WO2024251108A1 - 可电离脂质化合物、包含其的脂质载体及应用 - Google Patents

可电离脂质化合物、包含其的脂质载体及应用 Download PDF

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WO2024251108A1
WO2024251108A1 PCT/CN2024/097253 CN2024097253W WO2024251108A1 WO 2024251108 A1 WO2024251108 A1 WO 2024251108A1 CN 2024097253 W CN2024097253 W CN 2024097253W WO 2024251108 A1 WO2024251108 A1 WO 2024251108A1
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lipid
alkyl
alkenyl
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independently selected
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French (fr)
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潘兴华
王成
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Innovec Biotherapeutics
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Innovec Biotherapeutics
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Priority to CN202480037638.4A priority patent/CN121311463A/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • the present invention belongs to the field of biomedicine, and in particular relates to an ionizable lipid compound and a lipid carrier, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation containing the same.
  • Lipid nanoparticles are widely used in the field of drug delivery.
  • ionizable lipids are not only excellent protein/peptide antigen carriers, but also a new type of immune adjuvant that can directly activate antigen-presenting cells and enhance vaccine-induced immune responses. Therefore, ionizable lipids are widely used in the vaccine field to encapsulate and transport nucleic acid molecules.
  • Ionizable lipids are the most critical link in the targeting and delivery of lipid nanoparticles. They bind to negatively charged nucleic acids, which helps to escape from endosomal endosomes and transfect nucleic acid molecules in vivo. They have many characteristics such as pH sensitivity. Ionizable lipids determine the delivery efficiency and transfection efficiency of lipid nanoparticle delivery systems. Therefore, designing ionizable lipids with good targeting and delivery properties is an essential key element of lipid nanoparticles.
  • MC3 DLin-MC3-DMA
  • Onpattro siRNA liposome products
  • siRNA and mRNA drugs (including emergency use authorization) certified by the FDA, all of which use new ionizable lipids. Therefore, new ionizable lipids have broad application prospects in nucleic acid drug delivery.
  • the present invention provides a series of compounds represented by formula (1), lipid carriers containing the compounds as ionizable lipids, nucleic acid lipid nanoparticle compositions and preparations thereof.
  • the lipid nanoparticles formed by the ionizable lipids can deliver nucleic acid molecules into the body, improve the transport rate of nucleic acid molecules, and thus improve the therapeutic effect of nucleic acid molecules.
  • the present invention provides a compound represented by formula (1) or a pharmaceutically acceptable salt thereof:
  • R 1 and R 2 are independently selected from C 1-8 alkyl, which is optionally substituted with one or more OH, NH 2 or halogen;
  • A is C 1-8 alkylene
  • G 3 is independently C 2-10 alkylene
  • R6 is C2-30 alkenyl.
  • R 1 and R 2 are independently selected from C 1-6 alkyl, which is optionally substituted with one or more OH, NH 2 , or halogen.
  • R 1 and R 2 are independently selected from C 1-4 alkyl, which is optionally substituted with one or more OH, NH 2 , or halogen.
  • R1 and R2 are independently selected from -CH3 , -CH2CH3 , -CH2CH2OH , or -CH2CH2CH3 ; alternatively , R1 and R2 are independently selected from -CH2CH3 or -CH2CH2OH .
  • A is C 1-6 alkylene.
  • A is C 1-4 alkylene.
  • A is C 2-4 alkylene.
  • A is selected from -CH2- , -CH2CH2- , or -CH2CH2CH2- ; alternatively, A is -CH2CH2- .
  • R 3 and R 4 are independently selected from -R 6 , -G 3 -OC( ⁇ O)-R 5 , -G 3 -C( ⁇ O)-OR 5 , -G 3 -NH-C( ⁇ O)-OR 5 , or -G 3 -NH-OC( ⁇ O)-R 5 .
  • G 3 is independently C 2-8 alkylene.
  • G 3 is independently C 2-6 alkylene.
  • G3 is independently selected from Alternatively, G3 is independently selected from
  • R5 is selected from
  • R5 is selected from
  • R5 is selected from
  • R 6 is C 2-24 alkenyl; alternatively, R 6 is C 6-24 alkenyl; alternatively, R 6 is C 8-24 alkenyl; alternatively, R 6 is C 10-20 alkenyl.
