WO2021089030A1 - 脂质在制备核酸递送试剂中的应用及其相关产品 - Google Patents

脂质在制备核酸递送试剂中的应用及其相关产品 Download PDF

Info

Publication number
WO2021089030A1
WO2021089030A1 PCT/CN2020/127393 CN2020127393W WO2021089030A1 WO 2021089030 A1 WO2021089030 A1 WO 2021089030A1 CN 2020127393 W CN2020127393 W CN 2020127393W WO 2021089030 A1 WO2021089030 A1 WO 2021089030A1
Authority
WO
WIPO (PCT)
Prior art keywords
linear
branched
nucleic acid
lipid
use according
Prior art date
Application number
PCT/CN2020/127393
Other languages
English (en)
French (fr)
Inventor
蒋澄宇
秦宇豪
李晓芸
张聪
Original Assignee
中国医学科学院基础医学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国医学科学院基础医学研究所 filed Critical 中国医学科学院基础医学研究所
Priority to EP20884183.3A priority Critical patent/EP4056552A4/en
Priority to CA3163934A priority patent/CA3163934A1/en
Priority to CN202080077628.5A priority patent/CN114650982A/zh
Priority to US17/775,283 priority patent/US20230088528A1/en
Publication of WO2021089030A1 publication Critical patent/WO2021089030A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/19—Acanthaceae (Acanthus family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/288—Taraxacum (dandelion)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/35—Caprifoliaceae (Honeysuckle family)
    • A61K36/355—Lonicera (honeysuckle)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/41—Crassulaceae (Stonecrop family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the invention relates to the field of biology. Specifically, the present application relates to the use of lipid compounds for the delivery of nucleic acids. These compounds or multiple combinations thereof can promote the absorption of various nucleic acids in vivo through oral administration, enter target sites in the body of a subject in need, and enter target cells.
  • the compounds involved in this application have been extracted and discovered in traditional Chinese medicine, and can also be obtained by synthetic methods.
  • nucleic acid molecules possess many properties that make them useful as therapeutic drugs. They can fold into complex conformations that allow them to bind to proteins, small molecules or other nucleic acids, and some can even form catalytic centers.
  • small interfering RNA siRNA
  • siRNA small interfering RNA
  • sRNAs small interfering RNA
  • nucleic acid molecules that can be used as drugs also include, for example, mRNA, antisense nucleic acid, and the like.
  • nucleic acid molecules such as RNA are easily degraded and have a relatively short half-life in the body, they are generally not considered as the optimal choice for therapeutic drugs. Therefore, how to effectively deliver nucleic acid molecules including small RNA, mRNA, etc. to target organs and target cells in vivo to achieve their biological activity and therapeutic or preventive effects is an urgent problem for those skilled in the art.
  • the application provides the use of a lipid composition in the preparation of a reagent for nucleic acid delivery, the lipid composition comprising one or more compounds having formula (I) or a salt, hydrate or Solvate:
  • L 1 is not present, -CH 2 -OC(O)-, -CH 2 -O- or -CR(OH)-;
  • L 2 is not present, -OC(O)- or -NH-C(O)-;
  • L 3 is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, linear or branched C 1-20 hetero Alkenyl or
  • A is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1-20 heteroalkene base;
  • B is -OH, linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1- 20 heteroalkenyl;
  • Q is -H, -COOH, hydroxy-substituted linear or branched C 1-20 alkyl, hydroxy-substituted linear or branched C 1-20 alkenyl, hydroxy-substituted C 3-20 cycloalkyl, hydroxyl Substituted C 3-20 cycloalkenyl, -N(R) 3 + or
  • R is each independently H or a linear or branched C 1-20 alkyl group
  • n 0, 1, or 2.
  • the salt of the compound includes a pharmaceutically acceptable salt.
  • the compound has a structure of the following formula:
  • the application also provides the use of a lipid composition in the preparation of a reagent for nucleic acid delivery, the lipid composition comprising one or more compounds selected from the following group, or salts, hydrates thereof Substances or solvates: lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 86, 87, 90, 91, 92, 95, 97, 98, 99, 100, 101 , 102, 107, 111, 112, 113, 114, 115, 116, 117, 118, and 119, wherein the compounds are shown in the following table:
  • Lipid compound number chemical name 72 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine 73
  • Three Linoleum 74
  • 1,2-palmitoleic acid-3-oleic acid glyceride 75
  • 1,2-palmitoleic acid-3-palmitic acid glyceride 76
  • 1,3-palmitic acid-2-myristic acid glyceride 77
  • 1,2-stearic acid-3-oleic acid glyceride 1,2-oleic acid-3-arachidic acid glyceride
  • 1,2-oleic acid-3-behenyl carbonate 80
  • 1,2-Myristic acid-3-palmitic acid glyceride 82 Q-10, Ubiquinone 50, Ubiquinone 10 83
  • 1-palmitoyl-2-hydroxy-sn-glyceryl-3-phosphoethanolamine 86
  • the lipid composition or the agent can deliver the nucleic acid by oral, inhalation or injection. In some embodiments, the lipid composition or the agent delivers the nucleic acid orally. In certain embodiments, the delivery includes in vivo digestive tract delivery.
  • the delivery includes in vitro cell delivery.
  • the lipid composition or the reagent can be used to prepare a lipid nucleic acid mixture.
  • the lipid nucleic acid mixture can be prepared by a suitable method, including but not limited to, heating method, reverse evaporation method, or mixing method.
  • the heating method includes adding a lipid organic solvent solution to an aqueous nucleic acid solution to obtain a mixed solution, and heating the mixed solution at an appropriate temperature. In some embodiments, the heating method further includes cooling the heated mixed solution, thereby obtaining a mixture of lipids and nucleic acids.
  • the mixed solution is heated at a temperature selected from: 25°C to 100°C, 30°C to 100°C, 40°C to 100°C, 50°C to 100°C, 60°C to 100°C, 70°C °C to 100 °C, 80 °C to 100 °C, 90 °C to 100 °C, and 95 °C to 100 °C.
  • the mixed solution is heated at a temperature selected from: 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C °C, 80°C, 85°C, 90°C, 95°C and 100°C.
  • the time for heating the mixed solution is from about 5 minutes to about 24 hours, from about 5 minutes to about 20 hours, from about 5 minutes to about 16 hours, from about 10 minutes to about 20 hours, from about 10 minutes to about 10 minutes.
  • the time for heating the mixed solution is about 5 minutes to about 1 hour, about 5 minutes to about 30 minutes, about 5 minutes to about 15 minutes, or about 10 minutes to about 15 minutes.
  • the time for heating the mixed solution is about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 20 hours or 24 hours.
  • the mixed solution is cooled at a temperature selected from: 25°C to -80°C, 20°C to -80°C, 15°C to -80°C, 10°C to -80°C, 4°C To -80°C, 0°C to -80°C, -10°C to -80°C, -20°C to -80°C, -30°C to -80°C, -40°C to -80°C. In some embodiments, the mixed solution is cooled at a temperature selected from: 25°C, 20°C, 15°C, 10°C, 4°C, or 0°C.
  • the reverse evaporation method includes mixing an aqueous solution of nucleic acid and an organic solvent solution of a lipid compound to obtain a mixed solution. In some embodiments, the reverse evaporation method further includes removing the organic solvent in the mixed solution, and then performing hydration to obtain a mixture of lipids and nucleic acids. In some embodiments, the mixed solution is sonicated and/or evaporated to remove the organic solvent. In some embodiments, the step of removing the organic solvent in the mixed solution is performed at an appropriate temperature.
  • the organic solvent in the mixed solution is removed at a temperature selected from: about 25°C to about 70°C, 30°C to about 70°C, about 30°C to about 65°C, about 40°C to about About 65°C, about 40°C to about 60°C, or about 50°C to about 60°C. In some embodiments, the organic solvent in the mixed solution is removed at a temperature selected from: about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C , About 60°C, about 65°C and about 70°C.
  • the present application provides a method for delivering nucleic acid to an individual in need, which comprises administering the lipid composition and the nucleic acid to the individual by oral, inhalation or injection.
  • the lipid composition and the nucleic acid are administered in the form of a lipid nucleic acid mixture.
  • the nucleic acid comprises DNA or RNA.
  • the DNA is, for example, non-coding DNA (such as antisense DNA) or coding DNA.
  • the RNA is, for example, antisense nucleic acid, mRNA, lncRNA, or small RNA (for example, miRNA, siRNA, piRNA, snoRNA, tsRNA), and the like.
  • the nucleic acid comprises a small nucleic acid with a length of 14-32bp, 16-28bp, or 18-24bp.
  • the nucleic acid is single-stranded or double-stranded.
  • the nucleic acid has a stem-loop structure.
  • the nucleic acid is used to treat diseases.
  • the nucleic acid is used to treat cancer, inflammation, fibrotic diseases, autoimmune diseases or autoinflammatory diseases, bacterial infections, behavioral and mental disorders, blood diseases, chromosomal diseases, congenital and genetic sexual diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, environmental diseases, eye diseases, female reproductive diseases, fungal infections, heart diseases, hereditary cancer syndromes, immune system diseases, kidney and urinary diseases Sexual diseases, lung diseases, new male reproductive diseases, metabolic disorders, oral diseases, musculoskeletal diseases, myelodysplastic syndromes, newborn screening, nutritional diseases, parasitic diseases, rare cancers, rare diseases, Skin diseases and viral infections.
  • the nucleic acid is used to treat hepatocellular carcinoma, corneal neovascularization, relapsed or refractory anaplastic astrocytoma (WHO grade III), or secondary glioblastoma (WHO Grade IV), advanced squamous cell lung cancer, acromegaly, psoriasis, Duchenne muscular dystrophy, advanced non-small cell lung cancer, metastatic castration-resistant prostate cancer, cytomegalovirus retinitis, HIV infection, Hepatitis B, hepatitis C, hyperlipoproteinemia, total knee arthroplasty, type II diabetes, familial amyloid polyneuropathy (FAP), wet macular degeneration (e.g.
  • neovascular age-related macular degeneration fovea Lower neovascular age-related macular degeneration, exudative age-related macular degeneration
  • hypercholesterolemia Crohn's disease, extensive liver fibrosis, infantile spinal muscular atrophy, melanoma
  • new coronary artery Diseases mild allergic asthma, chronic lymphocytic leukemia, and hypertriglyceridemia, small hepatic obliterans with renal or lung dysfunction after hematopoietic stem cell transplantation, and hereditary transthyretin amyloid lesions.
  • the application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid composition and a nucleic acid
  • the lipid composition comprises one or more compounds of formula (I) or pharmaceutically acceptable compounds thereof Accepted salt, hydrate or solvate.
  • the lipid composition and the nucleic acid are in the form of a lipid nucleic acid mixture.
  • the application also provides a pharmaceutical composition, which comprises a lipid composition and a nucleic acid, wherein the lipid composition comprises one or more compounds selected from the following group, or salts, hydrates thereof Substances or solvates: lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 86, 87, 90, 91, 92, 95, 97, 98, 99, 100, 101 , 102, 107, 111, 112, 113, 114, 115, 116, 117, 118, and 119.
  • the lipid composition and the nucleic acid are in the form of a lipid nucleic acid mixture.
  • the application also provides the use of the pharmaceutical composition described in the application in the preparation of medicines for the prevention and/or treatment of diseases that can be prevented and/or treated with nucleic acid, or for the use of The nucleic acid is delivered in vivo to a subject in need.
  • the application also provides a kit comprising: one or more compounds of formula (I) or pharmaceutically acceptable salts or hydrates thereof placed in a first container Or a solvate, and a nucleic acid placed in a second container.
  • the present application also provides a kit comprising: one or more compounds or a pharmaceutically acceptable salt, hydrate or solvate thereof placed in a first container, and placed in the first container The nucleic acid in the second container, wherein the one or more compounds are selected from the group consisting of lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 86, 87, 90, 91, 92, 95, 97, 98, 99, 100, 101, 102, 107, 111, 112, 113, 114, 115, 116, 117, 118, and 119.
  • the present application provides the use of the kit described in the present application in the preparation of a medicament for the prevention and/or treatment of diseases that can be prevented and/or treated with nucleic acid, or for the use of nucleic acid in vivo Deliver to those who need it.
  • the present application provides a method for delivering nucleic acid to target cells, comprising administering to the target cell the pharmaceutical composition described in this application, or the lipid formulated by the kit described in this application Nucleic acid mixture.
  • the present application provides a method for delivering nucleic acid in vivo to a subject in need thereof, comprising administering to the subject the pharmaceutical composition described in this application, or formulated by the kit described in this application The lipid nucleic acid mixture.
  • Figure 1 shows the blank group, free intake group and lipid 72 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 1a), lung tissue (Figure 1b), spleen tissue (Figure 1c) and blood ( Figure 1d) )the result of.
  • Figure 2 shows the results of the blank group, free intake group and lipid 73 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 2a), spleen tissue (Figure 2b) and blood (Figure 2c).
  • Figure 3 shows the blank group, free intake group and lipid 74 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 3a), lung tissue (Figure 3b), spleen tissue (Figure 3c) and blood ( Figure 3d) )the result of.
  • Figure 4 shows the blank group, free intake group and lipid 75 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 4a), lung tissue (Figure 4b), spleen tissue (Figure 4c) and blood ( Figure 4d) )the result of.
  • Figure 5 shows the blank group, the free intake group and the lipid 76 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 5a), lung tissue (Figure 5b), spleen tissue (Figure 5c) and blood ( Figure 5d). )the result of.
  • Figure 6 shows the blank group, free intake group and lipid 77 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 6a), lung tissue (Figure 6b), kidney tissue (Figure 6c), and spleen tissue (Figure 6a). 6d) and blood ( Figure 6e) results.
  • Figure 7 shows the blank group, free intake group and lipid 78 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 7a), lung tissue (Figure 7b), kidney tissue (Figure 7c) and spleen tissue ( Figure 7a). The result of 7d).
  • Figure 8 shows the results of the blank group, free intake group and lipid 79 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 8a), kidney tissue ( Figure 8b) and spleen tissue (Figure 8c).
  • Figure 9 shows the blank group, free intake group and lipid 80 delivery of PGY-sRNA-26 into mouse heart tissue (Figure 9a), lung tissue (Figure 9b), kidney tissue (Figure 9c) and spleen tissue ( Figure 9a). The result of 9d).
  • Figure 10 shows the results of the blank group, free intake group and lipid 81 delivering PGY-sRNA-26 into mouse heart tissue (Figure 10a), kidney tissue (Figure 10b) and spleen tissue (Figure 10c), respectively.
  • Figure 11 shows the results of the blank group, free intake group and lipid 82 delivery of PGY-sRNA-26 into mouse lung tissue (Figure 11a), spleen tissue (Figure 11b) and heart tissue (Figure 11c).
  • Figure 12 shows the results of the blank group, free intake group and lipid 83 delivering PGY-sRNA-26 into mouse spleen tissue (Figure 12a), kidney tissue (Figure 12b) and intestinal tissue (Figure 12c), respectively.
  • Figure 13 shows the results of the blank group, free intake group and lipid 84 delivery of PGY-sRNA-26 into mouse lung (Figure 13a) tissue and spleen tissue ( Figure 13b).
  • Figure 14 shows the results of the blank group, free intake group and lipid 85 delivery of PGY-sRNA-26 into mouse lung tissue (Figure 14a), spleen tissue (Figure 14b) and kidney tissue (Figure 14c), respectively.
  • Figure 15 shows the results of the blank group, free intake group and lipid 86 delivery of PGY-sRNA-26 into mouse intestinal tissue (Figure 15a) and kidney tissue ( Figure 15b).
  • Figure 16 shows the results of the blank group, free intake group and lipid 87 delivery of PGY-sRNA-26 into mouse kidney tissue (Figure 16a), lung tissue (Figure 16b) and spleen tissue (Figure 16c), respectively.
  • Figure 17 shows the blank group, free intake group and lipid 88 delivery of PGY-sRNA-26 into mouse blood (Figure 17a), lung tissue (Figure 17b), spleen tissue (Figure 17c) and kidney tissue ( Figure 17d). )the result of.
  • Figure 18 shows the results of the blank group, free intake group and lipid 89 delivery of PGY-sRNA-26 into mouse blood (Figure 18a), lung tissue (Figure 18b) and kidney tissue (Figure 18c).
  • Figure 19 shows the blank group, free intake group and lipid 90 delivery of PGY-sRNA-26 into mouse blood (Figure 19a), lung tissue (Figure 19b), spleen tissue (Figure 19c) and kidney tissue ( Figure 19d). )the result of.
  • Figure 20 shows the blank group, free intake group and lipid 91 delivery of PGY-sRNA-26 into mouse blood (Figure 20a), lung tissue (Figure 20b), spleen tissue (Figure 20c) and kidney tissue ( Figure 20d). )the result of.
  • Figure 21 shows the blank group, free intake group and lipid 92 delivery of PGY-sRNA-26 into mouse blood (Figure 21a), lung tissue (Figure 21b), spleen tissue (Figure 21c) and kidney (Figure 21d) the result of.
  • Figure 22 shows the blank group, free intake group and lipid 93 delivery of PGY-sRNA-26 into mouse blood (Figure 22a), lung tissue (Figure 22b), spleen tissue (Figure 22c) and kidney tissue ( Figure 22d). )the result of.
  • Figure 23 shows the blank group, free intake group and lipid 94 delivery of PGY-sRNA-26 into mouse blood (Figure 23a), lung tissue (Figure 23b), spleen tissue (Figure 23c) and kidney tissue ( Figure 23d). )the result of.
  • Figure 24 shows the blank group, free intake group and lipid 95 delivery of PGY-sRNA-26 into mouse blood (Figure 24a), lung tissue (Figure 24b), kidney tissue (Figure 24c) and spleen tissue ( Figure 24d). )the result of.
  • Figure 25 shows the blank group, free intake group and lipid 96 delivery of PGY-sRNA-26 into mouse blood (Figure 25a), lung tissue (Figure 25b), kidney tissue (Figure 25c) and spleen tissue ( Figure 25d). )the result of.
  • Figure 26 shows the blank group, free intake group and lipid 97 delivery of PGY-sRNA-26 into mouse blood (Figure 26a), lung tissue (Figure 26b), kidney tissue (Figure 26c) and spleen tissue ( Figure 26d). )the result of.
  • Figure 27 shows the blank group, free intake group and lipid 98 delivery of PGY-sRNA-26 into mouse blood (Figure 27a), lung tissue (Figure 27b), kidney tissue (Figure 27c) and spleen tissue (Figure 27d). )the result of.
  • Figure 28 shows the blank group, free intake group and lipid 99 delivery of PGY-sRNA-26 into mouse blood (Figure 28a), lung tissue (Figure 28b), kidney tissue (Figure 28c) and spleen tissue ( Figure 28d). )the result of.
  • Figure 29 shows the blank group, free intake group and lipid 100 delivery of PGY-sRNA-26 into mouse blood (Figure 29a), lung tissue (Figure 29b), kidney tissue (Figure 29c) and spleen tissue (Figure 29d). )the result of.
  • Figure 30 shows the blank group, free intake group and lipid 101 delivery PGY-sRNA-26 into mouse blood (Figure 30a), lung tissue (Figure 30b), kidney tissue (Figure 30c) and spleen tissue (Figure 30d) )the result of.
  • Figure 31 shows the results of the blank group, free intake group and lipid 102 delivering PGY-sRNA-26 into mouse blood (Figure 31a), kidney tissue ( Figure 31b) and spleen tissue (Figure 31c), respectively.
  • Figure 32 shows the results of the blank group, free intake group and lipid 103 delivering PGY-sRNA-26 into mouse blood (Figure 32a), kidney tissue ( Figure 32b) and lung tissue (Figure 32c), respectively.
  • Figure 33 shows the blank group, free intake group and lipid 104 delivery of PGY-sRNA-26 into mouse blood (Figure 33a), kidney tissue (Figure 33b), lung tissue (Figure 33c) and spleen tissue ( Figure 33d). )the result of.
  • Figure 34 shows the results of the blank group, free intake group and lipid 105 delivering PGY-sRNA-26 into mouse blood (Figure 34a), kidney tissue (Figure 34b) and lung tissue (Figure 34c), respectively.
  • Figure 35 shows the results of the blank group, free intake group and lipid 107 delivering PGY-sRNA-26 into mouse blood (Figure 35a), kidney tissue (Figure 35b) and spleen tissue (Figure 35c), respectively.
  • Figure 36 shows the blank group, free intake group and lipid 109 delivery of PGY-sRNA-26 into mouse blood (Figure 36a), lung tissue (Figure 36b), kidney tissue (Figure 36c) and spleen tissue ( Figure 36d). )the result of.
  • Figure 37 shows the results of the blank group, free intake group and lipid 111 delivering PGY-sRNA-26 into mouse blood (Figure 37a) and kidney tissue ( Figure 37b), respectively.
  • Figure 38 shows the results of the blank group, free intake group and lipid 112 delivering PGY-sRNA-26 into mouse lung (Figure 38a), kidney tissue ( Figure 38b) and spleen tissue (Figure 38c), respectively.
  • Figure 39 shows the results of the blank group, free intake group and lipid 113 delivering PGY-sRNA-26 into mouse lung tissue (Figure 39a), kidney tissue ( Figure 39b) and spleen tissue (Figure 39c), respectively.
  • Figure 40 shows the blank group, free intake group and lipid 114 delivery of PGY-sRNA-26 into mouse blood (Figure 40a), lung tissue (Figure 40b), kidney tissue (Figure 40c) and spleen tissue ( Figure 40d). )the result of.
  • Figure 41 shows the blank group, free intake group and lipid 115 to deliver PGY-sRNA-26 into mouse blood (Figure 41a), lung tissue (Figure 41b), kidney tissue (Figure 41c) and spleen tissue ( Figure 41d). )the result of.
  • Figure 42 shows the results of the blank group, free intake group and lipid 116 delivering PGY-sRNA-26 into mouse lung tissue (Figure 42a), kidney tissue ( Figure 42b) and spleen tissue (Figure 42c), respectively.
  • Figure 43 shows the results of the blank group, free intake group and lipid 117 delivering PGY-sRNA-26 into mouse lung tissue (Figure 43a), kidney tissue ( Figure 43b) and spleen tissue (Figure 43c), respectively.
  • Figure 44 shows the results of the blank group, free intake group and lipid 118 delivering PGY-sRNA-26 into mouse lung tissue (Figure 44a), kidney tissue ( Figure 44b) and spleen tissue (Figure 44c), respectively.
  • Figure 45 shows the results of the blank group, free intake group and lipid 119 delivering PGY-sRNA-26 into mouse blood (Figure 45a) and kidney tissue (Figure 45b), respectively.
  • Figure 46 respectively shows that lipid 93 delivers PGY-sRNA-26 into mouse brain tissue (Figure 46a), gastric tissue (Figure 46b), intestinal tissue (Figure 46c), kidney tissue ( Figure 46a) by means of intragastric administration. 46d), lung tissue (Figure 46e) and spleen tissue (Figure 46f) at 0 hours (blank group) / 3 hours / 6 hours / 9 hours / 12 hours / 24 hours into the results.
  • Figure 47 shows that lipid 94 delivers PGY-sRNA-26 by gavage into mouse brain tissue (Figure 47a), heart tissue (Figure 47b), spleen tissue (Figure 47c), lung tissue (Figure 47a), respectively. 47d), kidney tissue (Figure 47e), intestinal tissue (Figure 47f) and stomach tissue (Figure 47g) are the results of the intake at 0 hours (blank group)/3 hours/6 hours/9 hours/12 hours/24 hours.
  • Figure 48 shows that lipid 96 delivers PGY-sRNA-26 into mouse intestinal tissue (Figure 48a) and kidney tissue (Figure 48b) by gavage administration at 0 hours (blank group)/3 hours/6 Hour / 9 hours / 12 hours / 24 hours into the result of the volume.
  • Figure 49 shows that Lipid 100 delivers PGY-sRNA-26 into mouse gastric tissue (Figure 49a) and intestinal tissue (Figure 49b) at 0 hours (blank group)/3 hours/6 by means of intragastric administration. Hour / 9 hours / 12 hours / 24 hours into the result of the volume.
  • Figure 50 respectively shows that lipid 94 delivers PGY-sRNA-26 into mouse spleen tissue (Figure 50a), kidney tissue (Figure 50b), brain tissue (Figure 50c), and stomach tissue (Figure 50a) by way of tail vein injection administration.
  • Figure 50d the results of intestinal tissue (Figure 50e) at 0 hours (blank group)/3 hours/6 hours/9 hours/12 hours/24 hours.
  • Figure 51 shows that lipid 96 delivers PGY-sRNA-26 into mouse brain tissue (Figure 51a), intestinal tissue (Figure 51b), kidney tissue (Figure 51c), and lung tissue (Figure 51a) by means of tail vein injection administration.
  • Figure 51d liver tissue ( Figure 51e), heart tissue (Figure 51f) at 0 hours (blank group) / 3 hours / 6 hours / 9 hours / 12 hours / 24 hours into the results.
  • Figure 52 shows that lipid 98 delivers PGY-sRNA-26 into mouse kidney tissue (Figure 52a), intestinal tissue (Figure 52b), stomach tissue (Figure 52c), brain tissue ( Figure 52d), liver tissue ( Figure 52e), heart tissue (Figure 52f) in 0 hours (blank group) / 3 hours / 6 hours / 9 hours / 12 hours / 24 hours into the results.
  • Figure 53 shows that Lipid 100 delivers PGY-sRNA-26 into mouse kidney tissue (Figure 53a) and stomach tissue (Figure 53b) at 0 hours (blank group)/3 hours/ 6 hours / 9 hours / 12 hours / 24 hours into the results.
  • Figure 54 shows that Lipid 101 delivers PGY-sRNA-26 into mouse kidney tissue (Figure 54a), intestinal tissue (Figure 54b), stomach tissue (Figure 54c), brain tissue ( Figure 54d) The result of the entry amount at 0 hour (blank group) / 3 hours / 6 hours / 9 hours / 12 hours / 24 hours.
  • the present invention is based at least in part on the unexpected discovery obtained by the inventors of the present application through a large number of experiments, that is, there are some lipid components in some Chinese medicines (for example, rhodiola, dandelion, andrographis, honeysuckle, etc.).
  • Lipids can promote the uptake of nucleic acids, such as small RNAs, into cells and/or target sites in the subject in need thereof.
  • these lipid components may also be synthetic.
  • linking substituents are described herein.
  • the Markush variables listed for this group are the linking groups.
  • alkyl means an alkylene linking group.
  • substituted means that one or more hydrogens in the specified group are replaced by suitable substituents.
  • a “substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be selected from the group When more than one substituent is substituted, the substituents may be the same or different in each position.
  • the combinations of substituents contemplated by the present invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to a compound when it is subjected to conditions that allow its production, detection, and (in certain embodiments) its recovery, purification, and use for one or more of the purposes disclosed herein At the time, it will basically not change.
  • unsubstituted the chemical moieties described herein should be understood to include substituents.
  • aryl it includes substituted aryl groups and unsubstituted aryl groups.
  • C ij represents a range of the number of carbon atoms, where i and j are integers and j is greater than i, and the range of the number of carbon atoms includes the endpoints (ie, i and j) and each integer point between the endpoints.
  • C 1-6 represents the range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms and 6 carbon atoms.
  • the term “C 1-12” means 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1. To 3, or especially 1 to 2 carbon atoms.
  • alkyl refers to a saturated straight or branched chain hydrocarbon group.
  • C ij alkyl refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 20 carbon atoms. In some embodiments, the alkyl group contains 5 to 20 carbon atoms.
  • the alkyl group contains 1 to 20 carbon atoms, 1 to 19 carbon atoms, 1 to 18 carbon atoms, 1 to 17 carbon atoms, 1 to 16 carbon atoms, 1 to 15 carbon atoms , 1 to 14 carbon atoms, 1 to 13 carbon atoms, 1 to 12 carbon atoms, 1 to 11 carbon atoms, 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl (n-propyl), 2-propyl (isopropyl), 1-butyl (n-butyl), 2-methyl-1 -Propyl (isobutyl), 2-butyl (neobutyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3 -Pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2- Hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl -3-pentyl, 2-methyl -3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl,
  • C 1-20 alkyl examples include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl Alkyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl.
  • alkenyl refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond, which may optionally be substituted by one or more The substituents are independently substituted, and include groups having "cis” and “trans” orientations, or "E” and “Z” orientations.
  • the alkenyl group contains 2 to 20 carbon atoms. In some embodiments, the alkenyl group contains 5 to 20 carbon atoms.
  • the alkenyl group contains 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms , 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms.
  • the alkenyl group contains more than 1 carbon-carbon double bond.
  • the alkenyl group contains more than 1 carbon-carbon double bond
  • the double bonds may be separated from each other or conjugated.
  • the alkenyl group contains 5 carbon-carbon double bonds, 4 carbon-carbon double bonds, 3 carbon-carbon double bonds, 2 carbon-carbon double bonds, or 1 carbon-carbon double bond.
  • cycloalkyl refers to saturated monocyclic and polycyclic ring systems in which all ring atoms are carbon, which contains at least 3 ring-forming carbons atom.
