化物时可能经历铜催化的环加成反应。这些反应可用于使复合物的脂质双层官能化以促进膜渗透或细胞识别，或将复合物与有用组分如靶向或成像部分(例如染料)偶联。The neutral lipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha linolenic acid, erucic acid, Phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, behenic acid Pentaenoic acid and docosahexaenoic acid. Also contemplated are non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization and alkynes. For example, 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). Under appropriate reaction conditions, 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).
可用于这些复合物中的中性脂质可以选自由以下组成的非限制性组：1,2二亚油酰基sn甘油3磷酸胆碱(DLPC)、1,2二肉豆蔻酰基sn甘油磷酸胆碱(DMPC)、1,2二油酰基sn甘油3磷酸胆碱(DOPC)、1,2二棕榈酰基sn甘油3磷酸胆碱(DPPC)、1,2二硬脂酰基sn甘油3磷酸胆碱(DSPC)、1,2双十一烷酰基sn甘油磷酸胆碱(DUPC)、1棕榈酰基2油酰基sn甘油3磷酸胆碱(POPC)、1,2二O十八碳烯基sn甘油3磷酸胆碱(18:0 Diether PC)、1油酰基2胆固醇基半琥珀酰基sn甘油3磷酸胆碱(OChemsPC)、1十六烷基sn甘油3磷酸胆碱(C16 Lyso PC)、1,2二亚麻酰基sn甘油3磷酸胆碱、1,2二花生四烯酰基sn甘油3磷酸胆碱、1,2双二十二碳六烯酰基sn甘油3磷酸胆碱、1,2二油酰基sn甘油3磷酸乙醇胺(DOPE)、1,2二植烷酰基sn甘油3磷酸乙醇胺(ME 16.0 PE)、1,2二硬脂酰基sn甘油3磷酸乙醇胺、1,2二亚油酰基sn甘油3磷酸乙醇胺、1,2二亚麻酰基sn甘油3磷酸乙醇胺、1,2二花生四烯酰基sn甘油3磷酸乙醇胺、1,2双二十二碳六烯酰基sn甘油3磷酸乙醇胺、1,2二油酰基sn甘油3磷酸rac(1甘油)钠盐(DOPG)、二棕榈酰基磷脂酰甘油(DPPG)、棕榈酰基油酰基磷脂酰乙醇胺(POPE)、二硬脂酰基磷脂酰乙醇胺(DSPE)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、1硬脂酰基2油酰基硬脂酰乙醇胺(SOPE)、1硬脂酰基2油酰基磷脂酰胆碱(SOPC)、鞘磷脂、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酸、棕榈酰基油酰基磷脂酰胆碱、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺(LPE)以及其混合物。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-succinyl sn glyceryl 3 phosphocholine (OChemsPC), 1 hexadecyl sn glyceryl 3 phosphocholine (C16 Lyso PC), 1,2 Dilinolenoyl sn glyceryl 3 phosphocholine, 1,2 diarachidonoyl sn glyceryl 3 phosphocholine, 1,2 didocosahexaenoyl sn glyceryl 3 phosphocholine, 1,2 dioleoyl sn Glyceryl 3-phosphoethanolamine (DOPE), 1,2 diphytanyl sn glycerol 3 phosphoethanolamine (ME 16.0 PE), 1,2 distearoyl sn glycerol 3 phosphoethanolamine, 1,2 dilinoleoyl sn glycerol 3 phosphate Ethanolamine, 1,2 dilinolenoyl sn glycerol 3 phosphoethanolamine, 1,2 diarachidonoyl sn glycerol 3 phosphoethanolamine, 1,2 didocosahexaenoyl sn glycerol 3 phosphoethanolamine, 1,2 diole Acyl sn glycerol 3 phosphate rac (1 glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl Acyl Phosphatidylethanolamine (DPPE), Dimyristoyl Phosphoethanolamine (DMPE), 1 Stearoyl 2 Oleoyl Stearoylethanolamine (SOPE), 1 Stearoyl 2 Oleoyl Phosphatidylcholine (SOPC), Sphingomyelin , phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.
在一些实施方案中，中性脂质包括DSPC。在某些实施方案中，中性脂质包括DOPE。In some embodiments, the neutral lipid includes DSPC. In certain embodiments, the neutral lipid includes DOPE.
在一些实施方案中，中性脂质包括DSPC和DOPE两种。In some embodiments, neutral lipids include both DSPC and DOPE.
包含阳离子脂质的复合物的载体还可以包括一种或多种结构性脂质，如类固醇。结构性脂质在本公开中是指通过填充脂质之间的间隙来增强纳米颗粒的稳定性的脂质。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.
在一种实施方案中，所述阳离子脂质与所述结构性脂质的摩尔比为约1:1～5:1，例如，约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。In one embodiment, 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.
结构性脂质可以选自但不限于由以下组成的组：胆固醇、非甾醇、谷固醇、麦角固醇、 菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、番茄碱、熊果酸、α生育酚、皮质类固醇以及其混合物。在一些实施方案中，结构性脂质是胆固醇。在一些实施方案中，结构性脂质包括胆固醇和皮质类固醇(如泼尼松龙(prednisolone)、地塞米松、泼尼松(prednisone)和氢化可的松(hydrocortisone))或其组合。Structural lipids may be selected from, but are not limited to, the group consisting of: cholesterol, nonsterols, sitosterol, ergosterol, Campesterol, stigmasterol, brassisterol, tomatine, tomatine, ursolic acid, alpha tocopherol, corticosteroids and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, structural lipids include cholesterol and corticosteroids (eg, prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.
包含阳离子脂质的复合物的载体还可以包括一种或多种聚合物共轭脂质，如聚乙二醇化脂质。聚合物共轭脂质主要是指聚乙二醇(PEG)修饰的脂质。亲水性PEG稳定LNP，通过限制脂质融合来调节纳米颗粒大小，并通过减少与巨噬细胞的非特异性相互作用来增加纳米颗粒的半衰期。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.
在一种实施方案中，所述聚合物共轭脂质选自以下中的一种或多种：PEG修饰的磷脂酰乙醇胺、PEG修饰的磷脂酸、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、PEG修饰的二烷基甘油。PEG修饰的PEG分子量通常为350 5000Da。In one embodiment, 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.
例如，所述聚合物共轭脂质选自以下中的一种或多种：二硬脂酰基磷脂酰乙醇胺聚乙二醇2000(DSPE PEG2000)，二肉豆蔻酰甘油3甲氧基聚乙二醇2000(DMG PEG2000)和甲氧基聚乙二醇双十四烷基乙酰胺(ALC 0159)。For example, 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).
在本公开的复合物/载体的一种实施方案中，所述聚合物共轭脂质是D M G PEG2000。In one embodiment of the complex/carrier of the present disclosure, the polymeric conjugated lipid is DMG PEG2000.
在本公开的复合物/载体的一种实施方案中，载体包括中性脂质、结构脂质以及聚合物共轭脂质，所述可电离脂质、所述中性脂质、所述结构脂质、以及所述聚合物共轭脂质的摩尔比为50:10:38.5:1。In one embodiment of the complex/carrier of the present disclosure, 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.
药物活性成分active pharmaceutical ingredients
药物活性成分并且替代地称为“活性剂”可以包括一种或多种治疗剂和/或预防剂。在一种实施方案中，所述可电离脂质与所述治疗剂或预防剂的质量比为0.1:1-1000:1。Pharmaceutically active ingredients and alternatively "active agents" may include one or more therapeutic and/or prophylactic agents. In one embodiment, the mass ratio of the ionizable lipid to the therapeutic or preventive agent is 0.1:1-1000:1.
所述治疗剂或预防剂包括但不限于核酸分子、小分子化合物、多肽或蛋白质中的一种或多种。The therapeutic or preventive agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins.
例如，所述治疗剂或预防剂是能够引起免疫响应的疫苗或化合物。For example, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.
本公开的载体可将治疗剂和/或预防剂递送至哺乳动物细胞或器官，因此本公开还提供治疗有需要哺乳动物的疾病或病症的方法，这些方法包括向哺乳动物施用包括治疗剂和/或预防剂的复合物、药物组合物和/或使哺乳动物细胞与该复合物或药物组合物接触。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.