  • the cycloalkyl group may contain 3 to 20 ring-forming carbon atoms, 3 to 19 ring-forming carbon atoms, 3 to 18 ring-forming carbon atoms, 3 to 17 ring-forming carbon atoms, 3 to 16 Ring carbon atoms, 3 to 15 ring carbon atoms, 3 to 14 ring carbon atoms, 3 to 13 ring carbon atoms, 3 to 12 ring carbon atoms, 3 to 11 ring carbon atoms , 3 to 10 ring carbon atoms, 3 to 9 ring carbon atoms, 3 to 8 ring carbon atoms, 3 to 7 ring carbon atoms, 3 to 6 ring carbon atoms, 3 to 5 Ring carbon atoms, 4 to 20 ring carbon atoms, 4 to 19 ring carbon atoms, 4 to 18 ring carbon atoms, 4 to 17 ring carbon atoms, 4 to 16 ring carbon atoms, 4 to 15 ring-forming carbon atoms, 4 to 14 ring-forming carbon atoms
  • Cycloalkyl groups may be optionally substituted at one or more ring positions with one or more substituents described herein. Cycloalkyl groups can be saturated, partially unsaturated or fully unsaturated. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be an unsaturated cyclic alkyl group containing at least one double bond or triple bond in the ring system.
  • cycloalkenyl refers to monocyclic and polycyclic ring systems containing 3-20 ring-forming carbon atoms and at least one carbon-carbon double bond, As long as the size of the cycloalkenyl ring allows, all ring atoms in it are carbon.
  • the cycloalkenyl group contains more than 1 carbon-carbon double bond. It should be understood that in the case where the alkenyl group contains more than 1 carbon-carbon double bond, the double bonds may be separated from each other or conjugated.
  • the cycloalkenyl group contains 5 carbon-carbon double bonds, 4 carbon-carbon double bonds, 3 carbon-carbon double bonds, 2 carbon-carbon double bonds, or 1 carbon-carbon double bond .
  • heteroatom refers to nitrogen, oxygen, sulfur, or phosphorus, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of basic nitrogen.
  • heteroalkyl refers to an alkyl group in which at least one carbon atom is replaced by a heteroatom selected from nitrogen, oxygen, sulfur, or phosphorus, wherein the hetero The atom can be located at the end or in the middle of the alkyl group.
  • heteroalkenyl refers to an alkenyl group in which at least one carbon atom is replaced by a heteroatom selected from nitrogen, oxygen, sulfur, or phosphorus, wherein the hetero The atom can be located at the end or in the middle of the alkenyl group.
  • hydroxyl refers to -OH.
  • the term "pharmaceutically acceptable salt” refers to that it is suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic reactions or other problems within the scope of reasonable medical judgment, and is compatible with reasonable medical judgment.
  • the salt or zwitterionic form of the compound described herein is commensurate with the beneficial effect/risk ratio.
  • composition refers to a composition comprising the lipid composition described in the present application and nucleic acid, and the composition may optionally further contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a compound, material, composition and/or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, within the scope of reasonable medical judgment, Irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable compounds, materials, compositions, and/or dosage forms refer to those used in animals (more specifically, in humans) by regulatory agencies (e.g., U.S.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which relates to The compounds provided herein are carried or transported from one location, body fluid, tissue, organ (internal or external), or part of the body to another location, body fluid, tissue, organ, or part of the body.
  • the pharmaceutically acceptable carrier can be a vehicle, diluent, excipient, or other material that can be used to contact animal tissues without excessive toxicity or side effects.
  • Exemplary pharmaceutically acceptable carriers include sugar, starch, cellulose, malt, tragacanth, gelatin, Ringer's solution, alginic acid, isotonic saline, buffers, and the like.
  • the pharmaceutically acceptable carriers that can be used in the present application include those commonly known in the art, such as those disclosed in "Remington Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which was introduced by Incorporated into this article.
  • delivery refers to the direct delivery of a therapeutic agent (such as a nucleic acid) to be delivered to a target site in a living body.
  • a therapeutic agent such as a nucleic acid
  • the agent can be delivered locally by direct injection into a target site (e.g., a disease site, such as a tumor or inflammation site) or a target organ (e.g., heart, spleen, lung, kidney, etc.).
  • Systemic delivery refers to the delivery that leads to a broad biodistribution of a therapeutic agent (such as a nucleic acid) in a living body, thereby exposing an effective amount of the therapeutic agent to most parts of the body.
  • lipid composition In order to obtain a broad biodistribution, a blood lifetime is usually required so that these therapeutic agents do not rapidly degrade or clear before reaching the target site far from the administration site.
  • the systemic delivery of the lipid composition can be carried out in any suitable manner, including, for example, oral, inhalation, intra-digestive, intravenous, subcutaneous, and intraperitoneal.
  • lipid refers to a class of organic compounds that include, but are not limited to, fatty acid esters, and are characterized by being insoluble in water (for example, the solubility in water is less than about 0.01% by weight) but soluble in many organic solvents .
  • Lipids can be, for example, simple lipids (e.g., fats, oils, waxes), compound lipids (e.g., phospholipids, glycolipids), and derivatized lipids (e.g., steroids).
  • Nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form.
  • the nucleic acid may have natural ribonucleotides and deoxyribonucleotides, and may also include non-natural ribonucleotides and non-natural deoxyribonucleotides.
  • Natural ribonucleotides include, for example, adenylic acid, guanylic acid, cytidine acid, uridine acid, pseudouric acid, inosinic acid, and xanthylic acid.
  • Natural deoxyribonucleotides include, for example, deoxyadenylic acid, deoxyguanylic acid, deoxycytidine acid, and deoxythymidylic acid.
  • Non-natural ribonucleotides and deoxyribonucleic acids usually have modified nucleobases, modified ribose or deoxyribose, and/or modified phosphate linkages.
  • Nucleotides include sugars (such as deoxyribose or ribose), bases, and phosphate groups. Nucleotides are linked together through phosphate groups to form polymers.
  • Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not Limited to modifications such as amines, alcohols, mercaptans, carboxylates, and alkyl halides.
  • the nucleic acid may also include nucleotide analogs or modified nucleotides, which may be synthetic, naturally occurring, and non-naturally occurring, and which have binding properties similar to those of natural nucleic acids.
  • nucleotide analogs or modified nucleotides include, but are not limited to, nucleotides with phosphorothioate, phosphoramidate, methyl phosphate, or chiral-methyl phosphate, 2 '-O-methyl ribonucleotides and peptide-nucleic acids (PNAs).
  • nucleic acids include DNA or RNA.
  • DNA includes coding DNA and non-coding DNA. Examples include, but are not limited to, antisense DNA, plasmid DNA, pre-concentrated DNA, PCR products, DNA vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, and chimeras Sequence, chromosomal DNA, or derivatives and combinations of these groups.
  • RNA examples include, but are not limited to, antisense RNA, siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, viral RNA (vRNA), long non-coding RNA (lncRNA), Piwi -Interacting RNA (piRNA), small nucleolar RNA (snoRNA), tRNA-derived small RNA (tsRNA) or a combination thereof.
  • aiRNA asymmetric interfering RNA
  • miRNA microRNA
  • mRNA microRNA
  • mRNA microRNA
  • mRNA microRNA
  • mRNA mRNA
  • tRNA tRNA
  • rRNA viral RNA
  • vRNA long non-coding RNA
  • piRNA Piwi -Interacting RNA
  • small nucleolar RNA small nucleolar RNA
  • tsRNA tRNA-derived small RNA
  • a specific nucleic acid sequence also inherently includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.
  • degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is mixed with basic and/or deoxygenated codons. Glycoside residues (Batzer et al., Nucleic Acid Res. (Nucleic Acid Research), 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. (Journal of Biological Chemistry), 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)).
  • nucleotide pairing includes the classic Watson-Crick pairing, namely G-C, A-T or A-U pairing.
  • the classic Watson-Crick pairing also covers the situation when one or two nucleotides have been modified (for example, by ribose modification or phosphate backbone modification).
  • complementary sequences may also include non-Watson-Crick base pairs and/or base pairs formed by non-natural and modified nucleotides.
  • treatment refers to the elimination, reduction or amelioration of a disease or condition and/or symptoms associated therewith. Although not excluded, treating the disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated.
  • treatment as used herein may include "prophylactic treatment”, which refers to subjects who do not have a disease, but are at risk or prone to recurrence of the disease or condition, or the disease or condition is at risk of recurrence or is prone to relapse To reduce the likelihood of recurring diseases or conditions, or reduce the likelihood of recurrence of previously controlled diseases or conditions.
  • treatment also includes relapse prevention or stage prevention, as well as treatment of acute or chronic signs, symptoms and/or dysfunctions.
  • Treatment can be symptomatic, for example, to suppress symptoms. It can work for a short period of time, for a medium period, or it can be a long-term treatment, such as in the case of maintenance therapy.
  • subjects refers to any organism to which the lipid composition described herein can be administered for therapeutic purposes.
  • subjects refer to primates (e.g., humans), dogs, rabbits, guinea pigs, pigs, rats, and mice.
  • the subject is a primate. In other embodiments, the subject is a human.
  • This application provides methods and uses of lipid compounds for the delivery of nucleic acids.
  • the present invention is based at least in part on the discovery of the outstanding effects of certain lipid compounds in nucleic acid delivery.
  • the present application provides a variety of lipid compounds, which are found to form a stable nucleic acid lipid mixture with nucleic acid and can deliver nucleic acid to cells, especially cells in the body.
  • the lipid compound provided in the present application can deliver nucleic acid molecules into the body through oral administration, and into the target organs in the body, so as to achieve a good therapeutic effect.
  • different lipid compounds have different effects in delivering nucleic acid into target organs, and show different preferences for different target organs.
  • Certain lipid compounds exhibit significant nucleic acid delivery effects on more than one target organ, and can efficiently deliver nucleic acids to multiple target organs, and therefore have a wide range of applications for nucleic acid delivery. Certain lipid compounds exhibit a particularly significant delivery effect on a certain target organ, and therefore can be used to deliver nucleic acids that act on the target organ.
  • the application provides the use of a lipid composition in the preparation of a reagent for nucleic acid delivery, or a method for the lipid composition to deliver nucleic acid, wherein the lipid composition comprises one or more The compound of formula (I) or its salt, hydrate or solvate:
  • L 1 is not present, -CH 2 -OC(O)-, -CH 2 -O- or -CR(OH)-;
  • L 2 is not present, -OC(O)- or -NH-C(O)-;
  • L 3 is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, linear or branched C 1-20 hetero Alkenyl or
  • A is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1-20 heteroalkene base;
  • B is -OH, linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1- 20 heteroalkenyl;
  • Q is -H, -COOH, hydroxy-substituted linear or branched C 1-20 alkyl, hydroxy-substituted linear or branched C 1-20 alkenyl, hydroxy-substituted C 3-20 cycloalkyl, hydroxyl Substituted C 3-20 cycloalkenyl, -N(R) 3 + or
  • R is each independently H or a linear or branched C 1-20 alkyl group
  • n 0, 1, or 2.
  • L 1 is -CH 2 -OC(O)-, -CH 2 -O- or -CR(OH)-;
  • L 2 is absent, -OC(O)- or -NH- C(O)-;
  • L 3 is A, B, Q, R and n are as defined above.
  • L 1 is absent, -CH 2 -OC(O)- or -CH 2 -O-;
  • L 2 is -OC(O)-;
  • L 3 is linear or branched C 1 -20 alkyl or A, B, Q, R and n are as defined above.
  • L 1 is -CH 2 -OC(O)-; L 2 is absent or -OC(O)-; L 3 is A, B, Q, R and n are as defined above.
  • L 1 is -CH 2 -O-; L 2 is absent or -OC(O)-; L 3 is
  • A, B, Q, R and n are as defined above.
  • L 1 is -CH 2 -OC(O)- or -CH 2 -O-; L 2 is absent; L 3 is A, B, Q, R and n are as defined above.
  • Q is -H, hydroxy substituted linear or branched C 1-20 alkyl, hydroxy substituted linear or branched C 1-20 alkenyl, hydroxy substituted C 3-20 ring Alkyl, hydroxy substituted C 3-20 cycloalkenyl, -N(R) 3 + or
  • L 1 is -CH 2 -OC(O)-;
  • L 2 is -OC(O)-;
  • L 3 is A is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1-20 heteroalkene Group;
  • B is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl or linear or branched C 1-20 Heteroalkenyl;
  • Q is -H, hydroxy-substituted linear or branched C 1-20 alkyl, hydroxy-substituted linear or branched C 1-20 alkenyl, hydroxy-substituted C 3-20 cycloalkyl, Hydroxy substituted C 3-20 cycloalkenyl, -N(R) 3 + or
  • Each R is independently H or a linear or branched C 1
  • L 1 is -CH 2 -OC(O)-;
  • L 2 is -OC(O)-;
  • L 3 is A is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1-20 heteroalkene Group;
  • B is -OH, linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl or linear or branched C 1-20 heteroalkenyl;
  • Q is -H, hydroxy substituted linear or branched C 1-20 alkyl, hydroxy substituted linear or branched C 1-20 alkenyl, hydroxy substituted C 3-20 ring Alkyl, hydroxy substituted C 3-20 cycloalkenyl, -N(R) 3 + or
  • Each R is independently H or a linear or branched C 1-20 alkyl group; and n is 0,
  • L 1 is -CR(OH)-;
  • L 2 is -NH-C(O)-;
  • L 3 is A is linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl, or linear or branched C 1-20 heteroalkene Group;
  • B is -OH, linear or branched C 1-20 alkyl, linear or branched C 1-20 alkenyl, linear or branched C 1-20 heteroalkyl or linear or branched C 1-20 heteroalkenyl;
  • Q is -H, hydroxy substituted linear or branched C 1-20 alkyl, hydroxy substituted linear or branched C 1-20 alkenyl, hydroxy substituted C 3-20 ring Alkyl, hydroxy substituted C 3-20 cycloalkenyl, -N(R) 3 + or
  • the compound has a structure of the following formula:
  • L 1 , L 2 , L 3 , A, B and Q are as described above.
  • A is a linear or branched C 1-20 alkyl group, or a linear or branched C 1-20 alkenyl group. In certain embodiments, A is a linear or branched C 5-20 alkyl group, or a linear or branched C 5-20 alkenyl group. In certain embodiments, A is a linear or branched C 5-20 alkyl group. In certain embodiments, A is a linear C 5-20 alkenyl group with 1-5 double bonds.
  • B is -OH, linear or branched C 1-20 alkyl, or linear or branched C 1-20 alkenyl. In certain embodiments, B is -OH, linear or branched C 5-20 alkyl, or linear or branched C 5-20 alkenyl. In certain embodiments, B is -OH. In certain embodiments, B is a linear or branched C 5-20 alkyl group.
  • B is a linear C 5-20 alkyl group. In certain embodiments, B is a linear or branched C 5-20 alkenyl group. In certain embodiments, B is a linear C 5-20 alkenyl group with 1-5 double bonds.
  • the double bond is in the Z configuration.
  • the position of the double bond in the linear C 5-20 alkenyl group is selected from the following: C1-C2 position, C2-C3 position, C3-C4 position, C4-C5 position, C5 -C6 position, C6-C7 position, C7-C8 position, C8-C9 position, C9-C10 position, C10-C11 position, C12-C13 position, C13-C14 position or a combination thereof.
  • Q is -H, -COOH, hydroxy-substituted linear or branched C 1-20 alkyl, hydroxy-substituted C 3-20 cycloalkyl, -N(R) 3 + or
  • Q is a linear or branched C 1-20 alkyl group substituted with a hydroxyl group.
  • Q is a linear or branched C 1-10 alkyl group substituted with a hydroxyl group.
  • Q is a linear C 1-5 alkyl substituted with a hydroxyl group.
  • Q is -CH(OH)-CH 2 OH.
  • Q is a hydroxy substituted C 3-20 cycloalkyl.
  • Q is a hydroxy substituted C 3-10 cycloalkyl group.
  • Q is
  • Q is -N(R) 3 +
  • R is each independently H or linear C 1-10 alkyl.
  • Q is -N(CH 3 ) 3 + .
  • Q is Each R is independently H or a linear C 1-10 alkyl group.
  • Q is R is H.
  • the compound has a structure of the following formula:
  • L 3 is a linear or branched C 1-20 alkyl group
  • A is a linear or branched C 1-20 alkyl group
  • B is a linear or branched C 1-20 alkyl group.
  • the compound contains a structure of the formula
  • A is a linear or branched C 1-20 alkyl group.
  • the compound has a structure of the following formula:
  • A is a linear or branched C 1-20 alkyl group
  • Q is -N(R) 3 +
  • R is each independently a linear or branched C 1-20 alkyl group.
  • the compound contains a structure of the following formula:
  • A is linear or branched C 1-20 alkyl
  • B is linear or branched C 1-20 alkyl, or linear or branched C 1-20 alkenyl
  • Q is -N(R) 3 +
  • R are each independently a linear or branched C 1-20 alkyl group.
  • A is a linear or branched C 1-20 alkyl group, or a linear or branched C 1-20 alkenyl group
  • B is a linear or branched C 1-20 alkenyl group
  • Q is a straight chain substituted by a hydroxyl group Or branched C 1-20 alkenyl, hydroxy substituted C 3-20 cycloalkyl, or
  • R are each independently a linear or branched C 1-20 alkyl group.
  • the compound has a structure of the following formula:
  • A is linear or branched C 1-20 alkenyl
  • B is linear or branched C 1-20 alkyl, or linear or branched C 1-20 alkenyl
  • Q is -N(R ) 3 +
  • R is each independently a linear or branched C 1-20 alkyl group.
  • the lipid composition comprises one or more compounds having the structure of formula (I) or salts, hydrates or solvates thereof, and the compounds are selected from the group consisting of lipids 106, 96 , 93, 94, 84, 85, 108, 81, 88, 89, 109, 110, 103, 104 and 105, wherein the chemical name and chemical structure of the lipid are shown in Table 1.
  • the application also provides the use of the lipid composition in the preparation of a reagent for nucleic acid delivery, or a method for the lipid composition to deliver nucleic acid, wherein the lipid composition comprises one or more A compound or a salt, hydrate or solvate thereof, the compound is selected from the group consisting of lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 86, 87, 90, 91, 92, 95, 97, 98, 99, 100, 101, 102, 107, 111, 112, 113, 114, 115, 116, 117, 118 and 119, wherein the chemical name and chemical structure of the lipid As shown in Table 1.
  • the compound is synthetic. According to the chemical structure of the compound, those skilled in the art can synthesize the above-mentioned compound by means of chemical synthesis. Alternatively, some of the compounds described in this application can also be obtained commercially.
  • the compound is derived from an extract of traditional Chinese medicine.
  • "Traditional Chinese medicine extract” in this application refers to an extract obtained from Chinese medicinal materials or medicinal plants by appropriate methods.
  • the Chinese medicinal material or medicinal plant is selected from Rhodiola rosea, dandelion, honeysuckle or andrographis, or Chinese herbal medicine pieces. Any suitable extraction method can be used to obtain the Chinese medicine extract.
  • the herbal medicine pieces of Rhodiola rosea, dandelion, honeysuckle or Andrographis paniculata can be soaked in water, and then decocted in a strong fire and a low fire in turn, the decoction of the traditional Chinese medicine liquid is concentrated, and then chloroform-methanol is added in turn , Chloroform and water are fully mixed, and the chloroform layer is taken to obtain the traditional Chinese medicine extract.
  • the compound monomer can be further separated or purified from the traditional Chinese medicine extract.
  • the traditional Chinese medicine extract is prepared by the Bligh&Dyer method (Bligh EG and Dyer, WJ, A rapid method for total lipid extraction and purification, Can. J. Biochem. Physiol., 1959, 37: 911-917) It can be obtained by extracting fat-soluble components, or by preparing and extracting traditional Chinese medicine by decoction.
  • the traditional Chinese medicine extract is prepared and extracted by decoction of traditional Chinese medicine selected from rhodiola, dandelion, andrographis and honeysuckle Chinese herbal medicine pieces.
  • the traditional Chinese medicine extract is obtained by soaking the traditional Chinese medicine in water, then sequentially performing high-fire and low-fire decoction, concentrating the decoction of the traditional Chinese medicine liquid, and then adding chloroform- Methanol, chloroform and water are stirred and processed, and the chloroform layer is extracted.
  • the lipid composition provided in the application comprises one or more of the compounds provided in the application or a salt, hydrate or solvate thereof.
  • the lipid composition may be one compound provided in the present application or a salt, hydrate or solvate thereof, or a mixture of two or more compounds provided in the present application.
  • the lipid composition may also include one or more lipid compounds other than the compounds provided in the application.
  • the other lipid compounds may be, for example, neutral lipids, charged lipids, steroids, and polymer-conjugated lipids.
  • Neutral lipid refers to a lipid compound that exists in an uncharged form or a neutral zwitterionic form at a selected pH value (e.g., physiological pH value).
  • a “charged lipid” refers to a lipid compound that is not limited by a pH value (for example, a pH of about 3 to about 9) in a useful physiological range, and exists in a positively or negatively charged form.
  • the lipid composition may further include one or more solvents, which can be mixed with the compounds provided in the present application or their salts, hydrates or solvates to form a homogeneous mixture.
  • the solvent contained in the lipid composition may include an organic solvent or solvent mixture, such as chloroform, dichloromethane, diethyl ether, cyclohexane, cyclopentane, benzene, toluene, methanol or other aliphatic alcohols, such as ethanol, Propanol, isopropanol, butanol, tert-butanol, isobutanol, pentanol and hexanol.
  • organic solvent or solvent mixture such as chloroform, dichloromethane, diethyl ether, cyclohexane, cyclopentane, benzene, toluene, methanol or other aliphatic alcohols, such as ethanol, Propanol, isopropanol, butanol, tert-butanol, isobutanol, pentanol and hexanol.
  • solvents can be used alone, in admixture
  • the choice of solvent can usually take into account the polarity of the solvent, the ease of removing the solvent in the later stage of the formation of the lipid nucleic acid mixture, and/or the pharmaceutically acceptable properties.
  • the solvent is non-toxic or pharmaceutically acceptable.
  • Exemplary pharmaceutically acceptable solvents include lower alcohols (1-6 carbon atoms), such as methanol, ethanol, n-propanol, isopropanol, and n-butanol.
  • an appropriate amount of solvent may be used to enable nucleic acid and lipid to form a clear single-phase mixture.
  • the nucleic acid comprises DNA or RNA.
  • the nucleic acid may include, for example, coding DNA, non-coding DNA, antisense nucleic acid, messenger RNA (mRNA), long non-coding RNA (lncRNA), and small RNA (such as microRNA (miRNA), Small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), tRNA-derived small RNA (tsRNA)), etc.
  • mRNA messenger RNA
  • lncRNA long non-coding RNA
  • small RNA such as microRNA (miRNA), Small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), tRNA-derived small RNA (tsRNA)
  • the nucleic acid is single-stranded or double-stranded.
  • Single-stranded nucleic acids for example, miRNA, mRNA, antisense DNA, antisense RNA, etc.
  • Double-stranded nucleic acid for example, siRNA, double-stranded DNA, double-stranded RNA, etc.
  • the nucleic acid has a stem-loop structure.
  • the stem-loop structure refers to the presence of reverse-complementary sequences in the two parts of a single-stranded nucleic acid. When base pairing is performed, this part of the reverse-complementary sequence forms a double-strand, and the middle of the two reverse-complementary regions The non-complementary part protrudes to form a ring structure.
  • the stem-loop structure is also called the hairpin structure.
  • the nucleic acid includes a small nucleic acid.
  • a small nucleic acid refers to a nucleic acid of relatively short length (for example, less than 200 nucleotides). Small nucleic acids may be non-coding, such as small RNAs (e.g. miRNA, siRNA, piRNA, snoRNA, tsRNA) and the like. It can be single-stranded or double-stranded.
  • the nucleic acid is a small nucleic acid with a length of 14-32 bp, 16-28 bp, or 18-24 bp.
  • the nucleic acid is a nucleic acid drug.
  • Nucleic acid drugs can be of various types, such as antisense nucleic acids, siRNA, CpG oligodeoxynucleotides, nucleic acid aptamers, mRNA or DNA encoding target proteins, or miRNAs.
  • nucleic acid drugs include, AEG35156, aganirsen, AP 12009, Apatorsen, ATL1103, AVT-02 UE, Bevasiranib Sodium, BMN 044, BMN 053, CpG 7909, Custirsen, Drisapersen, Eteplirsen, Fomivirotplirsen, Defibresen, Pegaptanib, Mipomer , Nusinersen, Patisiran, Tegsedi and Fovista.
  • the nucleic acid can be used to treat diseases.
  • the nucleic acid is used to treat cancer, inflammation, fibrotic diseases, autoimmune diseases or autoinflammatory diseases, bacterial infections, behavioral and mental disorders, blood diseases, chromosomal diseases, congenital and genetic sexual diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, environmental diseases, eye diseases, female reproductive diseases, fungal infections, heart diseases, hereditary cancer syndromes, immune system diseases, kidney and urinary diseases Sexual diseases, lung diseases, new male reproductive diseases, metabolic disorders, oral diseases, musculoskeletal diseases, myelodysplastic syndromes, newborn screening, nutritional diseases, parasitic diseases, rare cancers, rare diseases, Skin diseases and viral infections.
  • the nucleic acid is used to treat hepatocellular carcinoma, corneal neovascularization, relapsed or refractory anaplastic astrocytoma (WHO grade III), or secondary glioblastoma (WHO Grade IV), advanced squamous cell lung cancer, acromegaly, psoriasis, Duchenne muscular dystrophy, advanced non-small cell lung cancer, metastatic castration-resistant prostate cancer, cytomegalovirus retinitis, HIV infection, Hepatitis B, hepatitis C, hyperlipoproteinemia, total knee arthroplasty, type II diabetes, familial amyloid polyneuropathy (FAP), wet macular degeneration (e.g.
  • neovascular age-related macular degeneration fovea Lower neovascular age-related macular degeneration, exudative age-related macular degeneration
  • hypercholesterolemia Crohn's disease, extensive liver fibrosis, infantile spinal muscular atrophy, melanoma
  • new coronary artery Diseases mild allergic asthma, chronic lymphocytic leukemia, and hypertriglyceridemia, small hepatic obliterans with renal or lung dysfunction after hematopoietic stem cell transplantation, and hereditary transthyretin amyloid lesions.
  • the lipid composition can form a lipid nucleic acid mixture with nucleic acid.
  • the nucleic acid can be delivered through the lipid nucleic acid mixture.
  • a "lipid nucleic acid mixture" in the present application refers to a lipid-based mixture that delivers nucleic acid, such as liposomes. Examples of liposomes include, for example, lipid particles or lipid vesicles encapsulating nucleic acid.
  • the lipid nucleic acid mixture can be prepared by an appropriate method, including, for example, a mixing method, a heating method, a reverse evaporation method, and the like.
  • the mixing method includes the step of mixing the compound or its salt, hydrate or solvate provided in the present application with the nucleic acid to be delivered.
  • the mixing may be direct mixing of the lipid composition provided in the present application and the nucleic acid to be delivered (for example, mixing the lipid composition and the dry powder of nucleic acid, and then adding an appropriate solvent to form a lipid-encapsulated nucleic acid. Lipid nucleic acid mixture).
  • the mixing may be mixing the lipid composition described in this application dissolved in a suitable solvent with nucleic acid, or combining nucleic acid dissolved in a suitable solvent with the lipid composition described in this application Or mix the lipid composition described in the present application dissolved in a suitable solvent with nucleic acid dissolved in a suitable solvent.
  • suitable solvents that can be used in the lipid composition described in this application are as described earlier in the specification of this application.
  • Suitable solvents that can be used for nucleic acids include water (DEPC-treated water, double-distilled water), buffer, physiological saline, or glucose solution.
  • the mixing step can be carried out in any suitable step.
  • the nucleic acid (or its solution) can be added to the lipid composition (or its solution), or the lipid composition (or its solution) can be added to the nucleic acid ( Or its solution).
  • the mixing may also include steps such as vortexing, sonication, etc., to help uniform mixing.
  • the mixing method includes adding the ethanol solution of the lipid composition described in the present application to an aqueous buffer of a suitable volume of the nucleic acid to be delivered, mixing by vortexing or ultrasound, and incubating to obtain the lipid nucleic acid. mixture.