治疗剂和/或预防剂可以是在递送至细胞或器官后在该细胞或器官中或者其它身体组织或系统中引起所希望的变化的物质。此类物种可用于治疗一种或多种疾病、病症或病况。在 一些实施方案中，治疗剂和/或预防剂是可用于治疗特定疾病、病症或病况的小分子药物。可用于复合物的药物的实例包括但不限于抗赘生剂(例如长春新碱(vincristine)、多柔比星(doxorubicin)、米托蒽醌(mitoxantrone)、喜树碱(camptothecin)、顺铂(cisplatin)、博莱霉素(bleomycin)、环磷酰胺(cyclophosphamide)、甲氨蝶呤和链脲佐菌素(streptozotocin))、抗肿瘤剂(例如放线菌素D(actinomycin D)、长春新碱、长春碱(vinblastine)、胞嘧啶阿拉伯糖苷(cytosine arabinoside)、蒽环霉素(anthracycline)、烷化剂、铂类化合物、抗代谢物以及核苷类似物，如甲氨蝶呤以及嘌呤和嘧啶类似物)、抗感染剂、局部麻醉剂(例如地布卡因(dibucaine)和氯丙嗪(chlorpromazine))、β肾上腺素能阻断剂(例如普萘洛尔(propranolol)、第莫洛(timolol)和拉贝洛尔(labetalol))、抗高血压剂(例如可乐定(clonidine)和肼酞嗪(hydralazine))、抗抑郁剂(例如丙咪嗪(imipramine)、阿米替林(amitriptyline)和多虑平(doxepin))、抗痉挛剂(例如苯妥英(phenytoin))、抗组胺(例如苯海拉明(diphenhydramine)、氯苯那敏(chlorpheniramine)和异丙嗪(promethazine))、抗生素/抗细菌剂(例如庆大霉素(gentamycin)、环丙沙星(ciprofloxacin)和头孢西丁(cefoxitin))、抗真菌剂(例如咪康唑(miconazole)、特康唑(terconazole)、益康唑(econazole)、异康唑(isoconazole)、布康唑(butaconazole)、克霉唑(clotrimazole)、伊曲康唑(itraconazole)、制霉菌素(nystatin)、奈替芬(naftifine)和两性霉素B(amphotericin B))、抗寄生虫剂、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼药、维生素、镇静剂以及成像剂。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. Examples of drugs that may be used in the complex 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 hydralazine), antidepressants (such as imipramine, amitriptyline amitriptyline and doxepin), antispasmodics (such as phenytoin), antihistamines (such as diphenhydramine, chlorpheniramine and promethazine) , antibiotics/antibacterials (such as gentamycin, ciprofloxacin, and cefoxitin), antifungals (such as miconazole, terconazole) , econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, and imaging agents.
在一些实施方案中，治疗剂和/或预防剂是细胞毒素、放射性离子、化学治疗剂、疫苗、引起免疫响应的化合物和/或另一治疗剂和/或预防剂。细胞毒素或细胞毒性剂包括对细胞有害的任何试剂。实例包括但不限于紫杉酚(taxol)、细胞松弛素B(cytochalasin B)、短杆菌肽D(gramicidin D)、溴化乙锭(ethidium bromide)、依米丁(emetine)、丝裂霉素(mitomycin)、依托泊苷(etoposide)、替尼泊苷(teniposide)、长春新碱、长春碱、秋水仙碱(colchicine)、多柔比星、柔红霉素(daunorubicin)、二羟基蒽二酮(dihydroxy anthracin dione)、米托蒽醌、光辉霉素(mithramycin)、放线菌素D、1去氢睾酮、糖皮质激素、普鲁卡因(procaine)、丁卡因(tetracaine)、利多卡因(lidocaine)、普萘洛尔、嘌呤霉素、类美登素(maytansinoid)如美登醇(maytansinol)、拉奇霉素(rachelmycin)(CC 1065)，以及其类似物或同系物。放射性离子包括但不限于碘(例如碘125或碘131)、锶89、磷、钯、铯、铱、磷酸根、钴、钇90、钐153和镨。疫苗包括能够提供针对与感染性疾病如流感、麻疹、人乳头瘤病毒(HPV)、狂犬病、脑膜炎、百日咳、破伤风、瘟疫、肝炎和肺结核相关的一种或多种病况的免疫性的化合物和制剂并且可以包括 编码感染性疾病源性抗原和/或表位的mRNA。疫苗还可以包括引导针对癌细胞的免疫响应的化合物和制剂并且可以包括编码肿瘤细胞源性抗原、表位和/或新表位的mRNA。引起免疫响应的化合物可以包括疫苗、皮质类固醇(例如地塞米松)和其它物种。在一些实施方案中，通过包括根据式(I)的复合物肌肉内施用能够引起免疫响应的疫苗和/或化合物。其它治疗剂和/或预防剂包括但不限于抗代谢物(例如甲氨蝶呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷In some embodiments, 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. 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. In some embodiments, 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
和5氟尿嘧啶达卡巴嗪(dacarbazine))、烷化剂(例如氮芥(mechlorethamine)、噻替哌(thiotepa)、苯丁酸氮芥(chlorambucil)、拉奇霉素(CC 1065)、美法兰(melphalan)、卡莫司汀(carmustine，BSNU)、罗莫司丁(lomustine，CCNU)、环磷酰胺、白消安(busulfan)、二溴甘露醇、链脲佐菌素、丝裂霉素C和顺二氯二胺络铂(II)(DDP)、顺铂)、蒽环霉素(例如柔红霉素(以前称为道诺霉素(daunomycin))和多柔比星)、抗生素(例如更生霉素(dactinomycin)(以前称为放线菌素)、博莱霉素、光辉霉素(mithramycin)和安曲霉素(anthramycin，AMC))以及抗有丝分裂剂(例如长春新碱、长春碱、紫杉酚和类美登素)。and 5-fluorouracil (dacarbazine), 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).
在其它实施方案中，治疗剂和/或预防剂是蛋白质。可用于本公开中的纳米粒子中的治疗性蛋白质包括但不限于庆大霉素、阿米卡星(amikacin)、胰岛素、促红细胞生成素(EPO)、粒细胞集落刺激因子(G CSF)、粒细胞巨噬细胞集落刺激因子(GM CSF)、因子VIR、黄体激素释放激素(LHRH)类似物、干扰素、肝素、乙型肝炎表面抗原、伤寒疫苗和霍乱疫苗。In other embodiments, 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.
在一些实施方案中，治疗剂是多核苷酸或核酸(例如核糖核酸或脱氧核糖核酸)。In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (eg, ribonucleic acid or deoxyribonucleic acid).
术语“多核苷酸”的最广泛含义包括呈寡核苷酸链或可以并入寡核苷酸链中的任何化合物和/或物质。根据本公开使用的示例性多核苷酸包括但不限于以下一种或多种：脱氧核糖核酸(DNA)；核糖核酸(RNA)，包括信使RNA(mRNA)、其杂交体；RNAi诱导因子；RNAi因子；siRNA；shRNA；miRNA；反义RNA；核糖酶；催化性DNA；诱导三股螺旋形成的RNA；适体等。在一些实施方案中，治疗剂和/或预防剂是RNA。可用于本文所描述的复合物和方法中的RNA可以选自由但不限于以下组成的组：shortmer、antagomir、反义RNA、核糖酶、小干扰RNA(siRNA)、不对称干扰RNA(aiRNA)、微RNA(miRNA)、Dicer底物RNA(dsRNA)、小发夹RNA(shRNA)、转运RNA(tRNA)、信使RNA(mRNA)及其混合物。在某些实施方案中，RNA是mRNA。The term "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. In some embodiments, 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. In certain embodiments, the RNA is mRNA.
在某些实施方案中，治疗剂和/或预防剂是mRNA。mRNA可以编码任何所关注多肽，包括任何天然或非天然存在或以其它方式修饰的多肽。由mRNA编码的多肽可以具有任何大小并且可以具有任何二级结构或活性。在一些实施方案中，由mRNA编码的多肽当在细胞中 表达时可以具有治疗作用。In certain embodiments, 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. In some embodiments, the polypeptide encoded by the mRNA when in the cell It can have therapeutic effects when expressed.
在其它实施方案中，治疗剂和/或预防剂是siRNA。siRNA能够选择性降低所关注基因的表达或下调该基因的表达。例如，siRNA的选择可以使得在将包括该siRNA的复合物施用给有需要受试者后使与特定疾病、病症或病况有关的基因沉默。siRNA可以包含与编码所关注基因或蛋白质的mRNA序列互补的序列。在一些实施方案中，siRNA可以是免疫调节性siRNA。In other embodiments, 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. For example, 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. In some embodiments, the siRNA can be an immunomodulatory siRNA.
在某些实施方案中，治疗剂和/或预防剂是sgRNA和/或cas9 mRNA。sgRNA和/或cas9 mRNA可以用作基因编辑工具。例如，sgRNA cas9复合物可以影响细胞基因的mRNA翻译。In certain embodiments, the therapeutic and/or prophylactic agent is sgRNA and/or cas9 mRNA. sgRNA and/or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.
在一些实施方案中，治疗剂和/或预防剂是shRNA或者其编码载体或质粒。shRNA可以在将适当构建体递送至核中后在目标细胞内部产生。与shRNA相关的构建体和机制是相关领域中众所周知的。In some embodiments, 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.
本公开的复合物/载体可以向受试者或患者递送活性成分，包括治疗剂或预防剂。所述治疗剂或预防剂包括但不限于核酸分子、小分子化合物、多肽或蛋白质中的一种或多种。因此，本公开的复合物可用于制备核酸药物、基因疫苗、小分子药物、多肽或蛋白质药物。由于上述治疗剂或预防剂的种类广泛，本公开的复合物可用于治疗或预防多种疾病或病症。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.
在一种实施方案中，所述疾病或病症以功能失常或异常的蛋白质或多肽活性为特征。In one embodiment, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.
例如，所述疾病或病症选自由以下组成的组：感染性疾病、癌症和增生性疾病、遗传性疾病、自体免疫性疾病、糖尿病、神经退化性疾病、心血管和肾血管疾病以及代谢性疾病。For example, 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 .
在一种实施方案中，所述感染性疾病选自由冠状病毒，流感病毒，或HIV病毒引起的疾病、小儿肺炎，裂谷热，黄热病，狂犬病，多种疱疹。In one embodiment, 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.
复合物可以包括一种或多种除前述部分中所描述的那些外的组分。例如，复合物可以包括一个或多个疏水性小分子，如维生素(例如维生素A或维生素E)或固醇。The complex may include one or more components in addition to those described in the preceding sections. For example, the complex may include one or more small hydrophobic molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.
复合物还可以包括一个或多个渗透性增强分子、碳水化合物、聚合物、表面改变剂或其它组分。渗透性增强分子可以是例如美国专利申请公布第2005/0222064号所描述的分子。碳水化合物可以包括简单糖(例如葡萄糖)和多糖(例如糖原以及其衍生物和类似物)。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).
表面改变剂可以包括但不限于阴离子性蛋白质(例如牛血清白蛋白)、表面活性剂(例如阳离子性表面活性剂，如二甲基双十八烷基溴化铵)、糖或糖衍生物(例如环糊精)、核酸、聚合物(例如肝素、聚乙二醇和泊洛沙姆)、粘液溶解剂(例如乙酰半胱氨酸、艾蒿、菠萝蛋白酶(bromelain)、木瓜蛋白酶、大青(clerodendrum)、溴己新(bromhexine)、羧甲司坦(carbocisteine)、 依普拉酮(eprazinone)、美司钠(mesna)、氨溴索(ambroxol)、索布瑞醇(sobrerol)、多米奥醇(domiodol)、来托司坦(letosteine)、司替罗宁(stepronin)、硫普罗宁(tiopronin)、凝溶胶蛋白(gelsolin)、胸腺肽(thymosin)β4、链球菌DNA酶α(dornase alfa)、奈替克新(neltenexine)和厄多司坦(erdosteine))和DNA酶(例如rhDNA酶)。表面改变剂可以被安置在复合物的纳米粒子内和/或表面上(例如通过涂布、吸附、共价连接或其它方法)。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. rhDNAse). Surface-altering agents may be disposed within and/or on the surface of the nanoparticles of the composite (eg, by coating, adsorption, covalent attachment, or other methods).
复合物还可以包含一种或多种官能化脂质。例如，脂质可以用炔基官能化，该炔基当在适当反应条件下暴露于叠氮化物时可能经历环加成反应。确切地说，脂质双层可以通过这一方式，用一个或多个可有效地促进膜渗透、细胞识别或成像的基团官能化。复合物的表面还可以与一种或多种有用抗体偶联。可用于靶向细胞递送、成像和膜渗透的官能团和偶联物是本领域中众所周知的。The complex may also contain one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups that may undergo cycloaddition reactions when exposed to azide under appropriate reaction conditions. Specifically, 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.
除这些组分外，复合物还可以进一步形成药物组合物。例如，药物组合物可以包括一种或多种药学上可接受的赋形剂或辅助成分，如但不限于一种或多种溶剂、分散介质、稀释剂、分散助剂、悬浮助剂、造粒助剂、崩解剂、填充剂、助流剂、液体媒剂、粘合剂、表面活性剂、等渗剂、增稠剂或乳化剂、缓冲剂、润滑剂、油、防腐剂、调味剂、着色剂等。赋形例如淀粉、乳糖或糊精。药学上可接受的赋形剂是本领域中众所周知的(参见例如Remington’s The Science and Practice of Pharmacy,第21版,A.R.Gennaro；Lippincott,Williams&Wilkins,Baltimore,MD,2006)。In addition to these components, the complex can further be formed into pharmaceutical compositions. For example, 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).
稀释剂的实例可以包括但不限于碳酸钙、碳酸钠、磷酸钙、磷酸二钙、硫酸钙、磷酸氢钙、磷酸钠、乳糖、蔗糖、纤维素、微晶纤维素、高岭土、甘露糖醇、山梨糖醇、肌醇、氯化钠、干淀粉、玉米淀粉、粉末状糖和/或其组合。Examples of 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.
在一些实施方案中，包括一种或多种本文所述的脂质的药物组合物还可以包括一种或多种佐剂，例如吡喃葡萄糖基脂质佐剂(GLA)、CpG寡聚脱氧核糖核苷酸(例如A类或B类)、多聚(I:C)、氢氧化铝和Pam3CSK4。In some embodiments, pharmaceutical 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.
本公开的药物组合物可以制成固体、半固体、液体或气体的形式的制剂，例如片剂、胶囊剂、软膏剂、酏剂、糖浆、溶液、乳液、悬浮液、注射剂、气溶胶。本公开的药物组合物可以通过制药领域熟知的方法来制备。例如，无菌注射溶液可通过将所需量的治疗剂或预防剂与所需的上述各种其它成分掺入适当的溶剂例如无菌蒸馏水中，然后过滤灭菌来制备。还可以加入表面活性剂以促进形成均匀的溶液或悬浮液。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. For example, 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.
例如，本公开的复合物、药物组合物可以经静脉内、肌肉内、皮内、皮下、鼻内或通过 吸入施用。For example, the complexes and pharmaceutical compositions of the present disclosure may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or via Administer by inhalation.
在一种实施方案中，所述药物组合物是静脉施用的。In one embodiment, the pharmaceutical composition is administered intravenously.
本公开的复合物、药物组合物以治疗有效量给药，所述量不仅可以随所选择的特定试剂而变化，还可以随给药途径，所治疗疾病的性质以及患者的年龄和状况而变化，并且最终可以由主治医师或临床医生自行决定。The complexes, pharmaceutical 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.
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。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。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.
实施例1可电离脂质化合物的合成Example 1 Synthesis of Ionizable Lipid Compounds
1、化合物I-1的合成1. Synthesis of compound I-1
合成路线如下：
The synthesis route is as follows:
(1)化合物1的合成：(1) Synthesis of compound 1:
在茄形瓶中加入无水哌嗪2a(1.0equiv.)和无水碳酸钾(5.0equiv.)，溶于超干二氯甲烷中。另取丙烯酰氯1b(3.0equiv.)溶于超干二氯甲烷，加入滴液漏斗。于冰水浴中，将滴液漏斗中的丙烯酰氯溶液逐滴加入茄形瓶中。冰水浴中反应6h后，停止反应，旋蒸除去反应溶剂。硅胶柱层析分离粗产物，洗脱剂为DCM/MeOH＝40:1(V/V)，纯化出白色固体产物，即化合物4，产率87％。取适量产物溶于氘代氯仿进行核磁分析。 Add anhydrous piperazine 2a (1.0 equiv.) and anhydrous potassium carbonate (5.0 equiv.) to an eggplant-shaped bottle, and dissolve them in ultra-dry dichloromethane. Dissolve acryloyl chloride 1b (3.0 equiv.) in ultra-dry dichloromethane and add it to the dropping funnel. In an ice-water bath, add the acryloyl chloride solution in the dropping funnel into the eggplant-shaped bottle dropwise. After reacting in an ice-water bath for 6 hours, the reaction was stopped and the reaction solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography, and the eluent was DCM/MeOH=40:1 (V/V). The white solid product, namely compound 4, was purified with a yield of 87%. Dissolve an appropriate amount of product in deuterated chloroform for nuclear magnetic analysis.
(2)化合物2的合成：(2) Synthesis of compound 2:
取化合物4(1.0equiv.)、2-(甲基氨基)乙基氨基甲酸叔丁酯2c(2.0equiv.)溶于无水乙醇，于油浴中80℃回流反应36h。反应结束后，旋蒸除去反应溶剂，进行硅胶柱层析，洗脱剂为DCM/MeOH＝20:1-15:1(V/V)(1％NH3·H2O)，纯化出白色固体产物，即化合物5，产率71％。取适量产物溶于氘代氯仿进行核磁分析。Dissolve compound 4 (1.0 equiv.) and 2-(methylamino)ethylcarbamic acid tert-butyl ester 2c (2.0 equiv.) in absolute ethanol, and conduct a reflux reaction at 80°C in an oil bath for 36 hours. After the reaction is completed, the reaction solvent is removed by rotary evaporation, and silica gel column chromatography is performed. The eluent is DCM/MeOH=20:1-15:1 (V/V) (1% NH3·H2O), and a white solid product is purified. That is, compound 5, with a yield of 71%. Dissolve an appropriate amount of product in deuterated chloroform for nuclear magnetic analysis.
(3)化合物I-1的合成：(3) Synthesis of compound I-1:
取化合物2(2mmol，1.0equiv.)、4mL TFA和4mL DCM，混合，室温搅拌反应2h，脱去Boc保护基。旋蒸除去TFA/DCM，得到淡黄色油状物，溶于异丙醇，加入无水碳酸钾，室温搅拌中和剩余TFA，至异丙醇溶液呈碱性。向溶液中加入1,2-环氧十四烷3a(12mmol，6.0equiv.)，于油浴中90℃回流36h。反应结束后，旋蒸除去反应溶剂，硅胶柱层析分离纯化，洗脱剂为DCM/MeOH＝25:1-15:1(V/V)(1％NH3·H2O)，旋干洗脱剂得到淡黄色半固体，即终产物I-1，产率35％。Take compound 2 (2mmol, 1.0equiv.), 4mL TFA and 4mL DCM, mix, stir and react at room temperature for 2h, and remove the Boc protecting group. Remove TFA/DCM by rotary evaporation to obtain a light yellow oil, dissolve it in isopropanol, add anhydrous potassium carbonate, and stir at room temperature to neutralize the remaining TFA until the isopropanol solution becomes alkaline. 1,2-Epoxytetradecane 3a (12 mmol, 6.0 equiv.) was added to the solution, and the solution was refluxed at 90°C for 36 hours in an oil bath. After the reaction is completed, the reaction solvent is removed by rotary evaporation, and the product is separated and purified by silica gel column chromatography. The eluent is DCM/MeOH=25:1-15:1 (V/V) (1% NH3·H2O). The eluent is spin-dried. A light yellow semi-solid was obtained, which was the final product I-1, with a yield of 35%.