  • the mixing method includes combining the ethanol solution of the lipid compound described in the present application with an ethanol solution of an appropriate volume of the nucleic acid to be delivered, mixing by vortex or ultrasound, and incubating to obtain the lipid nucleic acid mixture and then removing Ethanol, and resuspend the resulting lipid nucleic acid mixture in an aqueous buffer.
  • the nucleic acid and the lipid compound may be mixed in a certain ratio.
  • the ratio depends on the nucleic acid to be delivered and its amount, as well as the lipid compound that needs to be used to achieve the effect of nucleic acid encapsulation.
  • the ratio can be any ratio that meets the needs of treatment, as long as a stable lipid nucleic acid mixture can be formed and the required amount of nucleic acid can be provided.
  • the ratio of nucleic acid to lipid compound may be, for example, 0.1 nmol: 100 ⁇ g to 10 nmol: 100 ⁇ g, 0.2 nmol: 100 ⁇ g to 10 nmol: 100 ⁇ g, 0.3 nmol: 100 ⁇ g to 10 nmol: 100 ⁇ g, 0.4 nmol: 100 ⁇ g To 10nmol: 100 ⁇ g, 0.5nmol: 100 ⁇ g to 10nmol: 100 ⁇ g, 1nmol: 100 ⁇ g to 10nmol: 100 ⁇ g, 2nmol: 100 ⁇ g to 10nmol: 100 ⁇ g, 3nmol: 100 ⁇ g to 10nmol: 100 ⁇ g, 4nmol: 100 ⁇ g to 10nmol: 100 ⁇ g, 5nmol: 100 ⁇ g to 10nmol: 100 ⁇ g, 6nmol: 100 ⁇ g to 10nmol, 7nmol: 100 ⁇ g to 10nmol, 8nmol: 100 ⁇ g to 10nmol, or 9nmol: 100 ⁇ g to 10nmol: 100 ⁇ g.
  • the ratio of nucleic acid to lipid compound may be 1nmol: 100 ⁇ g, 2nmol: 100 ⁇ g, 3nmol: 100 ⁇ g, 4nmol: 100 ⁇ g, 5nmol: 100 ⁇ g, 6nmol: 100 ⁇ g, 7nmol: 100 ⁇ g, 8nmol: 100 ⁇ g, 9nmol: 100 ⁇ g or 10nmol: 100 ⁇ g. In some embodiments, the ratio of nucleic acid to lipid compound may be 10 nmol: 100 ⁇ g.
  • the mixing method can be performed at any appropriate temperature, as long as a lipid-nucleic acid mixture capable of delivering nucleic acid can be formed.
  • the nucleic acid and the lipid compound can be mixed at an appropriate temperature, for example, but not limited to, 0°C to 100°C, 4°C to 100°C, 10°C to 100°C, 15°C to 100°C , 20°C to 100°C, 25°C to 100°C, 30°C to 100°C, 35°C to 100°C, 40°C to 100°C, 45°C to 100°C, 50°C to 100°C, 55°C to 100°C, 60 °C to 100 °C, 65 °C to 100 °C, 70 °C to 100 °C, 75 °C to 100 °C, 80 °C to 100 °C, 85 °C to 100 °C, 90 °C to 100 °C, or 95 °C to 100 °C.
  • the temperature is 0°C, 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C °C, 90°C. In some embodiments, the temperature is 90°C.
  • the lipid nucleic acid mixture is prepared by a heating method.
  • the heating method includes mixing a solution of the compound or its salt, hydrate or solvate provided in the present application in an appropriate solvent with an aqueous solution of nucleic acid to be delivered to obtain a mixed solution, and heating the mixed solution at an appropriate temperature A step of.
  • the step of heating the mixed solution is performed at a temperature selected from 25°C to 100°C, 30°C to 100°C, 40°C to 100°C, 50°C to 100°C, 60°C to 60°C 100°C, 70°C to 100°C, 80°C to 100°C, 90°C to 100°C, or 95°C to 100°C.
  • the step of heating the mixed solution is performed at a temperature selected from about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, and about 55°C. , About 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, and about 100°C. In some embodiments, the step of heating the mixed solution is performed at 90°C.
  • the heating method includes heating the mixed solution for an appropriate time.
  • Those skilled in the art can select an appropriate heating time according to the properties of the lipid compound and nucleic acid used and the desired lipid nucleic acid mixture.
  • the heating time may be, for example, about 5 minutes to about 24 hours, about 5 minutes to about 20 hours, about 5 minutes to about 16 hours, about 10 minutes to about 20 hours, about 10 minutes to about 16 hours, about 15 minutes Minutes to about 24 hours, about 15 minutes to about 20 hours, about 30 minutes to about 24 hours, about 30 minutes to about 20 hours, about 40 minutes to about 16 hours, about 50 minutes to about 12 hours, about 1 hour to About 8 hours, or about 2 hours to about 4 hours.
  • the heating time may be, for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours. , About 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 16 hours, about 20 hours, or about 24 hours. In some embodiments, the heating time is about 15 minutes.
  • the heating method further includes a cooling step.
  • the cooling may be performed at an appropriate temperature after the step of heating the mixed solution, as long as the formed lipid nucleic acid mixture is not destroyed.
  • Exemplary cooling temperatures include, but are not limited to, 25°C to -80°C, 20°C to -80°C, 15°C to -80°C, 10°C to -80°C, 4°C to -80°C, 0°C to- 80°C, -10°C to -80°C, -20°C to -80°C, -30°C to -80°C, -40°C to -80°C.
  • the mixed solution is naturally cooled at room temperature after the step of heating the mixed solution, and the room temperature is, for example, 25°C or 20°C.
  • the lipid nucleic acid mixture is prepared by a reverse evaporation method.
  • the reverse evaporation method includes adding an aqueous solution of nucleic acid to a solution of a lipid compound dissolved in a suitable solvent, and then removing the solvent by methods such as ultrasound and evaporation.
  • the reverse evaporation method further includes hydration after removing the solvent to obtain a lipid nucleic acid mixture.
  • the hydration includes adding water or a suitable medium to the system.
  • the suitable medium is, for example, OPTI-MEM medium.
  • the step of removing the solvent is performed at a suitable temperature, for example, about 25°C to about 70°C, 30°C to about 70°C, about 30°C to about 65°C, about 40°C to about 65°C , About 40°C to about 60°C, or about 50°C to about 60°C.
  • the step of removing the solvent is at about 25°C, 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C. Or at a temperature of about 70°C.
  • the step of removing the solvent is performed at a temperature of about 55°C.
  • the nucleic acid delivery is delivered by oral, inhalation or injection. In some embodiments, the nucleic acid delivery is oral. In some embodiments, the agent is used to deliver the nucleic acid orally. In certain embodiments, the nucleic acid delivery is in vivo digestive tract delivery.
  • the nucleic acid can be delivered to the target organ or target tissue of interest of the individual.
  • the nucleic acid can be delivered to the heart tissue of the individual.
  • the lipid nucleic acid mixture contains lipid 73 and/or lipid 80.
  • the nucleic acid can be delivered to the lung tissue of the individual.
  • the lipid nucleic acid mixture contains one or more lipid compounds selected from the group consisting of lipid 72, lipid 76, lipid 92, lipid 94, lipid 95, lipid 97, lipid 98 and lipid 103.
  • the nucleic acid can be delivered to the kidney tissue of the individual.
  • the lipid nucleic acid mixture contains one or more lipid compounds selected from the group consisting of lipid 88, lipid 91, lipid 93, lipid 94, lipid 96, lipid 97 and lipid 98.
  • the nucleic acid is delivered to the liver. In some embodiments, after the lipid nucleic acid mixture is taken orally by the individual, the nucleic acid can be delivered to the spleen tissue of the individual. In some embodiments, the lipid nucleic acid mixture contains one or more lipid compounds selected from the group consisting of lipid 76, lipid 77, lipid 79, lipid 80, lipid 81, lipid 95, lipid 96, lipid 98, lipid 99, lipid 100, lipid 101 and lipid 102.
  • the nucleic acid can be delivered to the blood of the individual.
  • the lipid nucleic acid mixture contains one or more lipid compounds selected from the group consisting of lipid 75, lipid 102, lipid 104, and lipid 105.
  • the nucleic acid can be delivered to the intestinal tissue of the individual.
  • the lipid nucleic acid mixture contains the lipid compound Lipid 83.
  • the nucleic acid is delivered to two or more tissues.
  • the lipid nucleic acid mixture contains a lipid compound selected from the group consisting of lipid 76, lipid 80, lipid 94, lipid 95, lipid 96, lipid 97, lipid 98 and Lipid 102.
  • the nucleic acid delivery includes in vitro cell delivery.
  • “In vitro” in this application means outside of a multicellular organism, such as outside the human or animal body.
  • In vitro cells include, for example, in vitro cell cultures, tissues or cells in vitro, and the like.
  • In vitro delivery includes the delivery of nucleic acids into cells outside the body. For example, it includes contacting the lipid nucleic acid mixture with cells in vitro under conditions that allow nucleic acid to enter the cells.
  • the application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid composition and a nucleic acid
  • the lipid composition comprises one or more compounds of formula (I) or pharmaceutically acceptable compounds thereof Accepted salt, hydrate or solvate.
  • the compound is selected from the group consisting of lipids 106, 96, 93, 94, 84, 85, 108, 81, 88, 89, 109, 110, 103, 104, and 105.
  • the application also provides a pharmaceutical composition comprising a lipid composition and a nucleic acid, wherein the lipid composition comprises one or more selected from lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 86, 87, 90, 91, 92, 95, 97, 98, 99, 100, 101, 102, 107, 111, 112, 113, 114, 115, A compound of the group consisting of 116, 117, 118, and 119, or a salt, hydrate or solvate thereof.
  • the pharmaceutical composition provided in this application can be in solid or liquid form, including semi-solid, semi-liquid, suspension and gel forms.
  • Solid form for example, tablet or powder form.
  • Liquid forms for example, oral solutions, oral syrups, injectable liquids, or aerosols, which are suitable for, for example, inhalation administration.
  • the pharmaceutical composition is formulated for oral administration, inhalation, digestive tract, or respiratory tract administration. In certain embodiments, the pharmaceutical composition is formulated to deliver the nucleic acid through the digestive tract in vivo. In certain embodiments, the pharmaceutical composition is an oral pharmaceutical composition. When used for oral administration, the pharmaceutical composition is preferably in solid or liquid form.
  • the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewing gums and the like.
  • Such solid compositions may also contain one or more inert diluents or edible carriers (such as starch, lactose or dextrin), and one or more excipients selected from the following group: binders (such as carboxyl Methyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum or gelatin), disintegrants (alginic acid, sodium alginate, Primogel, corn starch, etc.), lubricants (such as magnesium stearate or Sterotex ), glidants (such as colloidal silicon dioxide), sweeteners (such as sucrose or saccharin), flavoring agents; and coloring agents.
  • binders such as carboxyl Methyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum or gelatin
  • disintegrants alginic acid, sodium
  • the pharmaceutical composition can be formulated as, for example, an elixir, syrup, solution, emulsion, or suspension.
  • the liquid pharmaceutical composition contains one or more excipients selected from the group consisting of sweeteners, preservatives, dyeing/coloring agents and flavor enhancers.
  • the pharmaceutical composition of the present invention can also be configured to be administered by inhalation through the respiratory tract.
  • Suitable inhalation formulations may include formulations that can be administered as an aerosol. Aerosols can be delivered in single-phase, two-phase systems, or three-phase systems. For example, aerosols can be delivered by liquefied gas and compressed gas, or by a suitable system for dispersing active ingredients.
  • the delivery of aerosol includes necessary containers, activators, valves, sub-containers, etc., which together can form a kit.
  • the pharmaceutical composition of the present application can also be configured for parenteral administration, such as injection administration.
  • the liquid composition for injection may be a solution, suspension or injection powder, and may include one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents. kind.
  • the pharmaceutical composition can also be used for delivery by cells in vitro.
  • the lipid composition and the nucleic acid are present in the pharmaceutical composition in the form of a mixture at least partially or completely.
  • the mixture is prepared by heating method, reverse evaporation method, or mixing method.
  • the application also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of diseases that can be prevented and/or treated with nucleic acid, or for the delivery of nucleic acid to in vivo Among the objects that need this.
  • the pharmaceutical composition provided in this application can be used to treat diseases that can be treated with nucleic acid.
  • diseases such as, but not limited to, cancer, inflammation, fibrotic diseases, autoimmune diseases or autoinflammatory diseases, bacterial infections, behavioral and mental disorders, blood diseases, chromosomal diseases, congenital and genetic diseases, connective Tissue disease, digestive disease, ear, nose and throat disease, endocrine disease, environmental disease, eye disease, female reproductive disease, fungal infection, heart disease, hereditary cancer syndrome, immune system disease, kidney and urinary disease, lung Ministry diseases, new male reproductive diseases, metabolic disorders, oral diseases, musculoskeletal diseases, myelodysplastic syndromes, newborn screening, nutritional diseases, parasitic diseases, rare cancers, rare diseases, skin diseases and viruses infection.
  • cancers include, but are not limited to, stomach cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, cervical cancer, breast cancer, leukemia, multiple myeloma.
  • inflammation examples include, but are not limited to, pneumonia, myocarditis, acute and chronic gastritis, acute and chronic enteritis, acute and chronic hepatitis, acute and chronic nephritis, dermatitis, encephalitis, lymphitis, conjunctivitis, keratitis, iridocyclitis, otitis media, Allergic rhinitis, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease, allergic dermatitis, sickle cell disease, multiple sclerosis, systemic lupus erythematosus, lupus nephritis.
  • nucleic acid drugs can treat many diseases.
  • the indications for known nucleic acid drugs include, for example, hepatocellular carcinoma, corneal neovascularization, relapsed or refractory anaplastic astrocytoma (WHO grade III) or secondary glioblastoma (WHO grade IV) ), advanced squamous cell lung cancer, acromegaly, psoriasis, Duchenne muscular dystrophy, advanced non-small cell lung cancer, metastatic castration-resistant prostate cancer, cytomegalovirus retinitis, HIV infection, type B Hepatitis, hepatitis C, hyperlipoproteinemia, total knee arthroplasty, type II diabetes, familial amyloid polyneuropathy (FAP), wet macular degeneration (e.g.
  • neovascular age-related macular degeneration subfoveal neoplasia
  • Vascular age-related macular degeneration exudative age-related macular degeneration
  • hypercholesterolemia Crohn’s disease
  • extensive liver fibrosis infantile spinal muscular atrophy
  • melanoma new coronary artery disease
  • Mild allergic asthma chronic lymphocytic leukemia
  • hypertriglyceridemia small hepatic obliterans with renal or pulmonary dysfunction after hematopoietic stem cell transplantation
  • hereditary transthyretin amyloidosis hereditary transthyretin amyloidosis.
  • the application also provides a kit comprising: one or more compounds of formula (I) or pharmaceutically acceptable salts or hydrates thereof placed in a first container Or a solvate, and a nucleic acid placed in a second container.
  • the compound is selected from the group consisting of lipids 106, 96, 93, 94, 84, 85, 108, 81, 88, 89, 109, 110, 103, 104, and 105.
  • the present application also provides a kit comprising: one or more compounds or a pharmaceutically acceptable salt, hydrate or solvate thereof placed in a first container, and placed in the first container The nucleic acid in the second container, wherein the one or more compounds are selected from lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, 82, 83, 86, 87, 90, 91, 92 , 95, 97, 98, 99, 100, 101, 102, 107, 111, 112, 113, 114, 115, 116, 117, 118, and 119.
  • the lipid composition and the nucleic acid are at least partially or fully formulated as a lipid nucleic acid mixture before use.
  • the mixture is prepared by a heating method, a reverse evaporation method, or a mixing method.
  • kit is formulated as an oral pharmaceutical composition.
  • kits wherein the kit is formulated for oral administration, inhalation, gastrointestinal tract, or respiratory tract administration.
  • the kit is formulated to deliver the nucleic acid via cell delivery in vitro or via the digestive tract in vivo.
  • the present application provides the use of the kit in the preparation of medicines for the prevention and/or treatment of diseases that can be prevented and/or treated with nucleic acids, or for the delivery of nucleic acids in vivo to those in need In the object.
  • the present application provides a method for delivering nucleic acid to target cells, comprising administering to the target cell the pharmaceutical composition described in this application, or the lipid formulated in the kit described in this application Nucleic acid mixture.
  • the present application provides a method for delivering nucleic acid in vivo to a subject in need thereof, comprising administering to the subject the pharmaceutical composition described in this application, or the kit described in this application is formulated The lipid nucleic acid mixture.
  • the subject is a human or animal, such as a mammal.
  • the nucleic acid is delivered in vivo to the blood circulation or target tissue/cell of the subject.
  • the method includes administering the drug by oral, inhalation, or injection.
  • the method includes administering the drug through the digestive tract or through the respiratory tract.
  • the method includes oral administration of the drug.
  • the efficient targeted delivery of nucleic acids can be significantly improved, which overcomes the low encapsulation rate, poor safety, poor stability, complex preparation process, and uneven product of nucleic acid liposomes in the prior art. Defects that are difficult to reproduce and need to be further improved in targeting.
  • Example 1 Extraction, identification and synthesis of lipids derived from traditional Chinese medicine
  • a) Positive mode heater temperature 300°C, sheath gas flow rate, 45arb, auxiliary gas flow rate, 15arb, purge gas flow rate, 1arb, nozzle voltage, 3.0KV, capillary temperature, 350°C, S-Lens RF Level, 30% .
  • Negative mode heater temperature 300°C, sheath gas flow rate, 45arb, auxiliary gas flow rate, 15arb, purge gas flow rate, 1arb, nozzle voltage, 2.5KV, capillary temperature, 350°C, S-Lens RF Level, 60% .
  • each lipid except lipids 106, 108, and 110 was purchased.
  • the concentration of the lipid chloroform solution was 10 mg/ml (stock solution), and the lipid compounds were grouped according to the number of the lipid compound.
  • Example 3 In vivo delivery study of lipid nucleic acid mixture
  • mice used in the experiment were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., about 6 weeks old, 20-24 g. It is housed in the SPF animal room of the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The mice were fasted for 12h before gavage.
  • the lipid nucleic acid mixture was prepared according to the method of Example 2.
  • the prepared lipid nucleic acid mixture was orally administered at a dose of 100 ⁇ g lipid per mouse: 1 nmol single-stranded small RNA PGY-sRNA-26.
  • RNA After intragastric administration of each group of mice for 12 hours, 500 ⁇ l of blood was taken from the eyeballs, and 1.5ml Trizol Reagent LS (purchased from Invitrogen) was added to mix and lyse thoroughly. Add 3ml Trizol Reagent LS to the tissue sample, homogenize it to fully lyse, and sample the tissue: lung/kidney/spleen/heart/intestine. Take 1ml of blood or tissue lysate to extract RNA according to the following steps:
  • RNA sample with 50-100 ⁇ L RNase-free H 2 O, and measure the OD value to quantify the RNA concentration.
  • stem-loop method By reverse transcription kits (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat. no. 4368813), the stem-loop method (stem-loop method) (see, for example, Real-time quantification of microRNAs by stem-loop RT-PCR) , Nucleic Acids Res.
  • the reverse transcription system is as follows: template RNA (200ng/ ⁇ L) 10 ⁇ L, 10X RT buffer 2.0 ⁇ L, 25X dNTP Mix (100mM) 0.8 ⁇ L, U6RT stem-loop primer 2.0 ⁇ L, PGY-sRNA-26RT stem-loop primer 2.0 ⁇ L, MultiScribe TM reverse transcriptase 1.0 ⁇ L, RNase inhibitor 1.0 ⁇ L, nuclease-free H 2 O 1.2 ⁇ L, after instant centrifugation, put it into the PCR machine for reaction, the reaction conditions are as follows: (1) 25°C, 10min; (2) 37°C, 120min; (3) 85°C, 5min; (4) 4°C, stop the reaction.
  • the total volume of qPCR reaction system is 10 ⁇ l, including: 5 ⁇ L 2 ⁇ SYBR Green Master Mix, 0.5 ⁇ l forward primer (10 ⁇ M), 0.5 ⁇ l reverse primer (10 ⁇ M), 1 ⁇ l cDNA obtained by reverse transcription, 3 ⁇ l RNase-free ddH2O.
  • the PCR reaction conditions are: 95°C, pre-denaturation for 5 minutes, and start to enter the PCR amplification cycle: (1) 95°C, 10s; (2) 55°C, 10s; (3) 72°C, 20s; a total of 40 cycles are carried out; finally, the temperature is lowered at 40°C for 10s.
  • the forward and reverse primers of the amplification reaction were designed and synthesized by Beijing Jinke Xinye Biotechnology Co., Ltd.
  • U6F primer GCGCGTCGTGAAGCGTTC (SEQ ID No. 6)
  • U6R primer GTGCAGGGTCCGAGGT (SEQ ID No. 7)
  • PGY -sRNA-26 forward primer TCGCGCTCCGGAATGATTGGG (SEQ ID No. 3)
  • reverse primer miR all rev GTGCACGCTCCGAGGT (SEQ ID No. 4)).
  • Figures 1-45 respectively show the results of the blank group, the free uptake group and the tested different lipid-delivered single-stranded small RNA PGY-sRNA-26 into different target tissues. All the data in the figure are calculated according to the two-tailed t test. The statistical test results are marked with asterisks in the figure, * means P ⁇ 0.05, ** means P ⁇ 0.01, the more asterisks, the stronger the significance. Part of the data due to large individual differences, although a clear trend can be seen, there is no significant difference after calculation.
  • mice used in the experiment were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., about 6 weeks old, 20-24 g. It is housed in the SPF animal room of the Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The mice were fasted for 12h before gavage.
  • the lipid nucleic acid mixture was prepared according to the method of Example 2.
  • the prepared lipid nucleic acid mixture is administered orally or by tail vein injection at a dose of 100 ⁇ g lipid per mouse: 1 nmol single-stranded small RNA PGY-sRNA-26.
  • the oral dose is half of tail vein injection.
  • mice were taken at each time point at 3 hours, 6 hours, 9 hours, 12 hours, and 24 hours after administration, and each was extracted according to the method of Example 3.
  • Organ RNA Organ RNA
  • RNA extracted from each organ was reverse transcribed into cDNA.
  • Figures 46-49 respectively show the results of lipid 93, 94, 96, 100 delivering single-stranded small RNA PGY-sRNA-26 into different target tissues at different time points by intragastric administration;
  • Figures 50-54 respectively show The results of lipids 94, 96, 98, 100, 101 delivered through tail vein injection to deliver single-stranded small RNA PGY-sRNA-26 into different target tissues at different time points.
  • Lipids 72, 73, 74, 75, 76, 77, 78, 79, 80, and 81 can effectively orally deliver sRNA single-stranded nucleic acid into mouse blood and lung, kidney, spleen, and heart tissues.
  • Lipids 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 109, 111, 112, 113, 114, 115, 116, 117, 118, and 119 can effectively orally deliver sRNA single-stranded nucleic acid into the blood and lung, kidney, spleen, heart, and intestine tissues of mice.
  • Lipid 72 can effectively orally deliver sRNA single-stranded nucleic acid into mouse lung tissue.
  • Lipid 73 can effectively orally deliver sRNA single-stranded nucleic acid into mouse heart tissue.
  • Lipid 75 can effectively orally deliver sRNA single-stranded nucleic acid into the blood of mice.
  • Lipid 76 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse lung tissue and spleen tissue.
  • Lipid 77 can effectively orally deliver sRNA single-stranded nucleic acid into mouse spleen tissue.
  • Lipid 79 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse spleen tissue.
  • Lipid 80 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse spleen and heart tissues.
  • Lipid 81 can effectively orally deliver sRNA single-stranded nucleic acid into mouse spleen tissue.
  • Lipid 83 can effectively orally deliver sRNA single-stranded nucleic acid into mouse intestinal tissues.
  • Lipid 88 can effectively orally deliver sRNA single-stranded nucleic acid into mouse kidney tissue.
  • Lipid 91 can effectively orally deliver sRNA single-stranded nucleic acid into mouse kidney tissue.
  • Lipid 92 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse lung tissue.
  • Lipid 93 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse kidney tissue.
  • Lipid 94 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse lung tissue and kidney tissue.
  • Lipid 95 can effectively orally deliver sRNA single-stranded nucleic acid into mouse lung tissue and spleen tissue.
  • Lipid 96 can effectively orally deliver sRNA single-stranded nucleic acid into mouse kidney tissue and spleen tissue.
  • Lipid 97 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into mouse lung tissue and kidney tissue.
  • Lipid 98 can effectively orally deliver sRNA single-stranded nucleic acid into mouse lung tissue, kidney tissue and spleen tissue.
  • Lipid 99 can effectively orally deliver sRNA single-stranded nucleic acid into mouse spleen tissue.
  • Lipid 100 can effectively orally deliver sRNA single-stranded nucleic acid into mouse spleen tissue.
  • Lipid 101 can effectively orally deliver sRNA single-stranded nucleic acid into mouse spleen tissue.
  • Lipid 102 can effectively orally deliver sRNA single-stranded nucleic acid into the blood and spleen tissues of mice.
  • Lipid 103 can effectively orally deliver sRNA single-stranded nucleic acid into mouse lung tissue.
  • Lipid 104 can significantly and effectively deliver sRNA single-stranded nucleic acid orally into the blood of mice.
  • Lipid 105 can effectively orally deliver sRNA single-stranded nucleic acid into the blood of mice.
  • lipids 73 and 80 can effectively orally deliver sRNA single-stranded nucleic acids into mouse heart tissues.
  • Lipids 72, 76, 92, 94, 95, 97, 98 and 103 can deliver sRNA single-stranded nucleic acid orally into mouse lung tissues significantly and effectively.
  • Lipids 88, 91, 93, 94, 96, 97 and 98 can deliver sRNA single-stranded nucleic acid orally into mouse kidney tissue significantly and effectively.
  • Lipids 76, 77, 79, 80, 81, 95, 96, 98, 99, 100, 101, and 102 can deliver sRNA single-stranded nucleic acid orally into mouse spleen tissues significantly and effectively.
  • Lipids 75, 102, 104, and 105 can effectively orally deliver sRNA single-stranded nucleic acid into the blood of mice.
  • Lipid 83 can effectively orally deliver sRNA single-stranded nucleic acid into
  • the three organs with the most significant effect of delivering nucleic acid into different lipid monomers are the spleen, lung, and kidney.
  • the spleen has the most significant lipids, followed by the lungs, and the kidneys last.
  • the effect of lipids in delivering nucleic acids into the blood is also very significant.
  • Lipid 98 has the most significant delivery effect, and its delivery of nucleic acid into the spleen, lung and kidney is significant, and it is expected to be a relatively broad-spectrum nucleic acid delivery carrier.
  • lipids 97, 76, 80, 94, 95, 96, and 102 are significant in delivering nucleic acids into the two organs.
  • lipids 93, 94, 96 and 100 deliver nucleic acid into different organs at different time points to reach the peak of the amount of entry.
  • the amount of lipid 93 delivering nucleic acid into intestinal tissue and stomach tissue is the highest at 3 hours
  • the amount entering lung tissue and kidney tissue is the highest at 6 hours
  • the amount entering brain tissue is the highest at 9 hours
  • the amount entering spleen tissue is at 12 hours.
  • the amount of nucleic acid delivered by lipid 94 into heart tissue, brain tissue, intestinal tissue, spleen tissue, and stomach tissue is the highest at 3 hours, and the amount of nucleic acid delivered into kidney tissue and lung tissue is the highest at 9 hours;
  • Lipid 96 delivers nucleic acid into the kidney
  • the amount of lipid 94 delivered nucleic acid into the stomach tissue was the highest in 3 hours, the amount into the spleen tissue, brain tissue, and intestine tissue was the highest in 6 hours, and the amount into the kidney tissue was 9 hours is the highest;
  • Lipid 96 delivers the highest amount of nucleic acid into kidney tissue and liver tissue in 3 hours, the amount that enters brain tissue, intestine tissue, and lung tissue is the highest in 6 hours, and the amount that enters heart tissue is the highest in 9 hours; lipids 98.
  • the amount of nucleic acid delivered into stomach tissue was the highest in 3 hours, and the amount of nucleic acid delivered into kidney tissue, intestinal tissue, brain tissue, liver tissue, and heart tissue was the highest in 6 hours; the amount of nucleic acid delivered by Lipid 100 into stomach tissue was the highest in 6 hours.
  • the amount entering kidney tissue was the highest at 9 hours; the amount of lipid 101 delivering nucleic acid into kidney tissue, intestinal tissue, and brain tissue was the highest at 3 hours, and the amount entering stomach tissue was the highest at 12 hours.