取适量产物溶于氘代氯仿进行核磁分析。取适量产物溶于氘代氯仿进行核磁分析。结果如下：1H NMR(400MHz,CDCl3)δ(ppm)＝5.19(s,2H),3.75–3.42(m,8H),3.23(d,J＝5.0,4H),2.88–2.16(m,30H),1.47–1.16(m,92H),0.89(t,J＝6.8,12H)。结果表明，I-1成功合成。Dissolve an appropriate amount of product in deuterated chloroform for nuclear magnetic analysis. Dissolve an appropriate amount of product in deuterated chloroform for nuclear magnetic analysis. The results are as follows: 1H NMR (400MHz, CDCl3) δ (ppm) = 5.19 (s, 2H), 3.75–3.42 (m, 8H), 3.23 (d, J = 5.0, 4H), 2.88–2.16 (m, 30H) ,1.47–1.16(m,92H),0.89(t,J=6.8,12H). The results showed that I-1 was successfully synthesized.
2、化合物II-1的合成2. Synthesis of compound II-1
合成路线如下：
The synthesis route is as follows:
(1)化合物1、化合物2的合成同化合物I-1的合成部分描述内容。 (1) The synthesis of compounds 1 and 2 is the same as that described in the synthesis of compound I-1.
(2)化合物II-1的合成：取化合物2(2mmol，1.0equiv.)、4mL TFA和4mL DCM，混合，室温搅拌反应2h，脱去Boc保护基。旋蒸除去TFA/DCM，得到淡黄色油状物，溶于乙腈，加入无水碳酸钾，室温搅拌中和剩余TFA，至乙腈溶液呈碱性。向溶液中加入溴代十四烷3b(12mmol，6.0equiv.)，于油浴中90℃回流48h。反应结束后，旋蒸除去反应溶剂，硅胶柱层析分离纯化，洗脱剂为DCM/MeOH＝25:1-20:1(V/V)(1％NH3·H2O)，旋干洗脱剂得到浅黄色半固体，即终产物II-1，产率34％。(2) Synthesis of compound II-1: Take compound 2 (2mmol, 1.0equiv.), 4mL TFA and 4mL DCM, mix, stir and react at room temperature for 2h, and remove the Boc protecting group. Remove TFA/DCM by rotary evaporation to obtain a light yellow oil, dissolve it in acetonitrile, add anhydrous potassium carbonate, and stir at room temperature to neutralize the remaining TFA until the acetonitrile solution becomes alkaline. Tetradecane bromide 3b (12 mmol, 6.0 equiv.) was added to the solution, and the solution was refluxed at 90°C for 48 hours in an oil bath. After the reaction is completed, the reaction solvent is removed by rotary evaporation, and the product is separated and purified by silica gel column chromatography. The eluent is DCM/MeOH=25:1-20:1 (V/V) (1% NH3·H2O). The eluent is spin-dried. A light yellow semi-solid, the final product II-1, was obtained with a yield of 34%.
取适量产物溶于氘代氯仿进行核磁分析，结果如下：1H NMR(400MHz,CDCl3)δ(ppm)＝3.57(dd,J＝48.8,18.3,8H),2.86–2.21(m,30H),1.55–1.18(m,96H),0.89(t,J＝6.8,12H)。结果表明，II-2成功合成。Dissolve an appropriate amount of the product in deuterated chloroform for nuclear magnetic analysis. The results are as follows: 1H NMR (400MHz, CDCl3) δ (ppm) = 3.57 (dd, J = 48.8, 18.3, 8H), 2.86–2.21 (m, 30H), 1.55 –1.18(m,96H),0.89(t,J＝6.8,12H). The results showed that II-2 was successfully synthesized.
实施例2阳性对照化合物合成Example 2 Synthesis of Positive Control Compound
本发明阳性对照选用C12-200(MW＝1136.96)、SM-102(MW＝710.18)、ALC-0315(MW＝766.29)、DLin-MC3-DMA(MC3,MW＝642.09)中的一种或多种。其中，C12-200、MC3是经典mRNA递送脂质可电离脂质，SM-102是Moderna已上市mRNA疫苗产品采用的可电离脂质，ALC-0315是CureVac和BioNTeach已上市mRNA疫苗产品采用的可电离脂质。The positive control of the present invention selects one or more of C12-200 (MW=1136.96), SM-102 (MW=710.18), ALC-0315 (MW=766.29), DLin-MC3-DMA (MC3, MW=642.09) kind. Among them, 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, and ALC-0315 is the ionizable lipid used in CureVac and BioNTeach’s marketed mRNA vaccine products. Ionized lipids.
其结构为：

Its structure is:

合成路线参照文献报道，委托第三方公司合成或市售获得。The synthesis route is based on literature reports, and can be synthesized by a third-party company or obtained commercially.
实施例3本发明其他示例性化合物的合成Example 3 Synthesis of other exemplary compounds of the present invention
本发明的化合物，其结构整体来说，可分为由4个三级胺(N)构成的亲水中心和4条饱和脂肪链(T)构成的疏水尾部，每种可电离脂质分子的正电性及亲疏水性质相当，保证了其对mRNA的负载能力相当，精细结构的不同构成具体化合物的不同。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.
本发明化合物的合成主要分为两步：第一，通过成酰胺反应和迈克尔加成(Michael addition)构建4N正电中心；第二，通过环氧开环反应、烷基化反应或迈克尔加成反应连接4T疏水尾部。本发明公开的其他示例性化合物通过实施例1中描述的类似路线，用不同的起始材料制备。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 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.
本发明的其他化合物结构式如下：






The structural formulas of other compounds of the present invention are as follows:






相对于本领域其他已公开的可电离脂质分子，比如文献报道C12-200的合成路线需要用到相对危险的雷尼镍(Raney Nickel)加氢还原反应，本发明的化合物合成路线简便易行，结构表征明确，化合物产量充足。Compared with other ionizable lipid molecules that have been disclosed in the field, such as the synthesis route of C12-200 reported in the literature that requires the relatively dangerous Raney Nickel hydrogenation reduction reaction, 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.
实施例4 LNPs@mRNA的制备和制剂学性质考察Example 4 Preparation and preparation of LNPs@mRNA and investigation of pharmaceutical properties
进一步地，发明人基于实施例1制得的可电离脂质，构建了mRNA递送系统LNPs@mRNA，并对其进行粒径、电位、包封率、TEM等制剂学性质的考察，以评估其成制剂性。Furthermore, 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.
1、LNPs@mRNA的制备1. Preparation of LNPs@mRNA
(1)溶液配制：将可电离脂质、DOPE(或DSPC)、Chol、DMG-PEG2000溶于无水乙醇中，使可电离脂质的浓度为10mg/mL。本发明实施例1制得的可电离脂质和C12-200、MC3、SM-102和ALC-0315的处方可电离脂质、DSPC、胆固醇(Chol)、DMG-PEG2000摩尔比为50:10:38.5:1.5。mRNA以PBS缓冲液(RNase-free水配制)稀释至适宜浓度待用。为确保阳性对照性能最大化，参考现有技术公开，C12-200、MC3、SM-102和ALC-0315配方选自已知文献公开较优配比。(1) 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. In order to ensure the maximum performance of the positive control, with reference to the prior art disclosures, the C12-200, MC3, SM-102 and ALC-0315 formulas were selected from the better proportions disclosed in known literature.
(2)LNP制备：将步骤(1)获得可电离脂质溶液与mRNA溶液混合。微流控工艺参数：乙醇相和水相的体积比为1:3，流速为9mL/min。(2) 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.
(3)超滤：以PBS缓冲液将LNP初制剂稀释25倍，经超滤杯超滤至初始体积即为LNP终制剂，超滤过程中初制剂中的乙醇被除去。超滤工艺参数：滤膜100kDa，气压0.2MPa，转速100-200rpm。(3) 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.
2、LNPs@mRNA制剂学性质考察2. Investigation of the pharmaceutical properties of LNPs@mRNA preparations
粒径电位测定：取适量LNPs@mRNA制剂以纯化水稀释10倍，以马尔文纳米粒度电位仪检测其粒径、粒径分布、ζ电位等制剂学性质。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, ζ potential and other pharmaceutical properties.
透射电镜表征：以纯化水稀释LNPs@mRNA使纳米颗粒质量浓度为2mg/mL，小心滴 至TEM专用铜网上，静置2min后用滤纸吸去多余液体，滴加2％磷钨酸负染3min后吸去多余染液，用洗耳球吹干后上样检测并拍照。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.
包封率检测：溶液准备：以RNase-free水稀释LNPs@mRNA至mRNA浓度为0.1μg/μL。在避光条件下，取适量RiboGreen染料用1×TE缓冲液稀释400倍，避光保存待用。破膜：样品孔中加入25μL稀释的mRNA制剂和75μL 1×TE缓冲液，总mRNA孔中加入25μL稀释的mRNA制剂、25μL 1×TE缓冲液和50μL 1％Triton。将上述96孔板37℃避光孵育10min。显色：10min后，在避光条件下向每孔中加入100μL稀释的RiboGreen染料，振荡使其均匀。酶标仪检测条件：激发波长485nm，发射波长528nm。计算公式：包封率(％)＝(1-样品孔荧光值/总mRNA荧光值)×100％。Encapsulation efficiency detection: solution preparation: dilute LNPs@mRNA with RNase-free water to an mRNA concentration of 0.1 μg/μL. 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 μL of diluted mRNA preparation and 75 μL of 1×TE buffer to the sample well, and add 25 μL of diluted mRNA preparation, 25 μL of 1×TE buffer and 50 μL of 1% Triton to the total mRNA well. Incubate the above 96-well plate at 37°C in the dark for 10 minutes. Color development: After 10 minutes, add 100 μL of diluted RiboGreen dye to each well in the dark, and shake to make it uniform. Microplate reader detection conditions: excitation wavelength 485nm, emission wavelength 528nm. Calculation formula: encapsulation rate (%) = (1-sample well fluorescence value/total mRNA fluorescence value) × 100%.
实验结果如图1显示：本发明实施例1制得的不同可电离脂质制备的LNP粒径相当，平均粒径均在100nm左右，Di(90)在500nm以内，PDI均在0.3以下，表明LNP粒径分布均匀。制剂电位在5mV左右。The experimental results are shown in Figure 1: 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.
本发明实施例1制得的不同可电离脂质制备的LNPs@mRNA呈实心球状，TEM下粒径在100nm左右，发现了指纹样结构，为LNP纳米结构的代表性特。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.
包封率检测结果如图2所示，相比于阳性对照MC3、ALC-0315、SM-102，由本发明实施例1的可电离脂质制备的LNPs@mRNA的包封率明显高于阳性对照。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. .
以上试验结果表明：本发明的可电离脂质制得的LNPs@mRNA具有良好的纳米制剂性质，尤其是包封率优于阳性对照MC3、ALC-0315、SM-102。The above test results show that the LNPs@mRNA prepared from the ionizable lipid of the present invention has good nanoformulation properties, especially the encapsulation rate is better than the positive controls MC3, ALC-0315, and SM-102.
实施例5 LNPs@FLuc mRNA的细胞转染及细胞毒性评价Example 5 Cell transfection and cytotoxicity evaluation of LNPs@FLuc mRNA
前述实施例验证了本发明所提供的可电离脂质纳米颗粒的制剂学性质，进一步地，发明人以EGFP mRNA和FLuc mRNA作为报告基因，通过对HEK293T和DC2.4的转染实验和毒性实验评价本发明所提供的LNPs@mRNA体外效果及安全性。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转染实验：HEK293T和DC2.4细胞铺板。然后根据实施例4描述的方法制备LNPs@EGFP mRNA、阳性对照制剂MC3-LNPs@EGFP mRNA、ALC-0315-LNPs@EGFP mRNA、SM-102-LNPs@EGFP mRNA；控制给药制剂mRNA浓度为0.02μg/μL，24孔细胞板每孔给药50μL，即1μg/孔，继续于37℃、5％CO2的培养箱中培养24h。24h后，流式细胞仪检测GFP阳性率及平均荧光强度(mean fluorescence intensity，MFI)。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 μg/μL, 50 μL was administered to each well of the 24-well cell plate, that is, 1 μg/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).
LNPs@FLuc mRNA转染实验：HEK293T和DC2.4细胞铺板。根据实施例4描述的方 法制备LNPs@FLuc mRNA、阳性对照制剂MC3-LNPs@FLuc mRNA、ALC-0315-LNPs@FLuc mRNA、SM-102-LNPs@FLuc mRNA；控制给药制剂mRNA浓度为0.02μg/μL，24孔细胞板每孔给药50μL，即1μg/孔，继续于37℃、5％CO2的培养箱中培养24h。24h后，每孔中加入10μL底物荧光素(荧光素钾盐，15mg/mL)，避光静置10min后酶标仪检测生物发光。LNPs@FLuc mRNA transfection experiment: HEK293T and DC2.4 cells were plated. According to the method described in Example 4 LNPs@FLuc mRNA, 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 μg/μL, and 24-well cells were used. Add 50 μL to each well of the plate, that is, 1 μg/well, and continue to culture in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, add 10 μL of substrate fluorescein (fluorescein potassium salt, 15 mg/mL) to each well, let it stand in the dark for 10 minutes, and then detect bioluminescence with a microplate reader.
细胞毒性实验：选择LNPs@FLuc mRNA、阳性对照制剂C12-200-LNPs@FLuc mRNA考察DC2.4细胞MTT毒性。关键实验参数如下：96孔板中每孔给药10μL；37℃、5％CO2的培养箱中培养24h；5mg/mL的MTT溶液，每孔加入上述MTT溶液40μL，37℃、5％CO2的培养箱中孵育4h。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 μL 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 μL of the above MTT solution to each well, 37°C, 5% CO2 Incubate in the incubator for 4 hours.
细胞转染实验结果如图3显示：在HEK-293T和DC2.4上，本发明的II-1@EGFP mRNA较阳性对照MC3、ALC-0315在细胞水平mRNA表达能力更强。The results of cell transfection experiments are shown in Figure 3: on HEK-293T and DC2.4, the II-1@EGFP mRNA of the present invention has stronger mRNA expression ability at the cellular level than the positive controls MC3 and ALC-0315.
MTT法细胞毒性实验结果如图4显示：LNPs@FLuc mRNA孵育24h对DC2.4细胞无明显毒性。The results of the MTT method cytotoxicity experiment are shown in Figure 4: LNPs@FLuc mRNA has no obvious toxicity to DC2.4 cells after incubation for 24 hours.
实施例6 LNPs@mRNA的体内表达及分布Example 6 In vivo expression and distribution of LNPs@mRNA
进一步地，考察了本发明的LNPs@mRNA体内递送mRNA的能力。Furthermore, the ability of the LNPs@mRNA of the present invention to deliver mRNA in vivo was examined.
静脉注射途径的体内表达考察：根据实施例4描述的方法制备LNPs@Fluc mRNA制剂、阳性对照制剂C12-200@Fluc mRNA，用PBS溶液(RNase-free水配制)调整制剂的mRNA浓度为0.05mg/mL，同时调整制剂的渗透压至等渗，每只C57BL/6小鼠尾静脉注射200μL，即10μg FLuc mRNA/只，每组3只，以PBS作为阴性Control。给药后保持小鼠正常饮食。给药6h后，腹腔注射200μL底物溶液(15mg/mL，荧光素钾盐)。注射底物后开始计时，10min后执行安乐死，快速解剖心、肝、脾、肺、肾，在IVIS仪器中检测各离体脏器的生物发光强度，曝光时间为60s。成像结束后统计各脏器的Total flux。In vivo expression investigation by intravenous injection route: Prepare LNPs@Fluc mRNA preparation and positive control preparation C12-200@Fluc mRNA according to the method described in Example 4. Use PBS solution (prepared with RNase-free water) to adjust the mRNA concentration of the preparation to 0.05 mg. /mL, and adjust the osmotic pressure of the preparation to isotonicity. Inject 200 μL, or 10 μg FLuc mRNA/mouse, into the tail vein of each C57BL/6 mouse. There are 3 mice in each group. PBS is used as the negative control. After administration, the mice were kept on a normal diet. 6 hours after administration, 200 μL of substrate solution (15 mg/mL, fluorescein potassium salt) was injected intraperitoneally. Start timing after injecting the substrate, perform euthanasia 10 minutes later, quickly dissect the heart, liver, spleen, lungs, and kidneys, and detect the bioluminescence intensity of each isolated organ in the IVIS instrument. The exposure time is 60 seconds. After imaging, the total flux of each organ was counted.
肌肉注射途径的体内表达考察：根据实施例4描述的方法制备LNP@Fluc mRNA、阳性对照制剂MC3-LNPs@Fluc mRNA、ALC-0315-LNPs@Fluc mRNA、SM-102-LNPs@Fluc mRNA，用含PBS溶液(RNase-free水配制)调整制剂的mRNA浓度为0.1mg/mL，同时调整制剂的渗透压至等渗，每只BALB/c小鼠后腿肌肉注射200μL，即20μg FLuc mRNA/只，每组3只，以PBS作为阴性Control。给药后保持小鼠正常饮食。给药8h后，腹腔注射200μL底物溶液(15mg/mL，荧光素钾盐)，即3mg/只。注射底物后开始计时，10min后将小鼠放入气麻装置，待完全麻醉后在IVIS仪器中检测活体全身的生物发光强度，曝光 时间为60s。复苏后正常饮食。给药24h后和48h后重复上述麻醉及检测。统计全身Total flux，绘制发光强度—时间曲线。In vivo expression investigation of intramuscular injection route: 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 μL into the hind leg muscle of each BALB/c mouse, that is, 20 μg FLuc mRNA/mouse. , 3 animals in each group, with PBS as negative control. After administration, the mice were kept on a normal diet. 8 hours after administration, 200 μL 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.
静脉注射途径的体内表达结果显示(如图5、图6所示)：出乎预料地，与对照C12-200相比，经静脉注射途径、本发明的LNPs@mRNA在肺部出乎预料地高表达，这将利好于靶向肺部的核酸药物；同时在脾脏也有一定的表达水平，提示本发明的LNPs@mRNA也可用于静脉给药途径的核酸药物。The in vivo expression results of the intravenous injection route (shown in Figures 5 and 6) 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.