Abstract

通过一种或多种脂质化合物递送核酸的用途,以及包含所述脂质化合物和核酸的脂质核酸混合物、药物组合物或试剂盒。所提供的脂质化合物能够促进核酸的吸收，特别是口服吸收，以及促进核酸进入有此需要的对象体内的靶部位。

Description

脂质在制备核酸递送试剂中的应用及其相关产品 技术领域
本发明涉及生物领域。具体地，本申请涉及脂质化合物用于递送核酸的用途，这些化合物或其多种组合能够促进各种核酸通过口服体内吸收，进入有此需要的对象体内的靶部位，并进入靶细胞中。本申请涉及的这些化合物在中药中被提取和发现，也可以通过合成的方法获得。
背景技术
在过去几十年中，用核酸分子作为治疗药物的理念从概念走向了临床现实。事实上，核酸分子拥有许多的属性使得它们可以作为治疗药物。它们能够折叠形成复杂的构象让它们可以与蛋白质、小分子或者其他核酸相互结合，有些甚至可以形成催化中心。例如，小干扰RNA(siRNA)作为RNAi的效应分子，其作为治疗药物具有越来越广阔的前景。目前已经有多种siRNA药物进入临床实验，预示着其良好的发展前景。通常，人们将siRNA，miRNA及其它非编码小RNA不加区分地称之为小核酸或小RNA(sRNA)。除了小RNA以外，可以作为药物的核酸分子还包括例如mRNA、反义核酸等。然而，由于核酸分子例如RNA容易降解，在体内有相对较短的半衰期，因此作为治疗药物来说，其通常不被认为是最优的选择。因此，如何将核酸分子包括小RNA、mRNA等有效递送至体内靶器官以及靶细胞中，实现其生物活性和治疗或预防作用，是本领域技术人员迫切需要解决的问题。
发明内容
在一方面，本申请提供了脂质组合物在制备用于核酸递送的试剂中的用途，所述脂质组合物包含一种或多种具有式(I)的化合物或其盐、水合物或溶剂化物：
Figure PCTCN2020127393-appb-000001
其中：
L 1为不存在、-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；
L 2为不存在、-O-C(O)-或-NH-C(O)-；
L 3为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基、直链或支链C 1-20杂烯基或
Figure PCTCN2020127393-appb-000002
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000003
R各自独立地为H或直链或支链C 1-20烷基；
n为0、1或2。
在某些实施方式中，所述化合物的盐包括药学上可接受的盐。
在某些实施方式中，所述化合物具有下式的结构：
Figure PCTCN2020127393-appb-000004
在另一方面，本申请还提供了脂质组合物在制备用于核酸递送的试剂中的用途，所述脂质组合物包含一种或多种选自下组的化合物，或其盐、水合物或溶剂化物：脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、102、107、111、112、113、114、115、116、117、118、和119，其中所述化合物如下表所示：
脂质化合物编号 化学名
72 1,2-二油酰基-sn-甘油基-3-磷脂酰乙醇胺
73 三亚麻精
74 1,2-棕榈油酸-3-油酸甘油酯
75 1,2-棕榈油酸-3-棕榈酸甘油酯
76 1,3-棕榈酸-2-肉豆蔻酸甘油酯
77 1,2-硬脂酸-3-油酸甘油酯
78 1,2-油酸-3-花生酸甘油酯
79 1,2-油酸-3-二十二碳酸甘油酯
80 1,2-肉豆蔻酸-3-棕榈酸甘油酯
82 Q-10，泛醌50，泛醌10
83 1-棕榈酰-2-羟基-sn-甘油基-3-磷酸乙醇胺
86 1-棕榈酰基-2-油酰基-sn-甘油基-3-磷酸胆碱
87 1-硬脂酰基-2-油酰基-sn-甘油基-3-磷酸胆碱
90 N-木香油酰基-D-赤型-神经鞘氨醇
91 N-神经酰基-D-赤型-神经鞘氨醇
92 1-花生四烯基-2-羟基-sn-甘油基-3-磷酸胆碱
95 1-油酰基-2-羟基-sn-甘油基-3-磷酸胆碱
97 D-赤型-神经鞘氨醇(C22基)
98 D-赤型-神经鞘氨醇
99 D-赤型-神经鞘氨醇(C14基)
100 D-赤型-神经鞘氨醇(C16基)
101 D-赤型-神经鞘氨醇(C20基)
102 D-赤型-神经鞘氨醇
107 1,2-二亚油酰基-sn-甘油基-3-磷酸胆碱
111 1-棕榈酸-3-硬脂酸甘油酯
112 二十烷酸甘油三酯
113 1-棕榈酰-2-羟基-sn-甘油基-3-磷脂酰胆碱
114 1-十八烷酰基-2-羟基-sn-甘油基-3-磷酸胆碱
115 1-十七碳烯酰基-sn-甘油基-3-磷酸乙醇胺
116 1-(9Z-十八烯酰基)-sn-甘油基-3-磷酸乙醇胺
117 1,2-二-(9Z-十八烯酰基)-sn-甘油基-3-磷酸胆碱
118 1-棕榈酰基-2-油酰基-sn-甘油基-3-磷脂酰胆碱
119 1,2-二十六烷酰基-sn-甘油基-3-磷酸胆碱
在某些实施方式中，所述脂质组合物或所述试剂可以通过口服、吸入或注射的方式递送所述核酸。在某些实施方式中，所述脂质组合物或所述试剂通过口服的方式递送所述核酸。在某些实施方式中，所述递送包括体内消化道递送。
在某些实施方式中，所述递送包括体外细胞递送。
在某些实施方式中，所述脂质组合物或所述试剂可以用于制备脂质核酸混合物。
脂质核酸混合物可以通过适当的方法制备，包括但不限于，加热法、逆向蒸发法，或者混合法。
在某些实施方式中，所述加热法包括将脂质的有机溶剂溶液加入到核酸的水溶液中得到混合溶液，并在适当的温度下加热所述混合溶液。在某些实施方式中，所述加热法进一步包括冷却所述加热的混合溶液，从而得到脂质与核酸的混合物。
在一些实施方案中，在选自如下的温度下加热所述混合溶液：25℃至100℃、30℃至100℃、40℃至100℃、50℃至100℃、60℃至100℃、70℃至100℃、80℃至100℃、90℃至100℃，和95℃至100℃。在一些实施方案中，在选自如下的温度下加热所述混合溶液：30℃、35℃、37℃、40℃、45℃、50℃、55℃、 60℃、65℃、70℃、75℃，80℃、85℃、90℃、95℃和100℃。
在一些实施方案中，加热所述混合溶液的时间为约5分钟至约24小时，约5分钟至约20小时，约5分钟至约16小时，约10分钟至约20小时，约10分钟至约16小时，约15分钟至约24小时，约15分钟至约20小时，约30分钟至约24小时，约30分钟至约20小时，约40分钟至约16小时，约50分钟至约12小时，约1小时至约8小时，或约2小时至约4小时。在一些实施方案中，加热所述混合溶液的时间为约5分钟至约1小时，约5分钟至约30分钟，约5分钟至约15分钟，或约10分钟至约15分钟。在一些实施方案中，加热所述混合溶液的时间为约5分钟、约10分钟、约15分钟、约20分钟、约25分钟、约30分钟、40分钟、50分钟、1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、12小时、16小时、20小时或24小时。
在某些实施方案中，在选自如下的温度下冷却所述混合溶液：25℃至-80℃，20℃至-80℃，15℃至-80℃，10℃至-80℃，4℃至-80℃，0℃至-80℃，-10℃至-80℃，-20℃至-80℃，-30℃至-80℃，-40℃至-80℃。在一些实施方案中，在选自如下的温度下冷却所述混合溶液：25℃、20℃、15℃、10℃、4℃或0℃。
在某些实施方案中，所述逆向蒸发法包括将核酸的水溶液与脂质化合物的有机溶剂溶液进行混合以得到混合溶液。在某些实施方案中，所述逆向蒸发法进一步包括去除所述混合溶液中的有机溶剂，然后进行水化，从而得到脂质与核酸的混合物。在某些实施方案中，将所述混合溶液进行超声和/或蒸发以去除所述有机溶剂。在某些实施方案中，去除所述混合溶液中的有机溶剂的步骤在适当的温度下进行。
在一些实施方案中，在选自如下的温度下去除所述混合溶液中的有机溶剂：约25℃至约70℃，30℃至约70℃、约30℃至约65℃，约40℃至约65℃，约40℃至约60℃、或约50℃至约60℃。在一些实施方案中，在选自如下的温度下去除所述混合溶液中的有机溶剂：约25℃、约30℃、约35℃、约40℃、约45℃、约50℃、约55℃、约60℃、约65℃和约70℃。
在另一方面，本申请提供了一种向有需要的个体递送核酸的方法，其包括通过口服、吸入或注射的方式向所述个体施用所述脂质组合物和所述核酸。在 某些实施方式中，所述脂质组合物和所述核酸以脂质核酸混合物的形式施用。
在某些实施方式中，所述核酸包含DNA或RNA。在某些实施方案中，所述DNA为例如非编码DNA(如反义DNA)或编码DNA等。在某些实施方案中，所述RNA为例如反义核酸、mRNA、lncRNA、或小RNA(例如miRNA、siRNA、piRNA、snoRNA、tsRNA)等。
在某些实施方式中，所述核酸包含长度为14-32bp、16-28bp或18-24bp的小核酸。
在某些实施方式中，所述核酸为单链或双链的。
在某些实施方式中，所述核酸具有茎环结构。
在某些实施方式中，所述核酸用于治疗疾病。
在某些实施方式中，所述核酸用于治疗癌症、炎症、纤维化疾病、自身免疫疾病或自身炎性疾病、细菌感染、行为性和精神性病症、血液疾病、染色体疾病、先天性和遗传性疫病、结缔组织疾病、消化性疾病、耳鼻喉疾病、内分泌疾病、环境性疾病、眼部疾病、女性生殖性疾病、真菌感染、心脏病、遗传性癌症综合征、免疫系统疾病、肾脏和泌尿性疾病、肺部疾病、男性生殖新疾病、代谢性病症、口部疾病、肌肉骨骼疾病、骨髓增生异常性综合征、新生儿筛查、营养性疾病、寄生虫疾病、罕见癌症、罕见疾病、皮肤疾病和病毒感染。
在某些实施方式中，所述核酸用于治疗肝细胞癌、角膜新生血管形成、复发性或难治性间变性星形细胞瘤(WHO III级)或继发性胶质母细胞瘤(WHO IV级)、晚期鳞状细胞肺癌、肢端肥大症、银屑病、杜氏肌营养不良症、晚期非小细胞肺癌、转移性去势抗性前列腺癌、巨细胞病毒性视网膜炎，HIV感染、乙型肝炎、丙型肝炎、高脂蛋白症、全膝关节置换术、II型糖尿病、家族性淀粉样多发性神经病(FAP)、湿性黄斑变性(例如新血管性年龄相关性黄斑变性、中央凹下新血管性年龄相关性黄斑变性、渗出性年龄相关性黄斑变性)、高胆固醇血症、克罗恩病、广泛性肝纤维化、婴儿型脊髓性肌萎缩症、黑色素瘤、新生冠状动脉病变、轻度过敏性哮喘、慢性淋巴细胞性白血病、和高甘油三酯血症、造血干细胞移植后伴肾或肺功能障碍的肝小细胞闭塞症、伴遗传性转甲状腺素蛋白淀粉样病变。
在另一方面，本申请还提供了一种药物组合物，其包含脂质组合物以及核酸，其中所述脂质组合物包含一种或多种具有式(I)的化合物或其药学上可接受的盐、水合物或溶剂化物。在某些实施方式中，所述脂质组合物和所述核酸以脂质核酸混合物的形式存在。
在另一方面，本申请还提供了一种药物组合物，其包含脂质组合物以及核酸，其中所述脂质组合物包含一种或多种选自下组的化合物，或其盐、水合物或溶剂化物：脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、102、107、111、112、113、114、115、116、117、118、和119组成的组。在某些实施方式中，所述脂质组合物和所述核酸以脂质核酸混合物的形式存在。
在另一方面，本申请还提供了本申请所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗能用核酸预防和/或治疗的疾病，或者用于将核酸体内递送至有此需要的对象中。
在另一方面，本申请还提供了一种试剂盒，其包含：置于第一容器中的一种或多种具有所述式(I)的化合物或其药学上可接受的盐、水合物或溶剂化物，和置于第二容器中的核酸。
在另一方面，本申请还提供了一种试剂盒，其包含：置于第一容器中的一种或多种化合物或其药学上可接受的盐、水合物或溶剂化物，和置于第二容器中的核酸，其中所述一种或多种化合物选自下组：脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、102、107、111、112、113、114、115、116、117、118、和119。
在另一方面，本申请提供了本申请所述的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗能用核酸预防和/或治疗的疾病，或者用于将核酸体内递送至有此需要的对象中。
在另一方面，本申请提供了一种将核酸递送至靶细胞中的方法，包括向所述靶细胞施用本申请所述的药物组合物，或者由本申请所述的试剂盒配制得到的脂质核酸混合物。
在另一方面，本申请提供了一种将核酸体内递送至有此需要的对象中的方法，包括向所述对象施用本申请所述的药物组合物，或者由本申请所述的试剂盒配制得到的脂质核酸混合物。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1分别示出了空白组、自由摄取组和脂质72递送PGY-sRNA-26进入小鼠心脏组织(图1a)、肺组织(图1b)、脾组织(图1c)及血液(图1d)的结果。
图2分别示出了空白组、自由摄取组和脂质73递送PGY-sRNA-26进入小鼠心脏组织(图2a)、脾组织(图2b)及血液(图2c)的结果。
图3分别示出了空白组、自由摄取组和脂质74递送PGY-sRNA-26进入小鼠心脏组织(图3a)、肺组织(图3b)、脾组织(图3c)及血液(图3d)的结果。
图4分别示出了空白组、自由摄取组和脂质75递送PGY-sRNA-26进入小鼠心脏组织(图4a)、肺组织(图4b)、脾组织(图4c)及血液(图4d)的结果。
图5分别示出了空白组、自由摄取组和脂质76递送PGY-sRNA-26进入小鼠心脏组织(图5a)、肺组织(图5b)、脾组织(图5c)及血液(图5d)的结果。
图6分别示出了空白组、自由摄取组和脂质77递送PGY-sRNA-26进入小鼠心脏组织(图6a)、肺组织(图6b)、肾组织(图6c)、脾组织(图6d)及血液(图6e)的结果。
图7分别示出了空白组、自由摄取组和脂质78递送PGY-sRNA-26进入小鼠心脏组织(图7a)、肺组织(图7b)、肾组织(图7c)及脾组织(图7d)的结果。
图8分别示出了空白组、自由摄取组和脂质79递送PGY-sRNA-26进入小鼠心脏组织(图8a)、肾组织(图8b)及脾组织(图8c)的结果。
图9分别示出了空白组、自由摄取组和脂质80递送PGY-sRNA-26进入小鼠心脏组织(图9a)、肺组织(图9b)、肾组织(图9c)及脾组织(图9d)的结果。
图10分别示出了空白组、自由摄取组和脂质81递送PGY-sRNA-26进入小鼠心脏组织(图10a)、肾组织(图10b)及脾组织(图10c)的结果。
图11分别示出了空白组、自由摄取组和脂质82递送PGY-sRNA-26进入小鼠肺组织(图11a)、脾组织(图11b)及心脏组织(图11c)的结果。
图12分别示出了空白组、自由摄取组和脂质83递送PGY-sRNA-26进入小鼠脾组织(图12a)、肾组织(图12b)及肠组织(图12c)的结果。
图13分别示出了空白组、自由摄取组和脂质84递送PGY-sRNA-26进入小鼠肺(图13a)组织及脾组织(图13b)的结果。
图14分别示出了空白组、自由摄取组和脂质85递送PGY-sRNA-26进入小鼠肺组织(图14a)、脾组织(图14b)及肾组织(图14c)的结果。
图15分别示出了空白组、自由摄取组和脂质86递送PGY-sRNA-26进入小鼠肠组织(图15a)及肾组织(图15b)的结果。
图16分别示出了空白组、自由摄取组和脂质87递送PGY-sRNA-26进入小鼠肾组织(图16a)、肺组织(图16b)及脾组织(图16c)的结果。
图17分别示出了空白组、自由摄取组和脂质88递送PGY-sRNA-26进入小鼠血(图17a)、肺组织(图17b)、脾组织(图17c)及肾组织(图17d)的结果。
图18分别示出了空白组、自由摄取组和脂质89递送PGY-sRNA-26进入小鼠血(图18a)、肺组织(图18b)及肾组织(图18c)的结果。
图19分别示出了空白组、自由摄取组和脂质90递送PGY-sRNA-26进入小鼠血(图19a)、肺组织(图19b)、脾组织(图19c)及肾组织(图19d)的结果。
图20分别示出了空白组、自由摄取组和脂质91递送PGY-sRNA-26进入小鼠血(图20a)、肺组织(图20b)、脾组织(图20c)及肾组织(图20d)的结果。
图21分别示出了空白组、自由摄取组和脂质92递送PGY-sRNA-26进入小鼠血(图21a)、肺组织(图21b)、脾组织(图21c)及肾(图21d)的结果。
图22分别示出了空白组、自由摄取组和脂质93递送PGY-sRNA-26进入小鼠血(图22a)、肺组织(图22b)、脾组织(图22c)及肾组织(图22d)的结果。
图23分别示出了空白组、自由摄取组和脂质94递送PGY-sRNA-26进入小鼠血(图23a)、肺组织(图23b)、脾组织(图23c)及肾组织(图23d)的结果。
图24分别示出了空白组、自由摄取组和脂质95递送PGY-sRNA-26进入小鼠血(图24a)、肺组织(图24b)、肾组织(图24c)及脾组织(图24d)的结果。
图25分别示出了空白组、自由摄取组和脂质96递送PGY-sRNA-26进入小鼠血(图25a)、肺组织(图25b)、肾组织(图25c)及脾组织(图25d)的结果。
图26分别示出了空白组、自由摄取组和脂质97递送PGY-sRNA-26进入小鼠血(图26a)、肺组织(图26b)、肾组织(图26c)及脾组织(图26d)的结果。
图27分别示出了空白组、自由摄取组和脂质98递送PGY-sRNA-26进入小鼠血(图27a)、肺组织(图27b)、肾组织(图27c)及脾组织(图27d)的结果。
图28分别示出了空白组、自由摄取组和脂质99递送PGY-sRNA-26进入小鼠血(图28a)、肺组织(图28b)、肾组织(图28c)及脾组织(图28d)的结果。
图29分别示出了空白组、自由摄取组和脂质100递送PGY-sRNA-26进入小鼠血(图29a)、肺组织(图29b)、肾组织(图29c)及脾组织(图29d)的结果。
图30分别示出了空白组、自由摄取组和脂质101递送PGY-sRNA-26进入小鼠血(图30a)、肺组织(图30b)、肾组织(图30c)及脾组织(图30d)的结果。
图31分别示出了空白组、自由摄取组和脂质102递送PGY-sRNA-26进入小鼠血(图31a)、肾组织(图31b)及脾组织(图31c)的结果。
图32分别示出了空白组、自由摄取组和脂质103递送PGY-sRNA-26进入小鼠血(图32a)、肾组织(图32b)及肺组织(图32c)的结果。
图33分别示出了空白组、自由摄取组和脂质104递送PGY-sRNA-26进入小鼠血(图33a)、肾组织(图33b)、肺组织(图33c)及脾组织(图33d)的结果。
图34分别示出了空白组、自由摄取组和脂质105递送PGY-sRNA-26进入小鼠血(图34a)、肾组织(图34b)及肺组织(图34c)的结果。
图35分别示出了空白组、自由摄取组和脂质107递送PGY-sRNA-26进入小鼠血(图35a)、肾组织(图35b)及脾组织(图35c)的结果。
图36分别示出了空白组、自由摄取组和脂质109递送PGY-sRNA-26进入小鼠血(图36a)、肺组织(图36b)、肾组织(图36c)及脾组织(图36d)的结果。
图37分别示出了空白组、自由摄取组和脂质111递送PGY-sRNA-26进入小鼠血(图37a)及肾组织(图37b)的结果。
图38分别示出了空白组、自由摄取组和脂质112递送PGY-sRNA-26进入小鼠肺(图38a)、肾组织(图38b)及脾组织(图38c)的结果。
图39分别示出了空白组、自由摄取组和脂质113递送PGY-sRNA-26进入小鼠肺组织(图39a)、肾组织(图39b)及脾组织(图39c)的结果。
图40分别示出了空白组、自由摄取组和脂质114递送PGY-sRNA-26进入小鼠血(图40a)、肺组织(图40b)、肾组织(图40c)及脾组织(图40d)的结果。
图41分别示出了空白组、自由摄取组和脂质115递送PGY-sRNA-26进入小鼠血(图41a)、肺组织(图41b)、肾组织(图41c)及脾组织(图41d)的结果。
图42分别示出了空白组、自由摄取组和脂质116递送PGY-sRNA-26进入小鼠肺组织(图42a)、肾组织(图42b)及脾组织(图42c)的结果。
图43分别示出了空白组、自由摄取组和脂质117递送PGY-sRNA-26进入小鼠肺组织(图43a)、肾组织(图43b)及脾组织(图43c)的结果。
图44分别示出了空白组、自由摄取组和脂质118递送PGY-sRNA-26进入小鼠肺组织(图44a)、肾组织(图44b)及脾组织(图44c)的结果。
图45分别示出了空白组、自由摄取组和脂质119递送PGY-sRNA-26进入小鼠血(图45a)及肾组织(图45b)的结果。
图46分别示出了脂质93以灌胃给药的方式递送PGY-sRNA-26进入小鼠脑组织(图46a)、胃组织(图46b)、肠组织(图46c)、肾组织(图46d)、肺组织(图46e)及脾组织(图46f)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图47分别示出了脂质94以灌胃给药的方式递送PGY-sRNA-26进入小鼠脑组织(图47a)、心组织(图47b)、脾组织(图47c)、肺组织(图47d)、肾组织(图47e)、肠组织(图47f)及胃组织(图47g)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图48分别示出了脂质96以灌胃给药的方式递送PGY-sRNA-26进入小鼠肠组织(图48a)及肾组织(图48b)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图49分别示出了脂质100以灌胃给药的方式递送PGY-sRNA-26进入小鼠胃组织(图49a)、肠组织(图49b)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图50分别示出了脂质94以尾静脉注射给药的方式递送PGY-sRNA-26进入小鼠脾组织(图50a)、肾组织(图50b)、脑组织(图50c)、胃组织(图50d)、肠组织(图50e)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图51分别示出了脂质96以尾静脉注射给药的方式递送PGY-sRNA-26进入小鼠脑组织(图51a)、肠组织(图51b)、肾组织(图51c)、肺组织(图51d)、肝组织(图51e)、心组织(图51f)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图52分别示出了脂质98以尾静脉注射给药的方式递送PGY-sRNA-26进入小鼠肾组织(图52a)、肠组织(图52b)、胃组织(图52c)、脑组织(图52d)、肝组织(图52e)、心组织(图52f)于0小时(空白组)/3小时/6小时/9小时/12 小时/24小时进入量的结果。
图53分别示出了脂质100以尾静脉注射给药的方式递送PGY-sRNA-26进入小鼠肾组织(图53a)、胃组织(图53b)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
图54分别示出了脂质101以尾静脉注射给药的方式递送PGY-sRNA-26进入小鼠肾组织(图54a)、肠组织(图54b)、胃组织(图54c)、脑组织(图54d)于0小时(空白组)/3小时/6小时/9小时/12小时/24小时进入量的结果。
具体实施方式
本发明至少部分地基于本申请的发明人经过大量实验获得的意外发现，即，一些中药(例如，红景天、蒲公英、穿心莲、金银花等)中存在一些脂质组分，这些源自中药的脂质能够促进核酸如小RNA吸收/进入细胞和/或有此需要的对象体内靶部位。为本发明的目的，这些脂质组分也可以是合成的。
如下将参照以示例性的方式示出结构和式的本申请的某些实施方案来详细描述本申请。尽管结合列举的实施方案对本申请进行了描述，但应了解其不旨在将本申请限定为所描述的那些实施方案。相反，本申请旨在涵盖落入权利要求书所限定的范围内的所有替代方式、修改方式和等同方式。本领域技术人员将会认识到可用于实施本申请的与本文描述的方法和材料类似或等同的其他方法和材料，本申请不以任何方式局限于所描述的方法和材料。当所引用的文献和类似材料不同于本申请的描述或与本申请的描述(包括限定的术语、术语使用、技术等)矛盾时，以本申请的描述为准。
还应理解在不同实施方案中描述的某些特征也可在单个实施方案中组合提供。反之，在单个实施方案中描述的多种特征也可以单独提供或以任何适当的亚组合提供。
还应理解本文所记载的数值点旨在包括各数值点本身，以及任意两个所记载的点之间的数值范围(如同这些数值范围已经单独列出)。
定义
如本文所用，除非另有说明，否则应适用以下定义。出于本申请的目的，根据Handbook of Chemistry and Physics(第75版)的元素周期表(CAS版)鉴定化学元素。另外，有机化学的一般原理以及特定的官能部分和反应活性描述于“Organic Chemistry”,Thomas Sorrell,University Science Books,Sausalito:1999和“March’s Advanced Organic Chemistry”,第5版，Smith,M.B.和March,J.,John Wiley&Sons,New York:2001中，其全部内容通过引用并入本申请。
在本文中描述了连接取代基。当结构明确需要连接基团时，应理解针对该基团所列出的马库什变量为连接基团。例如，如果结构需要连接基团，且针对该变量的马库什基团定义列出了“烷基”，则应理解“烷基”表示亚烷基连接基团。
本文所用的术语“取代的”，无论前面是否有术语“任选地”，都是指指定基团中的一个或多个氢被合适的取代基取代。除非另有说明，否则“取代的”基团可以在所述基团的每个可取代的位置具有合适的取代基，并且当任何给定结构中一个以上的位置可以被选自特定基团的一个以上的取代基取代时，所述取代基可以在每个位置上都相同或不同。本发明所设想的取代基组合优选地是导致形成稳定的或化学上可行的化合物的那些取代基组合。本文所用的术语“稳定的”是指这样的化合物：当其经受允许其生产、检测、以及(在某些实施方案中)其回收、纯化的条件、以及用于本文公开的一个或多个目的时，其基本上不会改变。除非特别指出为“未取代的”，否则本文所述的化学部分应被理解为包括取代基。例如，当提及“芳基”时，其包括取代的芳基和未取代的芳基。
当连接至取代基的键显示为与环中连接两个原子的键交叉时，该取代基可键连至环中的任意原子。当列出取代基但未指明该取代基通过何种原子键连至给定式的化合物的其余部分时，该取代基可通过所述式中的任意原子键连。取代基和/或变量的组合是允许的，但前提是该组合得到稳定的化合物。
本文所用的术语“C i-j”表示碳原子数的范围，其中i和j为整数且j大于i，并且碳原子数的范围包括端点(即i和j)以及端点之间的每个整数点。例如，C 1-6表示1至6个碳原子的范围，包括1个碳原子，2个碳原子，3个碳原子，4个碳原子，5个碳原子和6个碳原子。在一些实施方案中，术语“C 1-12”表示1至 12，特别地1至10，特别地1至8，特别地1至6，特别地1至5，特别地1至4，特别1至3，或特别地1至2个碳原子。
本文所用的术语“烷基”，无论作为另一术语的一部分还是独立地使用，是指饱和的直链或支链烃基。术语“C i-j烷基”指具有i至j个碳原子的烷基。在一些实施方案中，烷基包含1至20个碳原子。在一些实施方案中，烷基包含5至20个碳原子。在一些实施方案中，烷基包含1至20个碳原子，1至19个碳原子，1至18个碳原子，1至17个碳原子，1至16个碳原子，1至15个碳原子，1至14个碳原子，1至13个碳原子，1至12个碳原子，1至11个碳原子，1至10个碳原子，1至9个碳原子，1至8个碳原子，1至7个碳原子，1至6个碳原子，1至5个碳原子，1至4个碳原子，1至3个碳原子，或1至2个碳原子。烷基的例子包括但不限于甲基、乙基、1-丙基(正丙基)、2-丙基(异丙基)、1-丁基(正丁基)、2-甲基-1-丙基(异丁基)、2-丁基(新丁基)、2-甲基-2-丙基(叔丁基)、1-戊基(正戊基)、2-戊基、3-戊基、2-甲基-2-丁基、3-甲基-2-丁基、3-甲基-1-丁基、2-甲基-1-丁基、1-己基、2-己基、3-己基、2-甲基-2-戊基、3-甲基-2-戊基、4-甲基-2-戊基、3-甲基-3-戊基、2-甲基-3-戊基、2,3-二甲基-2-丁基、3,3-二甲基-2-丁基、1-庚基、1-辛基、1-壬基、1-癸基等。“C 1-20烷基”的例子包括但不限于甲基、乙基、丙基、丁基、戊基、己基、庚基、辛基、壬基、癸基、十一烷基、十二烷基、十三烷基、十四烷基、十五烷基、十六烷基、十七烷基、十八烷基、十九烷基、二十烷基。
本文所用的术语“烯基”，无论作为另一术语的一部分还是独立地使用，是指具有至少一个碳-碳双键的直链或支链烃基，其可任选地被一个或多个本文所述的取代基独立地取代，并且包括具有“顺式”和“反式”取向，或者“E”和“Z”取向的基团。在一些实施方案中，烯基含有2至20个碳原子。在一些实施方案中，烯基含有5至20个碳原子。在一些实施方案中，烯基含有2至20个碳原子，2至19个碳原子，2至18个碳原子，2至17个碳原子，2至16个碳原子，2至15个碳原子，2至14个碳原子，2至13个碳原子，2至12个碳原子，2至11个碳原子，2至10个碳原子，2至9个碳原子，2至8个碳原子，2至7个碳原子，2至6个碳原子，2至5个碳原子，2至4个碳原子，2至3个碳原子。在一些实施方案中，烯基包含超过1个碳-碳双键。应理解，在所述烯基包含超过1个碳-碳双 键的情况下，所述双键可以互相分离或共轭。在一些实施方案中，烯基包含5个碳-碳双键，4个碳-碳双键，3个碳-碳双键，2个碳-碳双键，或1个碳-碳双键。
本文所用的术语“环烷基”，无论作为另一术语的一部分还是独立地使用，是指饱和的单环和多环环系，其中所有的环原子为碳，其包含至少3个成环碳原子。在一些实施方案中，环烷基可含有3至20个成环碳原子，3至19个成环碳原子，3至18个成环碳原子，3至17个成环碳原子，3至16个成环碳原子，3至15个成环碳原子，3至14个成环碳原子，3至13个成环碳原子，3至12个成环碳原子，3至11个成环碳原子，3至10个成环碳原子，3至9个成环碳原子，3至8个成环碳原子，3至7个成环碳原子，3至6个成环碳原子，3至5个成环碳原子，4至20个成环碳原子，4至19个成环碳原子，4至18个成环碳原子，4至17个成环碳原子，4至16个成环碳原子，4至15个成环碳原子，4至14个成环碳原子，4至13个成环碳原子，4至12个成环碳原子，4至11个成环碳原子，4至10个成环碳原子，4至9个成环碳原子，4至8个成环碳原子，4至7个成环碳原子，4至6个成环碳原子，4至5个成环碳原子。环烷基可任选地在一个或多个环位置处被一个或多个本文所述的取代基取代。环烷基可为饱和的、部分不饱和的或完全不饱和的。在一些实施方案中，环烷基可为饱和环状烷基。在一些实施方案中，环烷基可为在环系中含有至少一个双键或三键的不饱和环状烷基。
本文所用的术语“环烯基”，无论作为另一术语的一部分还是独立地使用，是指包含3-20个成环碳原子以及至少一个碳-碳双键的单环和多环环系，只要所述环烯基环的大小允许，其中所有的环原子为碳。在一些实施方案中，环烯基包含超过1个碳-碳双键。应理解，在所述烯基包含超过1个碳-碳双键的情况下，所述双键可以互相分离或共轭。在一些实施方案中，环烯基包含5个碳-碳双键，4个碳-碳双键，3个碳-碳双键，2个碳-碳双键，或1个碳-碳双键。
本文所用的术语“杂原子”是指氮、氧、硫或磷，并包括氮或硫的任意氧化形式，以及碱性氮的任意季化形式。
本文所用的术语“杂烷基”，无论作为另一术语的一部分还是独立地使用，是指至少一个碳原子被选自氮、氧、硫或磷的杂原子替换的烷基，其中所述杂原子 可位于烷基的端部或中间。
本文所用的术语“杂烯基”，无论作为另一术语的一部分还是独立地使用，是指至少一个碳原子被选自氮、氧、硫或磷的杂原子替换的烯基，其中所述杂原子可位于烯基的端部或中间。
本文所用的术语“羟基”是指-OH。
本文所用的术语“药学上可接受的盐”是指在合理的医学判断范围内适合用于与人或动物的组织接触而没有过度的毒性、刺激性、过敏性反应或其它问题，并且与合理的有益效果/风险比相称的本文所述化合物的盐或两性离子形式。
本文所用的术语“药物组合物”指包含本申请所述的脂质组合物和核酸的组合物，该组合物可任选地进一步含有药学上可接受的运载体。
本文所用的术语“药学上可接受的”是指这样的化合物、材料、组合物和/或剂型：其在合理的医学判断范围内适合用于与人和动物的组织接触而无过多毒性、刺激性、过敏反应或其他问题或并发症，与合理的利益/风险比相称。在一些实施方案中，药学上可接受的化合物、材料、组合物和/或剂型是指用于动物(更特别地，用于人)的由管理机构(例如美国食品和药物管理局、中国食品药品管理局或欧洲药品管理局)批准的或通常在公认的药典(例如美国药典、中国药典或欧洲药典)中列出的化合物、材料、组合物和/或剂型。
本文所用的术语“药学上可接受的运载体”是指药学上可接受的材料、组合物或媒介物，例如液体或固体填充剂、稀释剂、赋形剂、溶剂或包封材料，其涉及将本文提供的化合物从一个位置、体液、组织、器官(内部或外部)或身体的一部分携带或运输至另一个位置、体液、组织、器官或身体的一部分。药学上可接受的运载体可以是媒介物、稀释剂、赋形剂或可用于接触动物的组织而无过多毒性或副作用的其他材料。示例性的药学上可接受的运载体包括糖、淀粉、纤维素、麦芽、黄蓍胶、明胶、林格氏溶液、海藻酸、等渗盐水、缓冲剂等。可用于本申请的药学上可接受的运载体包括本领域通常已知的运载体，例如在“Remington Pharmaceutical Sciences”Mack Pub.Co.,New Jersey(1991)中公开的运载体，该文章通过引入并入本文。
本文所用的术语“递送”涵盖局部和全身递送两者。“局部递送”是指将待递送的治疗剂(例如核酸)直接递送至生物体内的靶部位。例如，试剂可以通过直接注射到靶部位(例如疾病部位，如肿瘤或炎症部位)或靶器官(例如心脏、脾、肺、肾等)而被局部递送。“全身递送”是指导致治疗剂(例如核酸)在生物体内广泛生物分布的递送，从而使有效量的治疗剂暴露于身体的大多数部位。为了获得广泛的生物分布，通常需要这样的血液寿命，使得这些治疗剂在到达远离给药部位的靶部位之前不迅速降解或清除。脂质组合物的全身递送可以是任何合适的方式，包括，例如，口服、吸入、消化道内、静脉内、皮下、和腹膜内来进行。
如本文所用的术语“脂质”是指一类有机化合物，其包括但不仅限于脂肪酸的酯，并且特征是不溶于水(例如水中的溶解度小于约0.01重量％)但可溶于许多有机溶剂中。脂质可以是，例如简单脂质(例如脂肪、油、蜡)，化合物脂质(例如磷脂、糖脂)，和衍生的脂质(例如类固醇)。
如本文所用的“核酸”是指含有处于单链或双链形式的至少两个脱氧核糖核苷酸或核糖核苷酸的聚合物。核酸可以具有天然的核糖核苷酸和脱氧核糖核苷酸，也可以包含非天然的核糖核苷酸和非天然的脱氧核糖核苷酸。天然的核糖核苷酸包括，例如腺苷酸、鸟苷酸、胞苷酸、尿苷酸、假尿甘酸、肌苷酸、黄苷酸。天然的脱氧核糖核苷酸包括，例如脱氧腺苷酸、脱氧鸟苷酸、脱氧胞苷酸、脱氧胸苷酸。非天然的核糖核苷酸和脱氧核糖核酸通常具有修饰的核碱基、修饰的核糖或脱氧核糖、和/或修饰的磷酸酯键。
“核苷酸”包含糖(例如脱氧核糖或核糖)，碱基，和磷酸基团。核苷酸通过磷酸基团连接在一起，形成聚合物。“碱基”包括嘌呤和嘧啶，其进一步包括天然化合物腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶、尿嘧啶、肌苷、和天然类似物，以及嘌呤和嘧啶的合成衍生物，其包括但不限于，具有胺、醇、硫醇、羧化物和烷基卤化物等的修饰。在本申请中，核酸还可以包含核苷酸类似物或修饰的核苷酸，其可以是合成的、天然存在的、和非天然存在的，且其具有与天然核酸类似的结合特性。所述核苷酸类似物或修饰的核苷酸的实例包括，但不仅限于，具有硫代磷酸酯、氨基磷酸酯、甲基磷酸酯、或手性-甲基磷酸酯的核苷酸、2’-O-甲基核糖核苷酸和肽-核酸(PNAs)。
核酸的例子包括DNA或RNA。DNA包括编码DNA和非编码DNA，例子包括，但不限于，反义DNA、质粒DNA、预浓缩DNA、PCR产物、DNA载体(P1，PAC，BAC，YAC，人工染色体)、表达盒、嵌合序列、染色体DNA、或这些组的衍生物和组合。RNA的例子包括，但不限于，反义RNA、siRNA、不对称干扰RNA(aiRNA)、微小RNA(miRNA)、mRNA、tRNA、rRNA、病毒RNA(vRNA)、长非编码RNA(lncRNA)、Piwi-相互作用RNA(piRNA)、核仁小RNA(snoRNA)、tRNA来源小RNA(tsRNA)或其组合。
除非另外指明，具体的核酸序列还固有地包括其保守修饰变体(例如，简并密码子取代)、等位基因、正交同源物、SNP、和互补序列以及明确指出的序列。特别地，简并密码子取代可以通过产生这样的序列实现，在所述序列中，一种或多种所选(或全部)密码子的第三个位置被混合的碱性和/或脱氧肌苷残基(Batzer等，NucleicAcidRes.(核酸研究)，19：5081(1991)；Ohtsuka等，J.Biol.Chem.(生物化学杂志)，260：2605-2608(1985)；Rossolini等，Mol.Cell.Probes(分子细胞探针)，8：91-98(1994))取代。
本文在核苷酸配对情况下所用的术语“互补”包括经典的沃森-克里克配对，即G-C、A-T或A-U配对。经典的沃森-克里克配对也涵盖当一个或两个核苷酸已被修饰(例如通过核糖修饰或磷酸酯主链修饰)的情形。本文所用的“互补的”序列还可包括非沃森-克里克碱基对和/或非天然和修饰的核苷酸形成的碱基对。
本文所用的术语“治疗”是指消除、减少或改善疾病或病况和/或与其相关的症状。尽管未排除，但治疗疾病或病况不要求所述疾病、病况或与其相关的症状被完全消除。本文所用的术语“治疗”可包括“预防性治疗”，其是指在未患有疾病，但有风险或易于重新发生疾病或病况或所述疾病或病况有复发风险或易于复发的受试者中，减少重新发生疾病或病况的可能性，或减少之前控制的疾病或病况的复发的可能性。本发明的含义内，“治疗”还包括复发预防或阶段预防，以及治疗急性或慢性征兆、症状和/或机能障碍。治疗可以是针对症状的，例如，抑制症状。其可在短时期内起作用，针对中等时期内，或可以是长期治疗，例如在维持疗法的情况下。
本文所用的术语“对象”是指为了治疗目的可对其施用本文所述的脂质组合 物的任何生物体。在一些实施方案中，对象是指灵长类(例如人)，狗，兔子，豚鼠，猪，大鼠和小鼠。在某些实施方案中，对象是灵长类动物。在其他实施方案中，对象是人。
脂质化合物用于递送核酸的方法和用途
本申请提供了脂质化合物用于递送核酸的方法和用途。本发明至少部分地基于对某些脂质化合物在核酸递送方面的突出效果的发现。本申请提供了多种脂质化合物，其被发现能够与核酸形成稳定的核酸脂质混合物，并且能够将核酸递送到细胞中，特别是在体内的细胞中。本申请提供的脂质化合物能够通过口服的方式，将核酸分子递送到体内，并且进入到体内的靶器官中，以实现良好的治疗效果。令人预想不到的是，不同的脂质化合物在递送核酸进入靶器官时的效果不尽相同，表现出对不同靶器官的不同的偏好。某些脂质化合物表现出对不止一种靶器官的显著的核酸递送效果，可以高效地递送核酸至多个靶器官，因此具有广泛的核酸递送的用途。某些脂质化合物表现出对某一种靶器官的特别显著的递送效果，因此可以用于递送作用于该靶器官的核酸。
在一方面，本申请提供了脂质组合物在制备用于核酸递送的试剂中的用途，或者脂质组合物用于递送核酸的方法，其中所述脂质组合物包含一种或多种具有式(I)的化合物或其盐、水合物或溶剂化物：
Figure PCTCN2020127393-appb-000005
其中：
L 1为不存在、-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；
L 2为不存在、-O-C(O)-或-NH-C(O)-；
L 3为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基、直链或支链C 1-20杂烯基或
Figure PCTCN2020127393-appb-000006
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000007
R各自独立地为H或直链或支链C 1-20烷基；
n为0、1或2。
在某些实施方式中，L 1为-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；L 2为不存在、-O-C(O)-或-NH-C(O)-；L 3为
Figure PCTCN2020127393-appb-000008
A，B，Q，R和n如上所定义。
在某些实施方式中，L 1为不存在、-CH 2-O-C(O)-或-CH 2-O-；L 2为-O-C(O)-；L 3为直链或支链C 1-20烷基或
Figure PCTCN2020127393-appb-000009
A，B，Q，R和n如上所定义。
在某些实施方式中，L 1为-CH 2-O-C(O)-；L 2为不存在或-O-C(O)-；L 3为
Figure PCTCN2020127393-appb-000010
A，B，Q，R和n如上所定义。
在某些实施方式中，其中L 1为-CH 2-O-；L 2为不存在或-O-C(O)-；L 3为
Figure PCTCN2020127393-appb-000011
A，B，Q，R和n如上所定义。
在某些实施方式中，L 1为-CH 2-O-C(O)-或-CH 2-O-；L 2为不存在；L 3为
Figure PCTCN2020127393-appb-000012
A，B，Q，R和n如上所定义。
在某些实施方式中，L 1为不存在；L 2为-O-C(O)-；L 3为直链或支链C 1-20烷基；A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000013
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，L 1为-CH 2-O-C(O)-；L 2为不存在；L 3为
Figure PCTCN2020127393-appb-000014
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000015
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，L 1为-CH 2-O-；L 2为不存在；L 3为
Figure PCTCN2020127393-appb-000016
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000017
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，L 1为-CH 2-O-；L 2为-O-C(O)-；L 3为
Figure PCTCN2020127393-appb-000018
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000019
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，L 1为-CH 2-O-C(O)-；L 2为-O-C(O)-；L 3为
Figure PCTCN2020127393-appb-000020
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000021
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，L 1为-CH 2-O-C(O)-；L 2为-O-C(O)-；L 3为
Figure PCTCN2020127393-appb-000022
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000023
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，L 1为-CR(OH)-；L 2为-NH-C(O)-；L 3为
Figure PCTCN2020127393-appb-000024
A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000025
R各自独立地为H或直链或支链C 1-20烷基；和n为0、1或2。
在某些实施方式中，所述化合物具有下式的结构：
Figure PCTCN2020127393-appb-000026
其中L 1、L 2、L 3、A、B和Q的定义如之前所述。
在某些实施方式中，A为直链或支链C 1-20烷基，或直链或支链C 1-20烯基。在某些实施方式中，A为直链或支链C 5-20烷基，或直链或支链C 5-20烯基。在某些实施方式中，A为直链或支链C 5-20烷基。在某些实施方式中，A为具有1-5个双键的直链C 5-20烯基。
在某些实施方式中，B为-OH、直链或支链C 1-20烷基，或直链或支链C 1-20烯基。在某些实施方式中，B为-OH、直链或支链C 5-20烷基，或直链或支链C 5-20烯基。在某些实施方式中，B为-OH。在某些实施方式中，B为直链或支链C 5-20烷基。
在某些实施方式中，B为直链C 5-20烷基。在某些实施方式中，B为直链或支链C 5-20烯基。在某些实施方式中，B为具有1-5个双键的直链C 5-20烯基。
在某些实施方式中，所述双键呈Z构型。在某些实施方式中，所述双键在所述直链C 5-20烯基中的位置选自如下：C1-C2位置、C2-C3位置、C3-C4位置、C4-C5位置、C5-C6位置、C6-C7位置、C7-C8位置、C8-C9位置、C9-C10位置、 C10-C11位置、C12-C13位置、C13-C14位置或其组合。
在某些实施方式中，Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的C 3-20环烷基、-N(R) 3 +或
Figure PCTCN2020127393-appb-000027
在某些实施方式中，Q为羟基取代的直链或支链C 1-20烷基。在某些实施方式中，Q为羟基取代的直链或支链C 1-10烷基。在某些实施方式中，Q为羟基取代的直链C 1-5烷基。在某些实施方式中，Q为-CH(OH)-CH 2OH。在某些实施方式中，Q为羟基取代的C 3-20环烷基。在某些实施方式中，Q为羟基取代的C 3-10环烷基。
在某些实施方式中，Q为
Figure PCTCN2020127393-appb-000028
在某些实施方式中，Q为-N(R) 3 +，R各自独立地为H或直链C 1-10烷基。
在某些实施方式中，Q为-N(CH 3) 3 +。
在某些实施方式中，Q为
Figure PCTCN2020127393-appb-000029
R各自独立地为H或直链C 1-10烷基。
在某些实施方式中，Q为
Figure PCTCN2020127393-appb-000030
R为H。
在某些实施方式中，所述化合物具有下式的结构：
Figure PCTCN2020127393-appb-000031
其中L 3为直链或支链C 1-20烷基；A为直链或支链C 1-20烷基；和B为直链或支链C 1-20烷基。
在某些实施方式中，所述化合物含有下式的结构
Figure PCTCN2020127393-appb-000032
其中A为直链或支链C 1-20烷基。
在某些实施方式中，所述化合物具有下式的结构：
Figure PCTCN2020127393-appb-000033
其中：A为直链或支链C 1-20烷基；Q为-N(R) 3 +；和R各自独立地为直链或支链C 1-20烷基。
在某些实施方式中，所述化合物含有下式的结构：
Figure PCTCN2020127393-appb-000034
其中：A为直链或支链C 1-20烷基；B为直链或支链C 1-20烷基，或直链或支链C 1-20烯基；Q为-N(R) 3 +；和R各自独立地为直链或支链C 1-20烷基。
Figure PCTCN2020127393-appb-000035
其中：A为直链或支链C 1-20烷基，或直链或支链C 1-20烯基；B为直链或支链C 1-20烯基；Q为羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基，或
Figure PCTCN2020127393-appb-000036
和R各自独立地为直链或支链C 1-20烷基。
在某些实施方式中，所述化合物具有下式的结构：
Figure PCTCN2020127393-appb-000037
其中：A为直链或支链C 1-20烯基；B为直链或支链C 1-20烷基，或直链或支链C 1-20烯基；和Q为-N(R) 3 +；R各自独立地为直链或支链C 1-20烷基。
在某些实施方式中，所述脂质组合物包含一种或多种具有式(I)结构的化合物或其盐、水合物或溶剂化物，所述化合物选自下组：脂质106、96、93、94、84、85、108、81、88、89、109、110、103、104和105，其中所述脂质的化学名和化学结构等信息如表1所示。
在另一方面，本申请还提供了脂质组合物在制备用于核酸递送的试剂中的用途，或者脂质组合物用于递送核酸的方法，其中所述脂质组合物包含一种或多种化合物或其盐、水合物或溶剂化物，所述化合物选自下组：脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、102、107、111、112、113、114、115、116、117、118和119，其中所述脂质的化学名和化学结构等信息如表1所示。
表1.脂质72-119的缩写、结构和化学名
Figure PCTCN2020127393-appb-000038
Figure PCTCN2020127393-appb-000039
Figure PCTCN2020127393-appb-000040
Figure PCTCN2020127393-appb-000041
Figure PCTCN2020127393-appb-000042
Figure PCTCN2020127393-appb-000043
Figure PCTCN2020127393-appb-000044
Figure PCTCN2020127393-appb-000045
Figure PCTCN2020127393-appb-000046
Figure PCTCN2020127393-appb-000047
在某些实施方式中，所述化合物是合成的。根据化合物的化学结构，本领域技术人员可以通过化学合成的方式合成得到上述化合物。或者也可以通过商购的方式获得本申请所述的某些化合物。
在某些实施方式中，所述化合物是来自于中药提取物。“中药提取物”在本申请中是指通过适当的方法从中药材或药用植物中提取得到的提取物。在某些实施方式中，所述中药材或药用植物选自红景天，蒲公英，金银花或穿心莲，或其中药饮片。可以使用任何适当的提取方法获得所述中药提取物。作为一个示例，可以将红景天，蒲公英，金银花或穿心莲的中药饮片在水中浸泡，然后依次进行强火煎煮和弱火煎煮，将煎煮后的中药药液浓缩，然后依次添加氯仿-甲醇、氯仿和水充分混合，取氯仿层获得所述中药提取物。在某些实施方式中，可以进一步地从所述中药提取物中分离或纯化出所述化合物单体。
在某些实施方式中，所述中药提取物通过Bligh&Dyer法(Bligh E.G.and Dyer,W.J.,A rapid method for total lipid extraction and purification,Can.J.Biochem.Physiol.,1959,37:911-917)提取脂溶性成分获得，或者通过中药的煎煮制备提取获得。
在某些实施方式中，所述中药提取物通过中药的煎煮制备提取获得，所述中药选自红景天，蒲公英，穿心莲和金银花中药饮片。
在某些实施方式中，所述中药提取物通过如下方式获得：将中药在水中浸泡，然后依次进行强火煎煮和弱火煎煮，将煎煮后的中药药液浓缩，然后依次添加氯仿-甲醇、氯仿和水搅拌处理，取氯仿层提取获得。
脂质组合物
在某些实施方式中，本申请提供的脂质组合物包含一种或多种本申请提供的化合物或其盐、水合物或溶剂化物。所述脂质组合物可以为一种本申请提供的化合物或其盐、水合物或溶剂化物，也可以为两种或更多种本申请提供的化合物的混合物。
在某些实施方式中，所述脂质组合物还可以包含一种或多种本申请提供的化合物以外的其他脂质化合物。所述其他脂质化合物可以是，例如中性脂质、带电脂质、甾族化合物和聚合物缀合的脂质。“中性脂质”是指在所选pH值(例如生理pH值)下以不带电形式或中性两性离子形式存在的脂质化合物。 “带电脂质”是指不受有用的生理范围内的pH值(例如pH约3至约9)的限制，以带正电或带负电的形式存在的脂质化合物。
在某些实施方式中，所述脂质组合物还可以进一步包含一种或多种溶剂，其能够与本申请提供的化合物或其盐、水合物或溶剂化物混合并形成均一的混合物。
所述脂质组合物中包含的溶剂可以包含有机溶剂或溶剂混合物，例如氯仿，二氯甲烷，二乙醚，环己烷，环戊烷，苯，甲苯，甲醇或其他脂族醇，例如乙醇，丙醇，异丙醇，丁醇，叔丁醇，异丁醇，戊醇和己醇。这些溶剂可以单独地，混合地和/或任选地与合适的缓冲剂一起作为脂质组合物中的溶剂。溶剂的选择通常可以考虑溶剂的极性，在脂质核酸混合物形成的后期除去溶剂的难易程度，和/或药学上可接受的性质。在某些实施方式中，所述溶剂是无毒的，或药学上可接受的。示例性的药学上可接受的溶剂包括低级醇(1-6个碳原子)，例如甲醇，乙醇，正丙醇，异丙醇和正丁醇。在某些实施方式中，可以使用适量的溶剂，以使核酸和脂质能够形成澄清的单相混合物。
本申请提供的脂质组合物可以用于递送核酸，或者制备用于核酸递送的试剂。在某些实施方式中，所述核酸包含DNA或RNA。在某些实施方式中，所述核酸可以包括，例如，编码DNA、非编码DNA、反义核酸、信使RNA(mRNA)、长非编码RNA(lncRNA)和小RNA(例如微RNA(miRNA)、小干扰RNA(siRNA)，Piwi-相互作用RNA(piRNA)，核仁小RNA(snoRNA)，tRNA来源小RNA(tsRNA))等。
在某些实施方式中，所述核酸为单链或双链的。单链的核酸例如，miRNA，mRNA，反义DNA、反义RNA等。双链的核酸例如，siRNA，双链DNA，双链RNA等。
在某些实施方式中，所述核酸具有茎环结构。茎环结构是指在单链核酸的两个部分分别存在反向互补的序列，当通过碱基配对时，这部分反向互补的序列形成双链，而在两个反向互补区的中间的非互补部分则突出形成环的结构。茎环结构也被称为发卡结构。
在某些实施方式中，所述核酸包括小核酸。小核酸是指长度较短的(例如小于200个核苷酸)核酸。小核酸可以是非编码的，例如小RNA(例如miRNA、siRNA、piRNA、snoRNA、tsRNA)等。可以是单链的，或者是双链的。在某些实施方式中，所述核酸为长度为14-32bp、16-28bp或18-24bp的小核酸。
在某些实施方式中，所述核酸是核酸药物。核酸药物可以有多种种类，例如反义核酸、siRNA、CpG寡脱氧核苷酸、核酸适配体、编码目标蛋白的mRNA或DNA、或miRNA等。示例性的核酸药物包括，AEG35156、aganirsen、AP 12009、Apatorsen、ATL1103、AVT-02 UE、Bevasiranib Sodium、BMN 044、BMN 053、CpG 7909、Custirsen、Drisapersen、Eteplirsen、Fomivirsen、Pegaptanib，Mipomersen，Eteplirsen，Defibrotide，Nusinersen，Patisiran，Tegsedi和Fovista。
在某些实施方式中，所述核酸可用于治疗疾病。
在某些实施方案中，所述核酸用于治疗癌症、炎症、纤维化疾病、自身免疫疾病或自身炎性疾病、细菌感染、行为性和精神性病症、血液疾病、染色体疾病、先天性和遗传性疫病、结缔组织疾病、消化性疾病、耳鼻喉疾病、内分泌疾病、环境性疾病、眼部疾病、女性生殖性疾病、真菌感染、心脏病、遗传性癌症综合征、免疫系统疾病、肾脏和泌尿性疾病、肺部疾病、男性生殖新疾病、代谢性病症、口部疾病、肌肉骨骼疾病、骨髓增生异常性综合征、新生儿筛查、营养性疾病、寄生虫疾病、罕见癌症、罕见疾病、皮肤疾病和病毒感染。
在某些实施方案中，所述核酸用于治疗肝细胞癌、角膜新生血管形成、复发性或难治性间变性星形细胞瘤(WHO III级)或继发性胶质母细胞瘤(WHO IV级)、晚期鳞状细胞肺癌、肢端肥大症、银屑病、杜氏肌营养不良症、晚期非小细胞肺癌、转移性去势抗性前列腺癌、巨细胞病毒性视网膜炎，HIV感染、乙型肝炎、丙型肝炎、高脂蛋白症、全膝关节置换术、II型糖尿病、家族性淀粉样多发性神经病(FAP)、湿性黄斑变性(例如新血管性年龄相关性黄斑变性、中央凹下新血管性年龄相关性黄斑变性、渗出性年龄相关性黄斑变 性)、高胆固醇血症、克罗恩病、广泛性肝纤维化、婴儿型脊髓性肌萎缩症、黑色素瘤、新生冠状动脉病变、轻度过敏性哮喘、慢性淋巴细胞性白血病、和高甘油三酯血症、造血干细胞移植后伴肾或肺功能障碍的肝小细胞闭塞症、伴遗传性转甲状腺素蛋白淀粉样病变。
在某些实施方式中，脂质组合物可以与核酸形成脂质核酸混合物。所述核酸可以通过所述脂质核酸混合物进行递送。“脂质核酸混合物”在本申请中是指，递送核酸的基于脂质的混合物，例如脂质体。脂质体的例子包括，例如包封了核酸的脂质颗粒或脂质囊泡。脂质核酸混合物可以通过适当的方法制得，例如包括混合法，加热法，逆向蒸发法等。
混合法包括将本申请提供的化合物或其盐、水合物或溶剂化物，与待递送的核酸进行混合的步骤。在某些实施方式中，所述混合可以是本申请提供的脂质组合物与待递送的核酸的直接混合(例如脂质组合物和核酸的干粉混合，再加入适当溶剂形成脂质包封核酸的脂质核酸混合物)。在某些实施方式中，所述混合可以是将溶于适当溶剂中的本申请所述的脂质组合物与核酸混合，或将溶于适当溶剂中的核酸与本申请所述的脂质组合物混合，或将溶于适当溶剂中的本申请所述的脂质组合物与溶于适当溶剂中的核酸混合。可用于本申请所述的脂质组合物的合适的溶剂的例子如本申请说明书之前所述。可用于核酸的合适的溶剂包括水(DEPC处理过的水、双蒸水)、缓冲液、生理盐水、或者葡萄糖溶液等。所述混合步骤可以以任何合适的步骤进行，例如可以将核酸(或其溶液)加入到脂质组合物(或其溶液)中，也可以是脂质组合物(或其溶液)加入到核酸(或其溶液)中。在某些实施方式中，所述混合还可以包括例如涡旋、超声等步骤，以帮助混合均匀。
在某些实施方式中，混合法包括将本申请所述脂质组合物的乙醇溶液添加到待递送的核酸的合适体积的水性缓冲液中，通过涡旋或超声混匀后孵育得到脂质核酸混合物。在某些实施方式中，混合法包括将本申请所述脂质化合物的乙醇溶液与待递送的核酸的合适体积的乙醇溶液合并，通过涡旋或超声混匀后孵育得到脂质核酸混合物后除去乙醇，并将所得脂质核酸混合物重悬于水性缓冲液中。