以上试验结果提示，本发明LNPs可进一步用于肺器官靶向的核酸药物制备。The above test results suggest that the LNPs of the present invention can be further used for the preparation of nucleic acid drugs targeting lung organs.
实施例7 LNPs@OVA mRNA的免疫抗肿瘤效果Example 7 Immunity and anti-tumor effects of LNPs@OVA mRNA
进一步构建了E.G7-OVA(Ovalbumin卵清蛋白)肿瘤模型，选择OVA-mRNA作为模式抗原，以评价LNPs@mRNA作为mRNA肿瘤疫苗递送系统的效能，进而筛选出本发明中具有高效抑瘤作用的mRNA肿瘤疫苗递送系统，并考察了本发明的可电离脂质化学结构对LNPs@mRNA肿瘤疫苗效果的潜在影响。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.
其中，E.G7-OVA肿瘤模型建立方法如下所述：E.G7-OVA细胞培养至对数生长期时，收集细胞，以无菌PBS洗涤细胞，离心弃上清液，用无菌PBS重悬细胞，调整细胞浓度至107/mL。每只雄性C57BL/6小鼠右侧肋部皮下接种100μL E.G7-OVA细胞，即106/只。观察小鼠的生长状态及皮下瘤的大小，接种5天后形成肉眼可见小瘤，即可开展免疫治疗实验。荷瘤小鼠与正常小鼠相比，活动、食欲、大小便等反应无明显差异。参考文献：Luo,X.；Li,B.；Zhang,X.；Zhao,W.；Bratasz,A.；Deng,B.；McComb,D.W.；Dong,Y.,Dual-functional lipid-like nanoparticles for delivery of mRNA and MRI contrast agents.Nanoscale 2017,9(4),1575-1579.Xiong,H.；Liu,S.；Wei,T.；Cheng,Q.；Siegwart,D.J.,Theranostic dendrimer-based lipid nanoparticles containing PEGylated BODIPY dyes for tumor imaging and systemic mRNA delivery in vivo.J Control Release 2020,325,198-205。Among them, 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 μL 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. Compared with normal mice, there were no significant differences in activity, appetite, urinary and defecation reactions between tumor-bearing mice. References: Luo, delivery of mRNA and MRI contrast agents.Nanoscale 2017,9(4),1575-1579.Xiong,H.;Liu,S.;Wei,T.;Cheng,Q.;Siegwart,DJ,Theranostic dendrimer-based lipid nanoparticles containing PEGylated BODIPY dyes for tumor imaging and systemic mRNA delivery in vivo. J Control Release 2020,325,198-205.
E.G7-OVA细胞(小鼠T淋巴瘤细胞)购自ATCC，用1640完全培养基(含10％FBS和1％P/S)在37℃、5％CO2的培养箱中培养，细胞密度达80-90％时传代。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%.
实验方案如下：通过微流控制剂技术制备了LNPs@OVA mRNA，考察了其对体外BMDC的激活作用及对细胞因子分泌的影响。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成熟活化考察方法：细胞铺板。根据实施例4描述的方法制备LNPs@OVA mRNA及阳性对照制剂C12-200-LNPs@OVA mRNA，控制给药制剂mRNA浓度为0.05μg/μL。每孔给药20μL，即1μg/孔，于37℃、5％CO2的培养箱中孵育24h。染色及检测：孵 育结束后，收集24孔板中细胞至流式管中，以预冷的1mL PBS 1500rpm离心5min洗细胞两次。向每只流式管中加入100μL上述细胞悬液和1μLAnti-mouse CD16/32，混匀后4℃孵育15min封闭非特异性结合。向对应的流式管中分别加入1μL PE-anti-mouse CD11c、FITC-anti-mouse CD80、APC/Cy7-anti-mouse CD86和APC-anti-mouse H-2Kb bound to SIINFEKL流式抗体，4℃孵育40min。孵育结束后，以预冷的1mL PBS 1500rpm离心5min洗细胞两次，而后加入300μL PBS混匀，流式细胞仪检测BMDC的成熟活化状态。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 μg/μL. Administer 20 μL per well, that is, 1 μg/well, and incubate for 24 h in an incubator at 37°C and 5% CO2. Staining and detection: incubation After the incubation, collect the cells in the 24-well plate into a flow tube, and wash the cells twice with pre-cooled 1 mL PBS at 1500 rpm for 5 min by centrifugation. Add 100 μL of the above cell suspension and 1 μL Anti-mouse CD16/32 to each flow tube, mix and incubate at 4°C for 15 min to block non-specific binding. Add 1 μL of PE-anti-mouse CD11c, FITC-anti-mouse CD80, APC/Cy7-anti-mouse CD86 and APC-anti-mouse H-2Kb bound to SIINFEKL flow cytometry antibodies to the corresponding flow tubes, 4°C Incubate for 40 minutes. After the incubation, the cells were washed twice by centrifugation at 1500 rpm for 5 min with 1 mL of pre-cooled PBS, then 300 μL of PBS was added and mixed, and the mature activation status of BMDC was detected by flow cytometry.
细胞因子检测:检测LNPs@OVA mRNA疫苗刺激BMDC成熟活化d同时在孵育结束后，收集细胞上清液以ELISA试剂盒中样品稀释液稀释5倍，待测。根据试剂盒说明书稀释标准品和酶标抗体，建立TNF-α的标准工作曲线，通过双抗夹心法检测细胞上清液中BMDC分泌TNF-α的水平.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-α, and detect the level of TNF-α secreted by BMDC in the cell supernatant using the double-antibody sandwich method.
结果显示：本发明的基于含哌嗪可电离脂质及其衍生物或类似物的mRNA疫苗对于BMDC成熟活化(如图7所示)、其细胞因子TNF-α的分泌均优于阳性对照C12-200(如图8所示)。The results show that the mRNA vaccine based on piperazine-containing ionizable lipids and its derivatives or analogues of the present invention is better than the positive control C12 in BMDC maturation activation (as shown in Figure 7) and secretion of cytokine TNF-α. -200 (as shown in Figure 8).
需要说明的是，本说明书附图中，如未特别说明，ns或未标注为无显著性差异；*，p<0.05；**，p<0.01；***，p<0.001；****，p<0.0001。It should be noted that in the drawings of this manual, unless otherwise stated, ns or not marked mean no significant difference; *, p<0.05; **, p<0.01; ***, p<0.001; *** *, p<0.0001.
需要说明的是，本说明书中描述的具体特征、结构、材料或者特点可以在任一个或多个实施例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。 It should be noted that the specific features, structures, materials or characteristics described in this specification can be combined in a suitable manner in any one or more embodiments. Furthermore, those skilled in the art may combine and combine the different embodiments described in this specification and the features of different embodiments unless they are inconsistent with each other.

Claims (21)

  1. 式Ⅰ所示的化合物，或其药学上可接受的盐、异构体、氘代物或前药：
    The compound represented by formula I, or its pharmaceutically acceptable salt, isomer, deuterated product or prodrug:
    其中，a为1,2或3；Among them, a is 1, 2 or 3;
    X1、X2分别独立地选自N或C；X 1 and X 2 are independently selected from N or C;
    L1、L2、L3、L4独立地选自-ReCH(OH)-、-ReC(＝O)-、-ReC(＝O)O-、-ReOC(＝O)-、-ReC(＝O)S-、-ReSC(＝O)-、-ReC(＝O)NRa-、-ReNRaC(＝O)-、-ReNRaC(＝O)O-、-ReOC(＝O)NRa-、-ReO-、-Re-O-O-、-ReS-、-Re-S-S-、-Re-S-S-S-、-ReCH(OH)CH2O-、-ReCH(OH)CH2S-或不存在，Re为或不存在，k为1以上的整数，Ra为-H、取代或未取代的烷基；L 1 , L 2 , L 3 , L 4 are independently selected from -R e CH(OH)-, -R e C(=O)-, -R e C(=O)O-, -R e OC( =O)-, -R e C(=O)S-, -R e SC(=O)-, -R e C(=O)NR a -, -R e NR a C(=O)-, -R e NR a C(=O)O-, -R e OC(=O)NR a -, -R e O-, -R e -OO-, -R e S-, -R e -SS- , -R e -SSS-, -R e CH(OH)CH 2 O-, -R e CH(OH)CH 2 S- or absent, R e is or does not exist, k is an integer above 1, R a is -H, substituted or unsubstituted alkyl;
    R1、R2、R3、R4独立地选自C1-C30直链烷基、C1-C30支化烷基、C2-C30直链烯基、C2-C30支化烯基、C2-C30直链炔基或C2-C30支化炔基；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;
    G1、G2、G3、G4独立地选自-Rc-、-RcCH(OH)Rd-、-RcC(＝O)Rd-、-RcC(＝O)ORd-、-RcOC(＝O)Rd-、-RcC(＝O)SRd-、-RcSC(＝O)Rd-、-RcC(＝O)N(Rb)Rd-、-RcN(Rb)C(＝O)Rd-、-RcN(Rb)C(＝O)ORd-、-RcOC(＝O)N(Rb)Rd-、-RcORd-、-Rc-O-O-Rd-、-RcSRd-、-Rc-S-S-Rd-、-Rc-S-S-S-Rd-或不存在，Rb为-H、取代或未取代的烷基，Rc、Rd独立地选自或不存在，n为1以上的整数；G 1 , G 2 , G 3 , G 4 are independently selected from -R c -, -R c CH(OH)R d -, -R c C(=O)R d -, -R c C(=O )OR d -, -R c OC(＝O)R d -, -R c C(＝O)SR d -, -R c SC(＝O)R d -, -R c C(＝O)N (R b )R d -, -R c N(R b )C(=O)R d -, -R c N(R b )C(=O)OR d -, -R c OC(=O) N(R b )R d -, -R c OR d -, -R c -OOR d -, -R c SR d -, -R c -SSR d -, -R c -SSSR d - or not present, R b is -H, substituted or unsubstituted alkyl, R c and R d are independently selected from or does not exist, n is an integer above 1;
    R5、R6独立地选自-H、取代或未取代的烷基；R 5 and R 6 are independently selected from -H, substituted or unsubstituted alkyl;
    Q1、Q2独立地选自O或S。Q 1 and Q 2 are independently selected from O or S.