在某些实施方式中，核酸和脂质化合物可以以一定的比例进行混合。所述比例取决于需要递送的核酸及其量，以及为达到核酸包封的效果所需要使用的脂质化合物。所述比例可以是满足治疗需求的任何比例，只要能形成稳定的脂质核酸混合物，并且提供所需量的核酸即可。在某些实施方式中，核酸与脂质化合物的比例可以是，例如，0.1nmol:100μg至10nmol:100μg，0.2nmol:100μg至10nmol:100μg，0.3nmol:100μg至10nmol:100μg，0.4nmol:100μg至10nmol:100μg，0.5nmol:100μg至10nmol:100μg，1nmol:100μg至10nmol:100μg，2nmol:100μg至10nmol:100μg，3nmol:100μg至10nmol:100μg，4nmol:100μg至10nmol:100μg，5nmol:100μg至10nmol:100μg，6nmol:100μg至10nmol，7nmol:100μg至10nmol，8nmol:100μg至10nmol，或9nmol:100μg至10nmol:100μg。在某些实施方式中，核酸与脂质化合物的比例可以为1nmol:100μg，2nmol:100μg，3nmol:100μg，4nmol:100μg，5nmol:100μg，6nmol:100μg，7nmol:100μg，8nmol:100μg，9nmol:100μg或10nmol:100μg。在某些实施方式中，核酸与脂质化合物的比例可以为10nmol:100μg。
所述混合法可以在任何适当的温度下进行，只要能形成能够递送核酸的脂质核酸混合物即可。在某些实施方式中，核酸和脂质化合物可以在适当的温度下进行混合，例如，但不限于，0℃至100℃，4℃至100℃，10℃至100℃，15℃至100℃，20℃至100℃，25℃至100℃，30℃至100℃，35℃至100℃，40℃至100℃，45℃至100℃，50℃至100℃，55℃至100℃，60℃至100℃，65℃至100℃，70℃至100℃，75℃至100℃，80℃至100℃，85℃至100℃，90℃至100℃，或95℃至100℃。在一些实施方式中，所述温度为0℃，4℃，10℃，20℃，30℃，40℃，50℃，55℃，60℃，65℃，70℃，75℃，80℃，85℃，90℃。在一些实施方式中，所述温度为90℃。
在某些实施方式中，所述脂质核酸混合物通过加热法制备。所述加热法包括将本申请提供的化合物或其盐、水合物或溶剂化物溶于适当溶剂中的溶液与待递送的核酸的水溶液混合得到混合溶液，并在适当的温度下加热所述混合溶液的步骤。在某些实施方式中，加热所述混合溶液的步骤在选自如下的温度下进行：25℃至100℃、30℃至100℃、40℃至100℃、50℃至100℃、60℃ 至100℃、70℃至100℃、80℃至100℃、90℃至100℃或95℃至100℃。在某些实施方式中，加热所述混合溶液的步骤在选自如下的温度下进行：约30℃、约35℃、约37℃、约40℃、约45℃、约50℃、约55℃、约60℃、约65℃、约70℃、约75℃，约80℃、约85℃、约90℃、约95℃和约100℃。在一些实施方式中，加热所述混合溶液的步骤在90℃下进行。
在某些实施方式中，所述加热法包括加热混合溶液达适当的时间。本领域技术人员可以根据所用的脂质化合物和核酸的性质以及所需的脂质核酸混合物，选择适当的加热时间。所述加热时间可为例如，约5分钟至约24小时，约5分钟至约20小时，约5分钟至约16小时，约10分钟至约20小时，约10分钟至约16小时，约15分钟至约24小时，约15分钟至约20小时，约30分钟至约24小时，约30分钟至约20小时，约40分钟至约16小时，约50分钟至约12小时，约1小时至约8小时，或约2小时至约4小时。在某些实施方式中，所述加热时间可为例如，约5分钟、约10分钟、约15分钟、约20分钟、约30分钟、约40分钟、约50分钟、约1小时、约2小时、约3小时、约4小时、约5小时、约6小时、约7小时、约8小时、约9小时、约10小时、约12小时、约16小时、约20小时或约24小时。在一些实施方案中，所述加热时间为约15分钟。
在某些实施方式中，所述加热法进一步包括冷却步骤。所述冷却可以在加热混合溶液的步骤之后在适当的温度下进行，只要不破坏形成的脂质核酸混合物即可。示例性的冷却温度包括，但不限于，25℃至-80℃，20℃至-80℃，15℃至-80℃，10℃至-80℃，4℃至-80℃，0℃至-80℃，-10℃至-80℃，-20℃至-80℃，-30℃至-80℃，-40℃至-80℃。在一些实施方案中，在加热混合溶液的步骤之后在室温下自然冷却所述混合溶液，所述室温例如为25℃或20℃。
在某些实施方式中，所述脂质核酸混合物通过逆向蒸发法制备。所述逆向蒸发法包括将核酸的水溶液加入到脂质化合物的溶于适当溶剂中的溶液，再通过超声、蒸发等方法除去溶剂。在某些实施方式中，所述逆向蒸发法进一步包括在去除溶剂之后进行水化，得到脂质核酸混合物。在一些实施方案中，所述 水化包括将水或适当的培养基加入体系中，在一些实施方案中，适当的培养基例如为OPTI-MEM培养基。
在某些实施方式中，除去溶剂的步骤在适当的温度下进行，例如，约25℃至约70℃，30℃至约70℃、约30℃至约65℃，约40℃至约65℃，约40℃至约60℃、或约50℃至约60℃。在某些实施方式中，除去溶剂的步骤在约25℃、30℃、约35℃、约37℃、约40℃、约45℃、约50℃、约55℃、约60℃、约65℃或约70℃的温度下进行。在某些实施方式中，除去溶剂的步骤在约55℃的温度下进行。
在某些实施方式中，所述核酸递送是通过口服、吸入或注射的方式递送。在某些实施方式中，所述核酸递送是通过口服的方式。在某些实施方式中，所述试剂用于通过口服的方式递送所述核酸。在某些实施方式中，所述核酸递送是体内消化道递送。
在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的感兴趣的靶器官或靶组织。
在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的心脏组织。在某些实施方式中，所述脂质核酸混合物含有脂质73和/或脂质80。
在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的肺组织。在某些实施方式中，所述脂质核酸混合物含有一种或多种选自下组的脂质化合物：脂质72，脂质76，脂质92，脂质94，脂质95，脂质97，脂质98和脂质103。
在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的肾组织。在某些实施方式中，所述脂质核酸混合物含有一种或多种选自下组的脂质化合物：脂质88，脂质91，脂质93，脂质94，脂质96，脂质97和脂质98。
在某些实施方式中，所述核酸被递送至肝。在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的脾组织。在某些实施 方式中，所述脂质核酸混合物含有一种或多种选自下组的脂质化合物：脂质76，脂质77，脂质79，脂质80，脂质81，脂质95，脂质96，脂质98，脂质99，脂质100，脂质101和脂质102。
在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的血液。在某些实施方式中，所述脂质核酸混合物含有一种或多种选自下组的脂质化合物：脂质75，脂质102，脂质104和脂质105。
在某些实施方式中，所述脂质核酸混合物被个体口服后，能够将所述核酸递送至个体的肠组织。在某些实施方式中，所述脂质核酸混合物含有脂质化合物脂质83。
在某些实施方式中，所述脂质核酸混合物被个体口服后，所述核酸被递送至两个或多个组织。某些实施方式中，所述脂质核酸混合物含有选自下组的脂质化合物：脂质76，脂质80，脂质94，脂质95，脂质96，脂质97，脂质98和脂质102。
在某些实施方式中，所述核酸递送包括体外细胞递送。“体外”在本申请中是指在多细胞的生物体以外，例如人体或动物体以外。体外细胞包括例如，体外细胞培养物、离体组织或细胞等。体外递送包括将核酸递送到体外的细胞内。例如，包括将所述脂质核酸混合物与体外细胞在允许核酸进入细胞的条件下接触。
药物组合物
在另一方面，本申请还提供了一种药物组合物，其包含脂质组合物以及核酸，其中所述脂质组合物包含一种或多种具有式(I)的化合物或其药学上可接受的盐、水合物或溶剂化物。在某些实施方式中，所述化合物选自脂质106、96、93、94、84、85、108、81、88、89、109、110、103、104、和105组成的组。
在另一方面，本申请还提供了一种药物组合物，其包含脂质组合物以及核酸，其中所述脂质组合物包含一种或多种选自脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、 102、107、111、112、113、114、115、116、117、118、和119组成的组的化合物，或其盐、水合物或溶剂化物。
本申请提供的药物组合物可以是固体或液体的形式，包括半固体、半液体、悬浮液和胶形式。固体形式例如，片剂或粉末形式。液体形式例如，口服溶液、口服糖浆、可注射液体、或气溶胶，所述气溶胶适用于，例如，吸入施用。
在某些实施方式中，所述药物组合物被配制成经口服、经吸入、经消化道、或经呼吸道施用。在某些实施方式中，所述药物组合物被配制成通过体内消化道递送的方式递送所述核酸。在某些实施方式中，所述药物组合物是口服药物组合物。当用于口服施用时，药物组合物优选为固体或液体形式。
作为用于口服施用的固体药物组合物，药物组合物可以配制成粉末、颗粒、压缩的片剂、丸剂、胶囊、咀嚼胶等形式。这类固体组合物通常还可以含有一种或多种惰性稀释剂或可食用载体(例如淀粉、乳糖或糊精)、以及一种或多种选自下组的辅料：粘合剂(例如羧甲基纤维素、乙基纤维素、微晶纤维素、黄蓍胶或明胶)、崩解剂(海藻酸、海藻酸钠、Primogel、玉米淀粉等)，润滑剂(例如硬脂酸镁或Sterotex)，助流剂(例如胶体二氧化硅)，甜味剂(例如蔗糖或糖精)，调味剂；以及着色剂。
作为用于口服施用的液体药物组合物，药物组合物可以配置成例如，酏剂、糖浆、溶液、乳液或悬浮液。当用于口服施用时，液体药物组合物含有一种或多种选自下组的辅料：甜味剂、防腐剂、染色/着色剂和增味剂。
本发明的药物组合物还可以配置成经呼吸道吸入施用。适当的吸入制剂可以包含可作为气溶胶施用的制剂。气溶胶可以以单相、双相系统或三相系统来递送。例如，气溶胶可以通过液化气和压缩气来递送，或者通过分散活性成分的适合的系统来递送。气溶胶的递送包括必要的容器、活化器、阀、子容器等，其在一起可以形成试剂盒。
在某些实施方式中，本申请的药物组合物还可以配置成通过胃肠外施用，例如注射施用。注射用液体组合物可以是溶液、悬浮液或注射粉针，可以包括 表面活性剂、防腐剂、润湿剂、分散剂、悬浮剂、缓冲剂、稳定剂和等渗剂中的一种或多种。
在某些实施方式中，所述药物组合物还可以用于通过体外细胞递送。
在某些实施方式中，所述脂质组合物和所述核酸在所述药物组合物中至少部分地或全部以混合物的形式存在。在某些实施方式中，所述混合物是通过加热法、逆向蒸发法，或混合法制得的。
在另一方面，本申请还提供了上述药物组合物在制备药物中的用途，所述药物用于预防和/或治疗能用核酸预防和/或治疗的疾病，或者用于将核酸体内递送至有此需要的对象中。
本申请提供的药物组合物可以用于治疗可用核酸治疗的疾病。常见的疾病例如，但不限于，癌症、炎症、纤维化疾病、自身免疫疾病或自身炎性疾病、细菌感染、行为性和精神性病症、血液疾病、染色体疾病、先天性和遗传性疫病、结缔组织疾病、消化性疾病、耳鼻喉疾病、内分泌疾病、环境性疾病、眼部疾病、女性生殖性疾病、真菌感染、心脏病、遗传性癌症综合征、免疫系统疾病、肾脏和泌尿性疾病、肺部疾病、男性生殖新疾病、代谢性病症、口部疾病、肌肉骨骼疾病、骨髓增生异常性综合征、新生儿筛查、营养性疾病、寄生虫疾病、罕见癌症、罕见疾病、皮肤疾病和病毒感染。
癌症的例子包括但不限于，胃癌、肺癌、结直肠癌、肝癌、胰腺癌、宫颈癌、乳腺癌、白血病、多发性骨髓瘤。
炎症的例子包括但不限于，肺炎、心肌炎、急慢性胃炎、急慢性肠炎、急慢性肝炎、急慢性肾炎、皮炎、脑炎、淋巴炎、结膜炎、角膜炎、虹膜睫状体炎、中耳炎、过敏性鼻炎、哮喘、肺纤维化慢性阻塞性肺疾病、过敏性皮炎、镰状细胞病、多发性硬化、系统性红斑狼疮、狼疮性肾炎。
已知的核酸药物可以治疗多种疾病。已知核酸药物的适应症包括，例如，肝细胞癌、角膜新生血管形成、复发性或难治性间变性星形细胞瘤(WHO III级)或继发性胶质母细胞瘤(WHO IV级)、晚期鳞状细胞肺癌、肢端肥大症、银屑病、杜氏肌营养不良症、晚期非小细胞肺癌、转移性去势抗性前列腺 癌、巨细胞病毒性视网膜炎，HIV感染、乙型肝炎、丙型肝炎、高脂蛋白症、全膝关节置换术、II型糖尿病、家族性淀粉样多发性神经病(FAP)、湿性黄斑变性(例如新血管性年龄相关性黄斑变性、中央凹下新血管性年龄相关性黄斑变性、渗出性年龄相关性黄斑变性)、高胆固醇血症、克罗恩病、广泛性肝纤维化、婴儿型脊髓性肌萎缩症、黑色素瘤、新生冠状动脉病变、轻度过敏性哮喘、慢性淋巴细胞性白血病、和高甘油三酯血症、造血干细胞移植后伴肾或肺功能障碍的肝小细胞闭塞症、伴遗传性转甲状腺素蛋白淀粉样病变。
试剂盒
在另一方面，本申请还提供了一种试剂盒，其包含：置于第一容器中的一种或多种具有所述式(I)的化合物或其药学上可接受的盐、水合物或溶剂化物，和置于第二容器中的核酸。在某些实施方式中，所述化合物选自脂质106、96、93、94、84、85、108、81、88、89、109、110、103、104、和105组成的组。
在另一方面，本申请还提供了一种试剂盒，其包含：置于第一容器中的一种或多种化合物或其药学上可接受的盐、水合物或溶剂化物，和置于第二容器中的核酸，其中所述一种或多种化合物选自由脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、102、107、111、112、113、114、115、116、117、118、和119组成的组。
在某些实施方式中，在所述试剂盒中，所述脂质组合物和所述核酸在使用前被至少部分地或全部地配制成脂质核酸混合物。在某些实施方式中，在所述试剂盒中，所述混合物是通过加热法、逆向蒸发法，或者混合法制得的。
在某些实施方式中，其中所述试剂盒被配制成口服药物组合物。
在某些实施方式中，其中所述试剂盒被配制成经口服、经吸入、经消化道、或经呼吸道施用。
在某些实施方式中，所述试剂盒被配制成通过体外细胞递送，或通过体内消化道递送的方式递送所述核酸。
在另一方面，本申请提供了试剂盒在制备药物中的用途，所述药物用于预防和/或治疗能用核酸预防和/或治疗的疾病，或者用于将核酸体内递送至有此需要的对象中。
递送核酸的方法
在另一方面，本申请提供了一种将核酸递送至靶细胞中的方法，包括向所述靶细胞施用本申请所述的药物组合物，或者本申请所述的试剂盒配制得到的脂质核酸混合物。
在另一方面，本申请提供了一种将核酸体内递送至有此需要的对象中的方法，包括向所述对象施用本申请所述的药物组合物，或者本申请所述的试剂盒配制得到的脂质核酸混合物。
在某些实施方式中，所述对象是人或动物，如哺乳动物。
在某些实施方式中，所述核酸被体内递送至所述对象的血液循环中或者靶组织/细胞中。
在某些实施方式中，所述方法包括通过口服、吸入或注射施用所述药物。
在某些实施方式中，所述方法包括经消化道、经呼吸道施用所述药物。
在某些实施方式中，所述方法包括经口服施用所述药物。
通过本申请所提供的以上技术方案，能够显著改进核酸的高效靶向递送，克服了现有技术中核酸脂质体存在包封率低、安全性差、稳定性差、制备工艺复杂、产品不均一、难以重现、以及靶向性有待进一步提高的缺陷。
本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1：中药来源脂质的提取、鉴定和合成
1.1中药的煎煮制备
1)取100g中药饮片(红景天，购自宁波海曙千草生物科技有限公司；蒲公英，金银花，穿心莲，购自北京同仁堂药店)，加入1000mL ddH 2O浸泡30min。
2)中药煎煮锅强火煎煮15min，弱火煎煮20min。
3)煎煮后的中药药液400mL加入旋转蒸发仪，60℃，60rpm，30min浓缩至100mL。
1.2中药来源脂质的提取
1)向160mL根据以上1.1项所得中药汤汁(旋转蒸发仪浓缩)中加入氯仿-甲醇混合液(氯仿:甲醇＝1:2，v/v)600mL，使得氯仿:甲醇:水＝1:2:0.8，搅拌混匀10-15min。
2)向锥形瓶中加入200mL氯仿，搅拌混匀10min。
3)向锥形瓶中加入200ml ddH 2O，使得氯仿:甲醇:水＝2:2:1.8，搅拌混匀10min。
4)除去上层液体和中间层的不溶性物质，取下层的氯仿层，-40℃冻存。
1.3中药来源的脂质鉴定
利用HPLC-MS/MS鉴定中药来源的脂质成分。
仪器条件
1)色谱条件：
仪器：Ultimate 3000；色谱柱：Kinetex C18(100×2.1mm,1.9μm)；柱温：45℃；流动相：A：乙腈:水(V/V，60:40)，溶液含10mmol/L甲酸铵，流动相B：乙腈：异丙醇(10:90，V/V)，溶液含10mmol/L甲酸铵和0.1％甲酸。流速：0.4mL/min；进样量：4μL。
2)质谱参数：
a)正模式：加热器温度300℃，鞘气流速，45arb，辅助气流速，15arb，吹扫气流速，1arb，喷嘴电压，3.0KV，毛细管温度，350℃，S-Lens RF Level,30％。扫描范围：200-1500。
b)负模式：加热器温度300℃，鞘气流速，45arb，辅助气流速，15arb，吹扫气流速，1arb，喷嘴电压，2.5KV，毛细管温度，350℃，S-Lens RF Level,60％。扫描范围：200-1500。
1.4中药来源的脂质合成
对于鉴定出的脂质72-119，购买得到除了脂质106、108和110之外的每种脂质。
实施例2：脂质核酸混合物的制备(加热法)：
脂质氯仿溶液浓度为10mg/ml(储存液)，并按照脂质化合物编号进行分组。
将小RNA 1nmol(如表2所示)用400μl DEPC处理过的水于玻璃管中溶解，加入10μl相应的脂质储存液溶液，使得体系中脂质的质量为100μg，充分混匀，90℃水浴加热15min后自然冷却，得脂质-小RNA的脂质核酸混合物。
表2.小RNA序列信息(订购于广州锐博生物科技有限公司)
Figure PCTCN2020127393-appb-000048
实施例3：脂质核酸混合物的体内递送研究
1.实验动物：
实验所用C57BL/6雄性小鼠购自北京维通利华实验动物技术有限公司，约6周龄，20-24g。饲养于中国医学科学院基础医学研究所动物中心SPF级动物房。小鼠灌胃前禁食12h。
2.脂质-小RNA混合物制备：
按照实施例2的方法制备脂质核酸混合物。将制备好的脂质核酸混合物以每只小鼠100μg脂质:1nmol单链小RNA PGY-sRNA-26的剂量进行口服给药。
3.相对进入量检测：
3.1实验分组：
1)空白组：小鼠(n＝4)灌胃500μl生理盐水，该组作为空白对照；
2)自由摄取组：小鼠(n＝4)直接灌胃sRNA溶液(1nmol/只，500μl)，该组作为阴性对照组；
3)脂质处理组：将步骤2中制备的500μl脂质核酸混合物以灌胃方式给药小鼠(n＝4)。
3.2组织取样提取RNA：
每组小鼠灌胃12h后，眼球取血500μl，加入1.5ml Trizol Reagent LS(购自Invitrogen)充分混匀裂解。组织样品加入3ml Trizol Reagent LS，匀浆使其充分裂解，组织取样：肺/肾/脾/心/肠。取1ml血液或组织裂解液按如下步骤提取RNA：
4℃，12,000rpm离心5min，弃沉淀，将Trizol转移到新的离心管中；
按200μL氯仿/mL Trizol加入氯仿，充分振荡，混匀后室温放置5min；
4℃，12,000rpm，离心15min；
吸取上层水相，至另一离心管中，按0.4mL异丙醇/mL Trizol加入异丙醇混匀，低温放置10-20min；
4℃，12,000rpm，离心15min，弃上清，RNA沉于管底；
加入1mL 75％乙醇，温和振荡离心管，悬浮沉淀；
4℃，12,000rpm，离心10min，弃上清，加入1mL 75％乙醇，温和振荡离心管，悬浮沉淀；
4℃，12,000rpm，离心10min，弃上清，室温晾干，用50-100μL RNase-free 的H 2O溶解RNA样品，测OD值定量RNA浓度。
3.3将sRNA逆转录为cDNA：
通过逆转录试剂盒(High-Capacity cDNA Reverse Transcription Kits，Applied Biosystems，cat.no.4368813)，用茎环法(stem-loop法)(参见例如Real-time quantification of microRNAs by stem-loop RT-PCR，Nucleic Acids Res.2005 Nov 27；33(20):e179，通过引用并入本文)将sRNA逆转录为cDNA，逆转录体系如下：模板RNA(200ng/μL)10μL，10X RT缓冲液2.0μL，25X dNTP Mix(100mM)0.8μL，U6RT stem-loop引物2.0μL，PGY-sRNA-26RT stem-loop引物2.0μL，MultiScribe TM逆转录酶1.0μL，RNA酶抑制剂1.0μL，无核酸酶H 2O 1.2μL，瞬时离心后，放入PCR仪反应，反应条件如下：(1)25℃，10min；(2)37℃，120min；(3)85℃，5min；(4)4℃，终止反应。反应结束后加入20μL无RNA酶ddH 2O，补足终体积至40μL。该逆转录过程中使用的茎环法引物由北京鸿迅生物技术有限公司合成(U6RT引物，因为RT-qPCR反应对小RNA的定量只能是相对定量，所以以U6作为标准的参考基因，计算其相对表达量)：
U6RT stem-loop引物：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAAATATG(SEQ ID No.5)；
PGY-sRNA-26RT stem-loop引物：
GTCGTATCCAGTGCACGCTCCGAGGTATTCGCACTGGATACGACACGCTT(SEQ ID No.2)。
3.4定量PCR扩增反应：
qPCR反应体系总体积10μl，包括：5μL 2×SYBR Green Master Mix，0.5μl正向引物(10μM)，0.5μl反向引物(10μM),1μl逆转录得到的cDNA,3μl无RNA酶ddH2O。使用LightCycler 480荧光定量PCR仪，PCR反应条件是：95℃，持续5min预变性，开始进入PCR扩增循环：(1)95℃，10s；(2)55℃，10s；(3)72℃，20s；总共进行40个循环；最后40℃持续10s降温。扩增反应正向引物和反向引物均由北京擎科新业生物技术有限公司设计和合成(U6F引物： GCGCGTCGTGAAGCGTTC(SEQ ID No.6)，U6R引物：GTGCAGGGTCCGAGGT(SEQ ID No.7)；PGY-sRNA-26正向引物：TCGCGCTCCGGAATGATTGGG(SEQ ID No.3)，反向引物miR all rev：GTGCACGCTCCGAGGT(SEQ ID No.4))。
3.5利用2-ΔCt法计算相对表达量。
4.实验结果以图的形式示出。
图1-45分别显示了空白组、自由摄取组和测试的不同脂质递送单链小RNA PGY-sRNA-26进入不同靶组织的结果。图中所有的数据都按照双尾t检验进行了差异水平计算。对于统计检验有显著性的结果，在图中以星号标记显示，*表示P<0.05，**表示P<0.01，星号越多表示显著性越强。部分数据由于个体差异较大，虽然能看出有明显趋势，但是经过计算没有显著性差异。
实施例4：脂质核酸混合物的药物代谢动力学研究
1.实验动物：
实验所用C57BL/6雄性小鼠购自北京维通利华实验动物技术有限公司，约6周龄，20-24g。饲养于中国医学科学院基础医学研究所动物中心SPF级动物房。小鼠灌胃前禁食12h。
2.脂质-小RNA混合物制备：
按照实施例2的方法制备脂质核酸混合物。将制备好的脂质核酸混合物以每只小鼠100μg脂质:1nmol单链小RNA PGY-sRNA-26的剂量进行口服或尾静脉注射给药，口服的剂量是尾静脉注射的一半。
3.相对进入量检测：
3.1实验分组：
1)空白组：小鼠(n＝6)灌胃500μl生理盐水，该组作为空白对照；
2)灌胃组：将步骤2中制备的200μl脂质核酸混合物以灌胃方式给药小鼠(n＝15)；
3)静脉注射组：将步骤2中制备的100μl脂质核酸混合物以尾静脉注射方式给药小鼠(n＝15)。
3.2组织取样提取RNA：
在灌胃组及静脉注射组给药后第3小时、第6小时、第9小时、第12小时和第24小时的每个时间点分别取3只小鼠，按照实施例3的方法提取各个器官的RNA。
3.3将sRNA逆转录为cDNA：
按照实施例3的方法将自各个器官中提取的RNA逆转录为cDNA。
3.4定量PCR扩增反应：
按照实施例3的方法进行定量PCR扩增反应。
3.5利用2-ΔCt法计算相对表达量。
4.实验结果以图的形式示出。
图46-49分别显示了脂质93、94、96、100通过灌胃给药的方式递送单链小RNA PGY-sRNA-26进入不同靶组织的不同时间点的结果；图50-54分别显示了脂质94、96、98、100、101通过尾静脉注射给药的方式递送单链小RNA PGY-sRNA-26进入不同靶组织的不同时间点的结果。
结论：
脂质72、73、74、75、76、77、78、79、80和81均可以有效口服递送sRNA单链核酸进入小鼠血液及肺、肾、脾、心脏组织。
脂质82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、107、109、111、112、113、114、115、116、117、118和119均可以有效口服递送sRNA单链核酸进入小鼠血液及肺、肾、脾、心、肠组织。
相比于对照具有显著性差异的实验结果如下：
1.脂质72可显著有效口服递送sRNA单链核酸进入小鼠肺组织。
2.脂质73可显著有效口服递送sRNA单链核酸进入小鼠心脏组织。
3.脂质75可显著有效口服递送sRNA单链核酸进入小鼠血液。
4.脂质76可显著有效口服递送sRNA单链核酸进入小鼠肺组织和脾组织。
5.脂质77可显著有效口服递送sRNA单链核酸进入小鼠脾组织。
6.脂质79可显著有效口服递送sRNA单链核酸进入小鼠脾组织。
7.脂质80可显著有效口服递送sRNA单链核酸进入小鼠脾组织和心脏组织。
8.脂质81可显著有效口服递送sRNA单链核酸进入小鼠脾组织。
9.脂质83可显著有效口服递送sRNA单链核酸进入小鼠肠组织。
10.脂质88可显著有效口服递送sRNA单链核酸进入小鼠肾组织。
11.脂质91可显著有效口服递送sRNA单链核酸进入小鼠肾组织。
12.脂质92可显著有效口服递送sRNA单链核酸进入小鼠肺组织。
13.脂质93可显著有效口服递送sRNA单链核酸进入小鼠肾组织。
14.脂质94可显著有效口服递送sRNA单链核酸进入小鼠肺组织和肾组织。
15.脂质95可显著有效口服递送sRNA单链核酸进入小鼠肺组织和脾组织。
16.脂质96可显著有效口服递送sRNA单链核酸进入小鼠肾组织和脾组织。
17.脂质97可显著有效口服递送sRNA单链核酸进入小鼠肺组织和肾组织。
18.脂质98可显著有效口服递送sRNA单链核酸进入小鼠肺组织、肾组织和脾组织。
19.脂质99可显著有效口服递送sRNA单链核酸进入小鼠脾组织。
20.脂质100可显著有效口服递送sRNA单链核酸进入小鼠脾组织。
21.脂质101可显著有效口服递送sRNA单链核酸进入小鼠脾组织。
22.脂质102可显著有效口服递送sRNA单链核酸进入小鼠血液和脾组织。
23.脂质103可显著有效口服递送sRNA单链核酸进入小鼠肺组织。
24.脂质104可显著有效口服递送sRNA单链核酸进入小鼠血液。
25.脂质105可显著有效口服递送sRNA单链核酸进入小鼠血液。
其中脂质73和80可以显著有效口服递送sRNA单链核酸进入小鼠心组织。脂质72、76、92、94、95、97、98和103可以显著有效口服递送sRNA单链核酸进入小鼠肺组织。脂质88、91、93、94、96、97和98可以显著有效口服递送sRNA单链核酸进入小鼠肾组织。脂质76、77、79、80、81、95、96、98、99、100、101和102可以显著有效口服递送sRNA单链核酸进入小鼠脾组织。脂质75、102、104和105可以显著有效口服递送sRNA单链核酸进入小鼠血液。脂质83可以显著有效口服递送sRNA单链核酸进入小鼠肠组织。
对于实验中所有的6个器官来看，不同脂质单体递送核酸进入效果最显著的为脾、肺、肾三个器官，进入脾具有显著性的脂质最多，肺次之，肾最后，脂质递送核酸进入血液的效果也十分显著。脂质98的递送效果最显著，其递送核酸进入脾、肺、肾均具有显著性，有望作为一种较为广谱的核酸递送载体。此外，脂质97、76、80、94、95、96和102递送核酸进入两种器官具有显著性。
药物代谢及动力学显示，脂质递送核酸进入不同器官达到进入量峰值的时间点可能不同。例如，当以灌胃给药的方式进行递送时，脂质93、94、96和100递送核酸进入不同器官达到进入量峰值的时间点不同。其中，脂质93递送核酸进入肠组织、胃组织的量在3小时最高，进入肺组织、肾组织的量在6小时最高，进入脑组织的量在9小时最高，进入脾组织的量在12小时最高；脂质94递送核酸进入心组织、脑组织、肠组织、脾组织、胃组织的量在3小时最高，进入肾组织、肺组织的量在9小时最高；脂质96递送核酸进入肾组织、肠组织的量在3小时最高；脂质100递送核酸进入胃组织、肠组织的量在6小时最高。当以尾静脉注射给药的方式进行递送时，脂质94递送核酸进入胃组织的量在3小时最高，进入脾组织、脑组织、肠组织的量在6小时最高，进入肾组织的量在9小时最高；脂质96递送核酸进入肾组织、肝组织的量在3小时最高，进入脑组织、肠组织、肺组织的量在6小时最高，进入心组织的量在9小时最高；脂质98递送核酸进入胃组织的量在3小时最高，进入肾组织、肠组织、脑组织、肝组织、心 组织的量在6小时最高；脂质100递送核酸进入胃组织的量在6小时最高，进入肾组织的量在9小时最高；脂质101递送核酸进入肾组织、肠组织、脑组织的量在3小时最高，进入胃组织的量在12小时最高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (104)