  2. 根据权利要求1所述的化合物，其特征在于，具有式II所示结构，或为式II所示结构的药学上可接受的盐、立体异构体、氘代物或前药，
    The compound according to claim 1, which has a structure represented by Formula II, or is a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug of the structure represented by Formula II,
  3. 如权利要求1或2所述的化合物，其特征在于，所述k为1，2，3，4，5，6，7，8，9或10；The compound of claim 1 or 2, wherein k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
    优选的，所述k为1。Preferably, the k is 1.
  4. 如权利要求1或2所述的化合物，其特征在于，所述L1、L2、L3、L4独立地选自-CH(OH)-、-C(＝O)-、-CH2C(＝O)O-、-C(＝O)O-、-OC(＝O)-、-C(＝O)S-、-SC(＝O)-、-CH2C(＝O)NRa-、-C(＝O)NRa-、-NRaC(＝O)-、-NRaC(＝O)O-、-OC(＝O)NRa-、-CH2O-、-O-、-CH2-O-O-、-CH2S-、-S-、-CH2-S-S-、-CH2-S-S-S-、-CH(OH)CH2O-、-CH(OH)CH2S-或不存在，Ra为-H、取代或未取代的烷基；The compound of claim 1 or 2, wherein said L1, L2, L3, and L4 are independently selected from -CH(OH)-, -C(=O)-, -CH2C(=O)O -, -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -CH2C(=O)NRa-, -C(=O )NRa-, -NRaC(=O)-, -NRaC(=O)O-, -OC(=O)NRa-, -CH2O-, -O-, -CH2-O-O-, -CH2S-, -S -, -CH2-S-S-, -CH2-S-S-S-, -CH(OH)CH2O-, -CH(OH)CH2S- or absent, Ra is -H, substituted or unsubstituted alkyl;
    优选的，所述L1、L2、L3、L4独立地选自-C(＝O)-、-C(＝O)NRa-、-CH2C(＝O)NRa-、-NRaC(＝O)-、-C(＝O)O-、-CH2C(＝O)O-、-OC(＝O)-、-CH2O-、-O-、-CH2S-、-S-、-CH(OH)-、-CH(OH)CH2O-、-CH(OH)CH2S-或不存在，Ra为-H或未取代的烷基。Preferably, the L 1 , L 2 , L 3 and L 4 are independently selected from -C(=O)-, -C(=O)NR a -, -CH 2 C(=O)NR a -, -NR a C(=O)-, -C(=O)O-, -CH 2 C(=O)O-, -OC(=O)-, -CH 2 O-, -O-, -CH 2 S-, -S-, -CH(OH)-, -CH(OH)CH 2 O-, -CH(OH)CH 2 S- or absent, R a is -H or unsubstituted alkyl.
  5. 如权利要求1～4任意一项所述的化合物，其特征在于，所述Ra为-H或未取代的C1～C6烷基；The compound according to any one of claims 1 to 4, wherein R a is -H or unsubstituted C 1 to C 6 alkyl;
    优选的，所述Ra为-H。Preferably, the R a is -H.
  6. 如权利要求1或2所述的化合物，其特征在于，所述L1、L2、L3、L4独立地选自-C(＝O)-、-C(＝O)NH-、-CH2C(＝O)NH-、-C(＝O)O-、-CH2C(＝O)O-、-CH2O-、-CH2S-、-CH(OH)-、-CH(OH)CH2O-或不存在；The compound according to claim 1 or 2, characterized in that said L 1 , L 2 , L 3 and L 4 are independently selected from -C(=O)-, -C(=O)NH-, - CH 2 C(=O)NH-, -C(=O)O-, -CH 2 C(=O)O-, -CH 2 O-, -CH 2 S-, -CH(OH)-, - CH(OH)CH 2 O-or absent;
    优选地，L1、L2、L3、L4独立地选自-C(＝O)NH-、-C(＝O)O-、-CH(OH)-、-CH(OH)CH2O-或不存在。Preferably, L 1 , L 2 , L 3 , L 4 are independently selected from -C(=O)NH-, -C(=O)O-, -CH(OH)-, -CH(OH)CH 2 O-or not present.
  7. 如权利要求1～6任意一项所述的化合物，其特征在于，所述L1和L2选自相同的基团和/或L3和L4选自相同的基团；The compound according to any one of claims 1 to 6, wherein L 1 and L 2 are selected from the same group and/or L 3 and L 4 are selected from the same group;
    任选的，所述L1和L3选自相同的基团和/或L2和L4选自相同的基团； Optionally, the L 1 and L 3 are selected from the same group and/or L 2 and L 4 are selected from the same group;
    优选的，所述L1、L2、L3和L4选自相同的基团。Preferably, said L 1 , L 2 , L 3 and L 4 are selected from the same group.
  8. 如权利要求1或2所述的化合物，其特征在于，所述R1、R2、R3、R4独立地选自C1-C30直链烷基、C2-C30直链烯基、C2-C30直链炔基；The compound according to claim 1 or 2, characterized in that R 1 , R 2 , R 3 and R 4 are independently selected from C 1 -C 30 linear alkyl, C 2 -C 30 linear alkene Base, C 2 -C 30 straight chain alkynyl group;
    优选的，所述R1、R2、R3、R4独立地选自未取代的C1～C30直链烷基；Preferably, the R 1 , R 2 , R 3 and R 4 are independently selected from unsubstituted C 1 to C 30 linear alkyl groups;
    优选的，所述R1、R2、R3、R4独立地选自未取代的C8～C18直链烷基；Preferably, the R 1 , R 2 , R 3 and R 4 are independently selected from unsubstituted C 8 to C 18 linear alkyl groups;
    优选的，所述R1、R2、R3、R4独立地选自未取代的C10～C14直链烷基。Preferably, the R 1 , R 2 , R 3 and R 4 are independently selected from unsubstituted C 10 to C 14 linear alkyl groups.
  9. 如权利要求1～8任意一项所述的化合物，其特征在于，R1和R2选自相同的基团和/或R3和R4选自相同的基团；The compound according to any one of claims 1 to 8, wherein R 1 and R 2 are selected from the same group and/or R 3 and R 4 are selected from the same group;
    任选的，所述R1和R3选自相同的基团和或R2和R4选自相同的基团；Optionally, the R 1 and R 3 are selected from the same group and or R 2 and R 4 are selected from the same group;
    优选的，所述R1、R2、R3和R4选自相同的基团。Preferably, the R 1 , R 2 , R 3 and R 4 are selected from the same group.
  10. 如权利要求1或2所述的化合物，其特征在于，所述G1、G2、G3、G4独立地选自或不存在，n3为1，2，3，4，5，6，7，8，9或10；The compound according to claim 1 or 2, characterized in that said 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;
    优选的，所述G1、G2独立地选自-CH2-或-CH2CH2-；Preferably, the G 1 and G 2 are independently selected from -CH 2 - or -CH 2 CH 2 -;
    优选的，所述G3、G4不存在。Preferably, the G 3 and G 4 do not exist.
  11. 如权利要求1或2所述的化合物，其特征在于，所述R5、R6独立地选自-H、未取代的C1～C6烷基或-OH取代的C1～C6烷基；The compound according to claim 1 or 2, wherein 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. base;
    优选的，所述R5、R6独立地选自-H、甲基、乙基、丙基、羟甲基、羟乙基或羟丙基；Preferably, the R 5 and R 6 are independently selected from -H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl or hydroxypropyl;
    优选的，所述R5、R6选自相同的基团。Preferably, the R 5 and R 6 are selected from the same group.
  12. 一种化合物，其特征在于，具有以下结构中的一种；





    A compound characterized by having one of the following structures;





    ，其药学上可接受的盐、立体异构体、氘代物或前药。, its pharmaceutically acceptable salts, stereoisomers, deuterated products or prodrugs.
  13. 权利要求1～12任意一项所述化合物，或其药学上可接受的盐、异构体、氘代物或前药在制备药物递送载体中的用途；进一步地，所述药物的活性成分任选自核酸、小分子化药、蛋白药物、多肽的至少一种；优选地，所述药物的活性成分选自核酸；优选地，所述药物具有心、肝、脾、肺或肾靶向性；优选地，所述药物靶向肺、脾脏。The use of the compound of any one of claims 1 to 12, or its pharmaceutically acceptable salt, isomer, deuterated product or prodrug in the preparation of a drug delivery carrier; further, the active ingredient of the drug is optional At least one of nucleic acids, small molecule drugs, protein drugs, and polypeptides; preferably, the active ingredient of the drug is selected from nucleic acids; preferably, the drug has heart, liver, spleen, lung, or kidney targeting; Preferably, the drug targets the lungs and spleen.
  14. 一种药物递送载体，其特征在于，包括含有权利要求1～12任意一项所述的化合物，或其药学上可接受的盐、立体异构体、氘代物或前药。A drug delivery carrier, characterized by containing the compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof.
  15. 一种药物复合物，其特征在于，包含载体以及活性成分，所述载体与所述活性成分相连，所述载体包括阳离子脂质，所述阳离子脂质包括权利要求1～12任意一项所述的化合物，或其药学上可接受的盐、立体异构体、氘代物或前药；任选地，所述药物的活性成分任选自核酸、小分子化药、蛋白药物、多肽、抗体中的至少一种；优选地，所述药物的活性成分选自核酸；任选地，所述核酸选自DNA、ASO、siRNA、miRNA、mRNA、适配体中至少一种；任选地，所述核酸为mRNA；任选地，所述递送载体与所述mRNA通过离子键相连；进一步地，所述药物靶向肺、脾脏；A drug complex, characterized in that it contains 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 any one of claims 1 to 12 A compound, or a pharmaceutically acceptable salt, stereoisomer, deuterated product or prodrug thereof; optionally, the active ingredient of the drug is selected from nucleic acids, small molecule drugs, protein drugs, polypeptides, and antibodies. At least one of; preferably, the active ingredient of the drug is selected from nucleic acid; optionally, the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamer; optionally, the The nucleic acid is mRNA; optionally, the delivery carrier is connected to the mRNA through an ionic bond; further, the drug targets the lungs and spleen;
    任选的，所述药物复合物的制剂形式选自脂质纳米颗粒LNP、PLGA纳米粒、胶束、脂质体、核-壳纳米粒、聚合物纳米颗粒中的一种；Optionally, the preparation form of the drug complex is selected from one of lipid nanoparticles LNP, PLGA nanoparticles, micelles, liposomes, core-shell nanoparticles, and polymer nanoparticles;
    优选的，所述药物复合物的制剂形式是脂质纳米颗粒LNP。 Preferably, the drug complex is in the form of lipid nanoparticles (LNP).
  16. 如权利要求15所述的药物复合物，其特征在于，所述药物组合物还含有中性磷脂、类固醇、聚乙二醇化脂质中至少一种赋形剂；The pharmaceutical complex according to claim 15, wherein the pharmaceutical composition further contains at least one excipient selected from the group consisting of neutral phospholipids, steroids, and pegylated lipids;
    任选的，所述中性磷脂选自DOPE、DSPC、DOPC、DSPE、DMPC、DMPE、DPPC、DPPE、DEPC、HSPC、POPC中至少一种；Optionally, the neutral phospholipid is selected from at least one of DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC, and POPC;
    优选的，所述中性磷脂为DOPE；Preferably, the neutral phospholipid is DOPE;
    任选的，所述类固醇选自胆固醇、谷甾醇、豆固醇、羊毛固醇、麦角固醇、岩藻甾醇中至少一种；Optionally, the steroid is selected from at least one of cholesterol, sitosterol, stigmasterol, lanosterol, ergosterol, and fucosterol;
    优选的，所述类固醇为胆固醇；Preferably, the steroid is cholesterol;
    任选的，所述聚乙二醇化脂质选自DMG-PEG、DSPE-PEG中至少一种；Optionally, the PEGylated lipid is selected from at least one of DMG-PEG and DSPE-PEG;
    优选地，所述聚乙二醇化脂质为DMG-PEG2000。Preferably, the pegylated lipid is DMG-PEG2000.
  17. 如权利要求15所述的药物复合物，其特征在于，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述中性磷脂的摩尔比为1:10～10:1。The drug complex according to claim 15, wherein the molar ratio of the compound, or its pharmaceutically acceptable salt, isosteromer, deuterated product or prodrug to the neutral phospholipid is 1: 10～10:1.
  18. 如权利要求15所述的药物复合物，其特征在于，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述聚乙二醇化脂质的摩尔比为5:1～1000:1；The drug complex according to claim 15, characterized in that the molar ratio of the compound, or a pharmaceutically acceptable salt, isosteromer, deuterated product or prodrug thereof to the PEGylated lipid It is 5:1~1000:1;
    优选地，所述化合物，或其药学上可接受的盐、异立体构体、氘代物或前药与所述聚乙二醇化脂质的摩尔比为10:1～20:1。Preferably, the molar ratio of the compound, or its pharmaceutically acceptable salt, isomer, deuterated product or prodrug to the PEGylated lipid is 10:1 to 20:1.
  19. 如权利要求15所述的药物复合物，其特征在于，所述核酸选自DNA、ASO、siRNA、miRNA、mRNA、适配体中至少一种；The drug complex according to claim 15, wherein the nucleic acid is selected from at least one of DNA, ASO, siRNA, miRNA, mRNA and aptamer;
    优选的，所述核酸为mRNA。Preferably, the nucleic acid is mRNA.
  20. 权利要求15～19任一项所述的药物复合物在制备药物中的用途，所述药物用于治疗或预防疾病；The use of the drug complex according to any one of claims 15 to 19 in the preparation of medicines, which are used to treat or prevent diseases;
    任选的，所述疾病为心、肝、脾、肺或肾相关疾病；Optionally, the disease is a heart, liver, spleen, lung or kidney related disease;
    优选地，所述疾病为脾或肺相关疾病；Preferably, the disease is a spleen- or lung-related disease;
    任选的，所述疾病为感染性疾病、癌症和增生性疾病、遗传性疾病、自体免疫性疾病、糖尿病、神经退化性疾病、心血管和肾血管疾病以及代谢性疾病；Optionally, the diseases are infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases and metabolic diseases;
    优选的，所述感染性疾病选自：由冠状病毒、流感病毒或HIV病毒引起的 疾病，小儿肺炎，裂谷热，黄热病，狂犬病，或多种疱疹；Preferably, the infectious disease is selected from: caused by coronavirus, influenza virus or HIV virus Disease, childhood pneumonia, Rift Valley fever, yellow fever, rabies, or herpes;
    优选的，所述癌症为实体肿瘤；Preferably, the cancer is a solid tumor;
    优选地，所述癌症为肝癌或肺癌。Preferably, the cancer is liver cancer or lung cancer.
  21. 根据权利要求20所述的用途，其特征在于，所述药物通过递呈抗原和/或激活免疫反应治疗或预防疾病。 The use according to claim 20, characterized in that the drug treats or prevents diseases by presenting antigens and/or activating immune responses.
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017112865A1 (en) * 2015-12-22 2017-06-29 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2019226650A1 (en) * 2018-05-23 2019-11-28 Modernatx, Inc. Delivery of dna
WO2021022173A1 (en) * 2019-07-31 2021-02-04 Modernatx, Inc. Compositions and methods for delivery of rna interference agents to immune cells
WO2021026358A1 (en) * 2019-08-07 2021-02-11 Moderna TX, Inc. Compositions and methods for enhanced delivery of agents
US20210198200A1 (en) * 2017-06-14 2021-07-01 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2021142280A1 (en) * 2020-01-10 2021-07-15 Modernatx, Inc. Methods of making tolerogenic dendritic cells
CN113164561A (en) * 2018-09-13 2021-07-23 摩登纳特斯有限公司 Polynucleotides encoding glucose-6-phosphatase for the treatment of glycogen storage disease
CN113939282A (en) * 2019-01-31 2022-01-14 摩登纳特斯有限公司 Method for preparing lipid nanoparticles
CN114044741A (en) * 2022-01-13 2022-02-15 北京悦康科创医药科技股份有限公司 Cationic lipid compound, composition containing same and application
CN114391008A (en) * 2020-08-20 2022-04-22 苏州艾博生物科技有限公司 Lipid compounds and lipid nanoparticle compositions

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017112865A1 (en) * 2015-12-22 2017-06-29 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US20210198200A1 (en) * 2017-06-14 2021-07-01 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2019226650A1 (en) * 2018-05-23 2019-11-28 Modernatx, Inc. Delivery of dna
CN113164561A (en) * 2018-09-13 2021-07-23 摩登纳特斯有限公司 Polynucleotides encoding glucose-6-phosphatase for the treatment of glycogen storage disease
CN113939282A (en) * 2019-01-31 2022-01-14 摩登纳特斯有限公司 Method for preparing lipid nanoparticles
WO2021022173A1 (en) * 2019-07-31 2021-02-04 Modernatx, Inc. Compositions and methods for delivery of rna interference agents to immune cells
WO2021026358A1 (en) * 2019-08-07 2021-02-11 Moderna TX, Inc. Compositions and methods for enhanced delivery of agents
WO2021142280A1 (en) * 2020-01-10 2021-07-15 Modernatx, Inc. Methods of making tolerogenic dendritic cells
CN114391008A (en) * 2020-08-20 2022-04-22 苏州艾博生物科技有限公司 Lipid compounds and lipid nanoparticle compositions
CN114044741A (en) * 2022-01-13 2022-02-15 北京悦康科创医药科技股份有限公司 Cationic lipid compound, composition containing same and application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Registry 16 March 1985 (1985-03-16), ANONYMOUS: "1,4-Piperazinedicarboxylic acid, 1,4-bis[4-(diethylamino)butyl] ester (CA INDEX NAME)", XP093121428, retrieved from STNext Database accession no. 95286-94-5 *

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