  1. 脂质组合物在制备用于核酸递送的试剂中的用途，所述脂质组合物包含一种或多种具有式(I)的化合物或其盐、水合物或溶剂化物：
    Figure PCTCN2020127393-appb-100001
    其中：
    L 1为不存在、-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；
    L 2为不存在、-O-C(O)-或-NH-C(O)-；
    L 3为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基、直链或支链C 1-20杂烯基或
    Figure PCTCN2020127393-appb-100002
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100003
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  2. 根据权利要求1所述的用途，其中所述脂质组合物用于通过口服、吸入或注射的方式递送所述核酸。
  3. 根据权利要求2所述的用途，其中所述脂质组合物用于通过口服的方式递送所述核酸。
  4. 根据前述权利要求中任一项所述的用途，其中所述递送包括体外细胞递送，或体内消化道递送。
  5. 根据前述权利要求中任一项所述的用途，其中所述脂质组合物与核酸混合制备成脂质核酸混合物，可选地，所述的脂质核酸混合物通过加热法、逆向蒸发法，或者混合法制得。
  6. 根据权利要求5所述的用途，其中所述加热法包括将所述脂质组合物与所述核酸混合获得混合物，并在选自25℃至100℃、30℃至100℃、40℃至100℃、50℃至100℃、60℃至100℃、70℃至100℃、80℃至100℃、90℃至100℃，和95℃至100℃的温度下加热所述混合物。
  7. 根据权利要求6所述的用途，其中在选自30℃、35℃、37℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃，80℃、85℃、90℃、95℃和100℃的温度下加热所述混合物。
  8. 根据权利要求5-7任一项所述的用途，其中将所述脂质组合物与所述核酸混合包括将所述脂质组合物的有机溶剂溶液加入到所述核酸的水溶液中。
  9. 根据权利要求5所述的用途，其中所述逆向蒸发法包括将所述核酸的水溶液与脂质化合物的有机溶剂溶液混合以得到混合溶液，然后去除所述混合溶液中的有机溶剂。
  10. 根据权利要求9所述的用途，其中在选自约25℃至约70℃，30℃至约70℃、约30℃至约65℃，约40℃至约65℃，约40℃至约60℃、或约50℃至约60℃的温度下去除所述混合溶液中的有机溶剂。
  11. 根据权利要求9所述的用途，其中将所述核酸的水溶液与脂质化合物的有机溶剂溶液混合包括将所述核酸的水溶液加入到所述脂质化合物的有机溶剂溶液中。
  12. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；
    L 2为不存在、-O-C(O)-或-NH-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100004
    A，B，Q，R和n如权利要求1所定义。
  13. 根据权利要求1-11中任一项所述的用途，其中
    L 1为不存在、-CH 2-O-C(O)-或-CH 2-O-；
    L 2为-O-C(O)-；
    L 3为直链或支链C 1-20烷基或
    Figure PCTCN2020127393-appb-100005
    A，B，Q，R和n如权利要求1所定义。
  14. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-C(O)-；
    L 2为不存在或-O-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100006
    A，B，Q，R和n如权利要求1所定义。
  15. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-；
    L 2为不存在或-O-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100007
    A，B，Q，R和n如权利要求1所定义。
  16. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-C(O)-或-CH 2-O-；
    L 2为不存在；
    L 3为
    Figure PCTCN2020127393-appb-100008
    A，B，Q，R和n如权利要求1所定义。
  17. 根据权利要求1-11中任一项所述的用途，其中
    L 1为不存在；
    L 2为-O-C(O)-；
    L 3为直链或支链C 1-20烷基；
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100009
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  18. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-C(O)-；
    L 2为不存在；
    L 3为
    Figure PCTCN2020127393-appb-100010
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100011
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  19. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-；
    L 2为不存在；
    L 3为
    Figure PCTCN2020127393-appb-100012
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100013
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  20. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-；
    L 2为-O-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100014
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100015
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  21. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-C(O)-；
    L 2为-O-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100016
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯 基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100017
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  22. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CH 2-O-C(O)-；
    L 2为-O-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100018
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100019
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  23. 根据权利要求1-11中任一项所述的用途，其中
    L 1为-CR(OH)-；
    L 2为-NH-C(O)-；
    L 3为
    Figure PCTCN2020127393-appb-100020
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或 直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100021
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  24. 根据前述权利要求中任一项所述的用途，其中所述化合物具有下式：
    Figure PCTCN2020127393-appb-100022
    其中L 1、L 2、L 3、A、B和Q如前述权利要求中任一项所述。
  25. 根据前述权利要求中任一项所述的用途，其中A为直链或支链C 1-20烷基，或直链或支链C 1-20烯基。
  26. 根据前述权利要求中任一项所述的用途，其中A为直链或支链C 5-20烷基，或直链或支链C 5-20烯基。
  27. 根据前述权利要求中任一项所述的用途，其中A为直链或支链C 5-20烷基。
  28. 根据前述权利要求中任一项所述的用途，其中A为具有1-5个双键的直链C 5-20烯基。
  29. 根据前述权利要求中任一项所述的用途，其中B为-OH、直链或支链C 1-20烷基，或直链或支链C 1-20烯基。
  30. 根据前述权利要求中任一项所述的用途，其中B为-OH、直链或支链C 5-20烷基，或直链或支链C 5-20烯基。
  31. 根据前述权利要求中任一项所述的用途，其中B为-OH。
  32. 根据前述权利要求中任一项所述的用途，其中B为直链或支链C 5-20烷基。
  33. 根据前述权利要求中任一项所述的用途，其中B为直链C 5-20烷基。
  34. 根据前述权利要求中任一项所述的用途，其中B为直链或支链C 5-20烯基。
  35. 根据前述权利要求中任一项所述的用途，其中B为具有1-5个双键的直链C 5-20烯基。
  36. 根据权利要求28或35所述的用途，其中所述双键呈Z构型。
  37. 根据权利要求28或35所述的用途，其中所述双键在所述直链C 5-20烯基中的位置选自如下：C1-C2位置、C2-C3位置、C3-C4位置、C4-C5位置、C5-C6位置、C6-C7位置、C7-C8位置、C8-C9位置、C9-C10位置、C10-C11位置、C12-C13位置、C13-C14位置或其组合。
  38. 根据前述权利要求中任一项所述的用途，其中Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的C 3-20环烷基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100023
  39. 根据前述权利要求中任一项所述的用途，其中Q为羟基取代的直链或支链C 1-20烷基。
  40. 根据前述权利要求中任一项所述的用途，其中Q为羟基取代的直链或支链C 1-10烷基。
  41. 根据前述权利要求中任一项所述的用途，其中Q为羟基取代的直链C 1-5烷基。
  42. 根据前述权利要求中任一项所述的用途，其中Q为-CH(OH)-CH 2OH。
  43. 根据前述权利要求中任一项所述的用途，其中Q为羟基取代的C 3-20环烷基。
  44. 根据前述权利要求中任一项所述的用途，其中Q为羟基取代的C 3-10环烷基。
  45. 根据前述权利要求中任一项所述的用途，其中Q为
    Figure PCTCN2020127393-appb-100024
  46. 根据前述权利要求中任一项所述的用途，其中Q为-N(R) 3 +，R各自独立地为H或直链C 1-10烷基。
  47. 根据前述权利要求中任一项所述的用途，其中Q为-N(CH 3) 3 +。
  48. 根据前述权利要求中任一项所述的用途，其中Q为
    Figure PCTCN2020127393-appb-100025
    R各自独立地为H或直链C 1-10烷基。
  49. 根据前述权利要求中任一项所述的用途，其中Q为
    Figure PCTCN2020127393-appb-100026
    R为H。
  50. 根据权利要求1-11中任一项所述的用途，其中所述脂质组合物包含具有下式的化合物：
    Figure PCTCN2020127393-appb-100027
    其中：
    L 3为直链或支链C 1-20烷基；
    A为直链或支链C 1-20烷基；
    B为直链或支链C 1-20烷基。
  51. 根据权利要求1-11中任一项所述的用途，其中所述脂质组合物包含具有下式的化合物：
    Figure PCTCN2020127393-appb-100028
    其中A为直链或支链C 1-20烷基。
  52. 根据权利要求1-11中任一项所述的用途，其中所述脂质组合物包含具有下式的化合物：
    Figure PCTCN2020127393-appb-100029
    其中：
    A为直链或支链C 1-20烷基；
    Q为-N(R) 3 +；
    R各自独立地为直链或支链C 1-20烷基。
  53. 根据权利要求1-11中任一项所述的用途，其中所述组合物包含具有下式的化合物：
    Figure PCTCN2020127393-appb-100030
    其中：
    A为直链或支链C 1-20烷基；
    B为直链或支链C 1-20烷基，或直链或支链C 1-20烯基；
    Q为-N(R) 3 +；
    R各自独立地为直链或支链C 1-20烷基。
  54. 根据权利要求1-11中任一项所述的用途，其中所述脂质组合物包含具有下式的化合物：
    Figure PCTCN2020127393-appb-100031
    其中：
    A为直链或支链C 1-20烷基，或直链或支链C 1-20烯基；
    B为直链或支链C 1-20烯基；
    Q为羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基，或
    Figure PCTCN2020127393-appb-100032
    R各自独立地为直链或支链C 1-20烷基。
  55. 根据权利要求1-11中任一项所述的用途，其中所述脂质组合物包含具有下式的化合物：
    Figure PCTCN2020127393-appb-100033
    其中：
    A为直链或支链C 1-20烯基；
    B为直链或支链C 1-20烷基，或直链或支链C 1-20烯基；
    Q为-N(R) 3 +；
    R各自独立地为直链或支链C 1-20烷基。
  56. 根据权利要求1-11中任一项所述的用途，其中所述脂质组合物包含一种或多种选自下组的化合物：
    Figure PCTCN2020127393-appb-100034
  57. 脂质组合物在制备用于核酸递送的试剂中的用途，所述脂质组合物包含一种或多种选自下组的化合物或其盐、水合物或溶剂化物：
    脂质化合物编号 化学名 72 1,2-二油酰基-sn-甘油基-3-磷脂酰乙醇胺 73 三亚麻精 74 1,2-棕榈油酸-3-油酸甘油酯 75 1,2-棕榈油酸-3-棕榈酸甘油酯 76 1,3-棕榈酸-2-肉豆蔻酸甘油酯 77 1,2-硬脂酸-3-油酸甘油酯 78 1,2-油酸-3-花生酸甘油酯 79 1,2-油酸-3-二十二碳酸甘油酯 80 1,2-肉豆蔻酸-3-棕榈酸甘油酯 82 Q-10，泛醌50，泛醌10 83 1-棕榈酰-2-羟基-sn-甘油基-3-磷酸乙醇胺 86 1-棕榈酰基-2-油酰基-sn-甘油基-3-磷酸胆碱，粉末 87 1-硬脂酰基-2-油酰基-sn-甘油基-3-磷酸胆碱 90 N-木香油酰基-D-赤型-神经鞘氨醇 91 N-神经酰基-D-赤型-神经鞘氨醇 92 1-花生四烯基-2-羟基-sn-甘油基-3-磷酸胆碱 95 1-油酰基-2-羟基-sn-甘油基-3-磷酸胆碱 97 D-赤型-神经鞘氨醇(C22基) 98 D-赤型-神经鞘氨醇 99 D-赤型-神经鞘氨醇(C14基) 100 D-赤型-神经鞘氨醇(C16基) 101 D-赤型-神经鞘氨醇(C20基) 102 D-赤型-神经鞘氨醇 107 1,2-二亚油酰基-sn-甘油基-3-磷酸胆碱 111 1-棕榈酸-3-硬脂酸甘油酯 112 二十烷酸甘油三酯 113 1-棕榈酰-2-羟基-sn-甘油基-3-磷脂酰胆碱 114 1-十八烷酰基-2-羟基-sn-甘油基-3-磷酸胆碱
    115 1-十七碳烯酰基-sn-甘油基-3-磷酸乙醇胺 116 1-(9Z-十八烯酰基)-sn-甘油基-3-磷酸乙醇胺 117 1,2-二-(9Z-十八烯酰基)-sn-甘油基-3-磷酸胆碱 118 1-棕榈酰基-2-油酰基-sn-甘油基-3-磷脂酰胆碱 119 1,2-二十六烷酰基-sn-甘油基-3-磷酸胆碱
  58. 根据前述权利要求中任一项所述的用途，其中所述盐是药学上可接受的盐。
  59. 根据前述权利要求中任一项所述的用途，其中所述化合物来自于中药提取物。
  60. 根据权利要求59所述的用途，其中所述中药提取物通过中药的煎煮制备提取获得，所述中药选自红景天，蒲公英，穿心莲和金银花中药饮片。
  61. 根据权利要求60所述的用途，其中所述中药提取物通过如下方式获得：将中药在水中浸泡，然后依次进行强火煎煮和弱火煎煮，将煎煮后的中药药液浓缩，然后依次添加氯仿-甲醇、氯仿和水搅拌处理，取氯仿层提取获得。
  62. 根据权利要求1-58中任一项所述的用途，其中所述化合物是合成的。
  63. 根据前述权利要求任一项所述的用途，其中所述核酸包含DNA或RNA，任选地，所述DNA选自编码DNA和非编码DNA，所述RNA选自反义核酸、mRNA、lncRNA和小RNA。
  64. 根据权利要求63所述的用途，其中所述核酸包含长度为14-32bp、16-28bp或18-24bp的小核酸。
  65. 根据前述权利要求任一项所述的用途，其中所述核酸为单链或双链的。
  66. 根据前述权利要求任一项所述的用途，其中所述核酸具有茎环结构。
  67. 根据前述权利要求任一项所述的用途，其中所述核酸用于治疗疾病。
  68. 根据权利要求67所述的用途，其中所述核酸用于治疗癌症、炎症、纤维化疾病、自身免疫疾病或自身炎性疾病、细菌感染、行为性和精神性病症、血 液疾病、染色体疾病、先天性和遗传性疫病、结缔组织疾病、消化性疾病、耳鼻喉疾病、内分泌疾病、环境性疾病、眼部疾病、女性生殖性疾病、真菌感染、心脏病、遗传性癌症综合征、免疫系统疾病、肾脏和泌尿性疾病、肺部疾病、男性生殖新疾病、代谢性病症、口部疾病、肌肉骨骼疾病、骨髓增生异常性综合征、新生儿筛查、营养性疾病、寄生虫疾病、罕见癌症、罕见疾病、皮肤疾病和病毒感染。
  69. 根据权利要求67所述的用途，其中所述核酸用于治疗肝细胞癌、角膜新生血管形成、复发性或难治性间变性星形细胞瘤(WHO III级)或继发性胶质母细胞瘤(WHO IV级)、晚期鳞状细胞肺癌、肢端肥大症、银屑病、杜氏肌营养不良症、晚期非小细胞肺癌、转移性去势抗性前列腺癌、巨细胞病毒性视网膜炎，HIV感染、乙型肝炎、丙型肝炎、高脂蛋白症、全膝关节置换术、II型糖尿病、家族性淀粉样多发性神经病(FAP)、湿性黄斑变性(例如新血管性年龄相关性黄斑变性、中央凹下新血管性年龄相关性黄斑变性、渗出性年龄相关性黄斑变性)、高胆固醇血症、克罗恩病、广泛性肝纤维化、婴儿型脊髓性肌萎缩症、黑色素瘤、新生冠状动脉病变、轻度过敏性哮喘、慢性淋巴细胞性白血病、和高甘油三酯血症、造血干细胞移植后伴肾或肺功能障碍的肝小细胞闭塞症、伴遗传性转甲状腺素蛋白淀粉样病变。
  70. 一种药物组合物，其包含脂质组合物以及核酸，其中所述脂质组合物包含一种或多种具有式(I)的化合物或其药学上可接受的盐、水合物或溶剂化物：
    Figure PCTCN2020127393-appb-100035
    其中：
    L 1为不存在、-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；
    L 2为不存在、-O-C(O)-或-NH-C(O)-；
    L 3为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基、 直链或支链C 1-20杂烯基或
    Figure PCTCN2020127393-appb-100036
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100037
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  71. 一种药物组合物，其包含脂质组合物和核酸，其中所述脂质组合物包含一种或多种选自脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、98、99、100、101、102、107、111、112、113、114、115、116、117、118、和119，或其盐、水合物或溶剂化物组成的组的化合物。
  72. 根据权利要求70或71所述的药物组合物，其中所述药物组合物被配制成经口服、经吸入、经消化道、或经呼吸道施用。
  73. 根据权利要求70或71所述的药物组合物，其中所述药物组合物用于通过体外细胞递送，或通过体内消化道递送的方式递送所述核酸。
  74. 根据权利要求70或71所述的药物组合物，其中所述药物组合物是口服药物组合物。
  75. 根据权利要求70-74任一项所述的药物组合物，其中所述脂质组合物和所述核酸至少部分地或全部以脂质核酸混合物的形式存在。
  76. 根据权利要求75所述的药物组合物，其中所述脂质核酸混合物是通过加热法、逆向蒸发法，或混合法制得的。
  77. 根据权利要求76所述的药物组合物，其中所述加热法包括将所述脂质组合物与所述核酸混合获得混合物，并在选自25℃至100℃，50℃至100℃，80℃至100℃，或95℃至100℃的温度下加热所述混合物。
  78. 根据权利要求77所述的药物组合物，其中在选自37℃，60℃，80℃或100℃的温度下加热所述混合物。
  79. 根据权利要求76-78中任一项所述的药物组合物，其中将所述脂质组合物与所述核酸混合包括将所述脂质组合物的有机溶剂溶液加入到所述核酸的水溶液中。
  80. 根据权利要求76所述的药物组合物，其中所述逆向蒸发法包括将所述核酸的水溶液与脂质化合物的有机溶剂溶液混合以得到混合溶液，然后在25℃至70℃，30℃至65℃，或40℃至60℃的温度下去除所述混合溶液中的有机溶剂。
  81. 根据权利要求80所述的药物组合物，其中在55℃的温度下去除所述混合溶液中的有机溶剂。
  82. 根据权利要求76和80-81中任一项所述的药物组合物，其中所述逆向蒸发法包括将所述核酸的水溶液加入到所述脂质组合物的有机溶剂溶液中。
  83. 根据权利要求70-82中任一项所述的药物组合物在制备药物中的用途，所述药物用于预防和/或治疗能用核酸预防和/或治疗的疾病，或者用于将核酸体内递送至有此需要的对象中。
  84. 一种试剂盒，其包含：
    置于第一容器中的一种或多种具有式(I)的化合物或其药学上可接受的盐、水合物或溶剂化物：
    Figure PCTCN2020127393-appb-100038
    置于第二容器中的核酸；
    其中：
    L 1为不存在、-CH 2-O-C(O)-、-CH 2-O-或-CR(OH)-；
    L 2为不存在、-O-C(O)-或-NH-C(O)-；
    L 3为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基、直链或支链C 1-20杂烯基或
    Figure PCTCN2020127393-appb-100039
    A为直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    B为-OH、直链或支链C 1-20烷基、直链或支链C 1-20烯基、直链或支链C 1-20杂烷基或直链或支链C 1-20杂烯基；
    Q为-H、-COOH、羟基取代的直链或支链C 1-20烷基、羟基取代的直链或支链C 1-20烯基、羟基取代的C 3-20环烷基、羟基取代的C 3-20环烯基、-N(R) 3 +或
    Figure PCTCN2020127393-appb-100040
    R各自独立地为H或直链或支链C 1-20烷基；
    n为0、1或2。
  85. 一种试剂盒，其包含：
    置于第一容器中的一种或多种化合物或其药学上可接受的盐、水合物或溶剂化物，和置于第二容器中的核酸，其中所述一种或多种化合物选自由脂质72、73、74、75、76、77、78、79、80、82、83、86、87、90、91、92、95、97、 98、99、100、101、102、107、111、112、113、114、115、116、117、118、和119组成的组。
  86. 根据权利要求84或85所述的试剂盒，其中所述脂质组合物和所述核酸在使用前被至少部分地或全部地配制成脂质核酸混合物。
  87. 根据权利要求86所述的试剂盒，其中所述脂质核酸混合物是通过加热法、逆向蒸发法，或者混合法制得的。
  88. 根据权利要求87所述的试剂盒，其中所述加热法在选自如下的温度下进行：25℃至100℃，50℃至100℃，80℃至100℃，或95℃至100℃的温度下进行。
  89. 根据权利要求88所述的试剂盒，其中所述加热法在选自如下的温度下进行：37℃，60℃，80℃或100℃的温度下进行。
  90. 根据权利要求87-89任一项所述的试剂盒，其中所述加热法包括将所述脂质组合物的有机溶剂溶液加到所述核酸的水溶液中。
  91. 根据权利要求87所述的试剂盒，所述逆向蒸发法在选自如下的温度下进行：25℃至70℃，30℃至65℃，或40℃至60℃的温度下进行。
  92. 根据权利要求91所述的试剂盒，所述逆向蒸发法在55℃的温度下进行。
  93. 根据权利要求87和91-92任一所述的试剂盒，其中所述逆向蒸发法包括将核酸的水溶液加入到所述脂质组合物的有机溶剂溶液中。
  94. 根据权利要求84-93任一项所述的试剂盒，其中所述试剂盒被配制成口服药物组合物。
  95. 根据权利要求84-93中任一项所述的试剂盒，其中所述试剂盒被配制成经口服、经吸入、经消化道、或经呼吸道施用。
  96. 根据权利要求84-93中任一项所述的试剂盒，其中所述试剂盒被配制成通过体外细胞递送，或通过体内消化道递送的方式递送所述核酸。
  97. 根据权利要求84-96中任一项所述的试剂盒在制备药物中的用途，所述药物用于预防和/或治疗能用核酸预防和/或治疗的疾病，或者用于将核酸体内递送至有此需要的对象中。
  98. 一种将核酸递送至靶细胞中的方法，包括向所述靶细胞施用如权利要求70-82中任一项所述的药物组合物，或者由权利要求84-96中任一项所述的试剂盒配制得到的脂质核酸混合物。
  99. 一种将核酸体内递送至有此需要的对象中的方法，包括向所述对象施用如权利要求70-82中任一项所述的药物组合物，或者由权利要求84-96中任一项所述的试剂盒配制得到的脂质核酸混合物。
  100. 根据权利要求99所述的方法，其中所述对象是人或动物，如哺乳动物。
  101. 根据权利要求99或100所述的方法，其中所述核酸被体内递送至所述对象的血液循环中或者靶组织/细胞中。
  102. 根据权利要求99-101中任一项所述的方法，其中包括通过口服、吸入或注射施用所述药物。
  103. 根据权利要求99-102中任一项所述的方法，其中包括经消化道、经呼吸道施用所述药物。
  104. 根据权利要求103中所述的方法，其中包括经口服施用所述药物。
PCT/CN2020/127393 2019-11-08 2020-11-08 脂质在制备核酸递送试剂中的应用及其相关产品 WO2021089030A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP20884183.3A EP4056552A4 (en) 2019-11-08 2020-11-08 USE OF LIPID IN THE PREPARATION OF A NUCLEIC ACID DELIVERY REAGENT AND ASSOCIATED PRODUCT
CA3163934A CA3163934A1 (en) 2019-11-08 2020-11-08 Use of lipid in preparation of nucleic acid delivery reagent and related product thereof
CN202080077628.5A CN114650982A (zh) 2019-11-08 2020-11-08 脂质在制备核酸递送试剂中的应用及其相关产品
US17/775,283 US20230088528A1 (en) 2019-11-08 2020-11-08 Use of lipid in preparation of nucleic acid delivery reagent and related product thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911087684 2019-11-08
CN201911087684.2 2019-11-08

Publications (1)

Publication Number Publication Date
WO2021089030A1 true WO2021089030A1 (zh) 2021-05-14

Family

ID=75848283

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/127393 WO2021089030A1 (zh) 2019-11-08 2020-11-08 脂质在制备核酸递送试剂中的应用及其相关产品

Country Status (5)

Country Link
US (1) US20230088528A1 (zh)
EP (1) EP4056552A4 (zh)
CN (1) CN114650982A (zh)
CA (1) CA3163934A1 (zh)
WO (1) WO2021089030A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024061211A1 (zh) * 2022-09-20 2024-03-28 中国医学科学院基础医学研究所 用于核酸递送的脂质化合物以及包含所述脂质化合物的相关应用及药物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051206A1 (en) * 1998-04-03 1999-10-14 Valentis, Inc. Cationic lipid formulation delivering nucleic acid to peritoneal tumors
WO2008103276A2 (en) * 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions and methods for potentiated activity of biologicaly active molecules
WO2018177383A1 (zh) * 2017-03-29 2018-10-04 中国医学科学院基础医学研究所 化合物或中药提取物在制备核酸递送试剂中的应用及其相关产品
WO2019184991A1 (zh) * 2018-03-29 2019-10-03 中国医学科学院基础医学研究所 化合物或中药提取物在制备核酸递送试剂中的应用及其相关产品

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003506398A (ja) * 1999-08-04 2003-02-18 イデア アクチェンゲゼルシャフト 脂質、高分子電解質、及び構造誘導性可溶性低原子価リンカーを含む周期的構造、並びにその生物学的使用方法
WO2006074546A1 (en) * 2005-01-13 2006-07-20 Protiva Biotherapeutics, Inc. Lipid encapsulated interfering rna
US20070293449A1 (en) * 2006-06-20 2007-12-20 Nastech Pharmaceutical Company Inc. Compositions and methods for delivery of double-stranded rna
AU2008308679B2 (en) * 2007-10-02 2015-01-29 Marina Biotech, Inc. Lipopeptides for delivery of nucleic acids
WO2009059201A2 (en) * 2007-11-02 2009-05-07 The Board Of Regents Of The University Of Texas System Id2 as a target in colorectal carcinoma
WO2009129385A1 (en) * 2008-04-16 2009-10-22 Abbott Laboratories Cationic lipids and uses thereof
WO2016145031A1 (en) * 2015-03-09 2016-09-15 Stc.Unm Cd 47 containing porous nanoparticle supported lipid bilayers (protocells)
EP3777891A4 (en) * 2018-03-29 2022-03-23 Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences ARTIFICIALLY SYNTHESIZED SPHINGOSINE-DERIVED LIPOID MONOMER AND USE FOR NUCLEIC ACID DELIVERY

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051206A1 (en) * 1998-04-03 1999-10-14 Valentis, Inc. Cationic lipid formulation delivering nucleic acid to peritoneal tumors
WO2008103276A2 (en) * 2007-02-16 2008-08-28 Merck & Co., Inc. Compositions and methods for potentiated activity of biologicaly active molecules
WO2018177383A1 (zh) * 2017-03-29 2018-10-04 中国医学科学院基础医学研究所 化合物或中药提取物在制备核酸递送试剂中的应用及其相关产品
WO2019184991A1 (zh) * 2018-03-29 2019-10-03 中国医学科学院基础医学研究所 化合物或中药提取物在制备核酸递送试剂中的应用及其相关产品

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Real-time quantification of microRNAs by stem-loop RT-PCR", NUCLEIC ACIDS RES., vol. 33, no. 20, 27 November 2005 (2005-11-27), pages el79
BATZER ET AL., NUCLEICACIDRES., vol. 19, 1991, pages 5081
BLIGH E.G.DYER, W. J.: "A rapid method for total lipid extraction and purification", CAN. J. BIOCHEM. PHYSIOL., vol. 37, 1959, pages 911 - 917, XP000998224
DU JIANCHAO, ZHU LIANG;JIANTAO XU;YAN ZHAO;XIAOYUN LI;YANLI ZHANG;DANDAN ZHAO;RUXUAN CHEN;YANG LIU;TRUPTI JOSHI;JIAHUI CHANG;ZHIQI: "Plant-Derived Phosphocholine Facilitates Cellular Uptake of Anti-Pulmonary Fibrotic HJT-sRNA-m7", SCIENCE CHINA LIFE SCIENCES, 1 January 2017 (2017-01-01), XP055810441 *
JI CHAO, KRIAUCIONIS SKIRMANTAS, KESSLER BENEDIKT M., JIANG CHENGYU: "From herbal small RNAs to one medicine", SCIENCE CHINA LIFE SCIENCES, ZHONGGUO KEXUE ZAZHISHE, CHINA, vol. 62, no. 3, 1 March 2019 (2019-03-01), China, pages 285 - 287, XP055810440, ISSN: 1674-7305, DOI: 10.1007/s11427-019-9513-y *
LI XIAOYUN, LIANG ZHU, DU JIANCHAO, WANG ZHIQING, MEI SONG, LI ZHIQING, ZHAO YAN, ZHAO DANDAN, MA YIMING, YE JUN, XU JIANTAO, ZHAO: "Herbal decoctosome is a novel form of medicine", SCIENCE CHINA LIFE SCIENCES, ZHONGGUO KEXUE ZAZHISHE, CHINA, vol. 62, no. 3, 1 March 2019 (2019-03-01), China, pages 333 - 348, XP055810439, ISSN: 1674-7305, DOI: 10.1007/s11427-018-9508-0 *
OHTSUKA ET AL., J.BIOL.CHEM., vol. 260, 1985, pages 2605 - 2608
ROSSOLINI ET AL., MOL.CELL.PROBES, vol. 8, 1994, pages 91 - 98
See also references of EP4056552A4
SMITH, M.B.MARCH, J.: "March's Advanced Organic Chemistry", 2001, JOHN WILEY & SONS
THOMAS SORRELL: "Handbook of Chemistry and Physics", 1999, article "Organic Chemistry"

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024061211A1 (zh) * 2022-09-20 2024-03-28 中国医学科学院基础医学研究所 用于核酸递送的脂质化合物以及包含所述脂质化合物的相关应用及药物

Also Published As

Publication number Publication date
US20230088528A1 (en) 2023-03-23
CA3163934A1 (en) 2021-05-14
CN114650982A (zh) 2022-06-21
EP4056552A1 (en) 2022-09-14
EP4056552A4 (en) 2023-11-29

Similar Documents

Publication Publication Date Title
EP3506909B1 (en) 4&#39;-phosphate analogs and oligonucleotides comprising the same
CN109890207B (zh) 包含可逆修饰的寡核苷酸的组合物及其用途
JP5952423B2 (ja) 新規オリゴヌクレオチド接合体およびその用途
CA2989682A1 (en) Oligonucleotide compositions and methods thereof
JP2008504840A (ja) 非リン酸骨格結合を含むオリゴヌクレオチド
US9701962B2 (en) Hydrophobically modified antisense oligonucleotides comprising a ketal group
JP7252132B2 (ja) 核酸送達試薬の調製における化合物または伝統的漢方薬抽出物の応用およびその関連生成物
EP3018208B1 (en) Improved nanoparticle type oligonucleotide structure having high efficiency and method for preparing same
JP7190794B2 (ja) 核酸医薬及び多分岐脂質の複合体
US20230277675A1 (en) Systemic delivery of oligonucleotides
BR122020018622A2 (pt) Molécula de ácido nucleico para redução de papd5 e papd7 de mrna para o tratamento de infecção hepatite b
CN105722979A (zh) 肝癌相关的基因特异性siRNA、包含所述siRNA的双链寡RNA分子和包含它们的用于预防或治疗癌症的组合物
WO2021089030A1 (zh) 脂质在制备核酸递送试剂中的应用及其相关产品
CN111448316A (zh) 新的硫代亚磷酰胺
WO2023005971A1 (zh) Fa类脂质化合物在制备核酸递送试剂中的应用及其相关产品
JP2022511055A (ja) 筋ジストロフィーに対するエクソンスキッピングオリゴマーコンジュゲート
WO2023005973A1 (zh) St类脂质化合物在制备核酸递送试剂中的应用及相关产品
WO2024002046A1 (en) Oligonucleotide delivery enhancing compounds, pharmaceutical compositions and methods using the same
WO2021020412A1 (ja) Ｍｕｒｆ１を標的とする核酸医薬
CN116615542A (zh) 寡核苷酸的全身递送
JP2021505175A (ja) Ｆｎｄｃ３ｂの発現を調節するためのオリゴヌクレオチド

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20884183

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3163934

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020884183

Country of ref document: EP

Effective date: 20220608