  • R is selected from
  • R3 and R4 are independently selected from
  • R3 and R4 are independently selected from
  • R3 and R4 are independently selected from
  • the present invention also provides the following compounds or pharmaceutically acceptable salts thereof:
  • the present invention provides a lipid carrier comprising a compound represented by formula (1) or a pharmaceutically acceptable salt thereof as an ionizable lipid.
  • the lipid carrier comprises a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, an auxiliary lipid, a structural lipid and a polymer-bound lipid.
  • the helper lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine DSPC, dioleoylphosphatidylserine DOPS, distearoylphosphatidylserine DSPS, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine DSPE, dipalmitoylphosphatidylserine DPPS, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine DPPC, 1 , at least one of 2-dioleoyl-sn-glycerol-3-phosphatidylcholine DOPC, dipalmitoylphosphatidylglycerol DPPG, oleoylphosphatidylcholine POPC, 1-palmitoyl-2-
  • the helper lipid is at least one selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine DSPC.
  • the structured lipid is at least one selected from cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, ⁇ -tocopherol, coproposterol and corticosteroids.
  • the structured lipid is cholesterol
  • the polymer-bound lipid is selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), DMG-PEG2000-mannose, cholesterol-PEG2000, 1,2-dimyristoyl-sn-glyceromethoxy-polyethylene glycol PEG-DMG, dimyristoylglycerol-polyethylene glycol PEG-c-DMG, polyethylene glycol-dimyristoylglycerol PEG-C14, PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEGylated phosphatidylethanolamine PEG-PE, P At least one of EG-modified ceramide, PEG-modified dialkylamine, PEG-modified di
  • the polymer-bound lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).
  • the molar ratio of ionizable lipids, auxiliary lipids, structural lipids and polymer-bound lipids is (20-75): (2-25): (15-55): (0-15); illustratively, the molar ratio of ionizable lipids, auxiliary lipids, structural lipids and polymer-bound lipids can be 45:10:42:3, 30:25:30:10, 46:15: 40:3, 50:10:38.5:1.5, 50:10:37:3, 50:9:38:3, 60:5:34.5:0.5, 75:5:19.5:0.5, 65:5:29.5:0.5, 55:8:36.5:0.5, 50:8:41.5:0.5, 50:10:39.5:0.5, 50:9.5:40:0.5, etc.
  • the present invention provides a nucleic acid lipid nanoparticle composition, which comprises a compound represented by formula (1) or a pharmaceutically acceptable salt thereof or the above-mentioned lipid carrier, and a nucleic acid.
  • the nucleic acid is at least one selected from DNA, RNA, a complex containing DNA or RNA (such as a complex of DNA and RNA, a complex of DNA and polypeptide/protein, a complex of RNA and polypeptide/protein), modified DNA, modified RNA, and a modified complex containing DNA or RNA.
  • a complex containing DNA or RNA such as a complex of DNA and RNA, a complex of DNA and polypeptide/protein, a complex of RNA and polypeptide/protein
  • modified DNA modified RNA
  • modified RNA and a modified complex containing DNA or RNA.
  • the nucleic acid is RNA.
  • the RNA is selected from mRNA, siRNA, dsRNA, rRNA, circRNA, saRNA, tRNA, snRNA or shRNA, preferably mRNA.
  • the mass ratio of the nucleic acid to any one of the compounds or pharmaceutically acceptable salts thereof is 1:(3-40).
  • the mass ratio of the nucleic acid to the lipid carrier is 1:(3-40).
  • the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.
  • the present invention provides a pharmaceutical preparation comprising any one of the above compounds or a pharmaceutically acceptable salt thereof, or the above lipid carrier, or the above nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable carrier.
  • the particle size of the pharmaceutical preparation is 30 to 500 nm.
  • the particle size can be 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 500 nm, etc.
  • the encapsulation rate of nucleic acid in pharmaceutical preparation is greater than 50%.
  • the encapsulation rate can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% etc.
  • the present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs or gene vaccines.
  • the present invention also provides a method for in vivo delivery of nucleic acid drugs or gene vaccines, comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.
  • the present invention also provides a method for treating or preventing a liver disease, comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof, wherein the liver disease is selected from hepatitis, fatty liver, liver fibrosis, cirrhosis, alcoholic/non-alcoholic liver damage or hepatocellular carcinoma.
  • the present invention also provides use of the above compound or a pharmaceutically acceptable salt thereof, the above lipid carrier, or the above nucleic acid lipid nanoparticle composition in preparing a drug for treating or preventing liver diseases.
  • the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal.
  • the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered, for example, via an enteral or parenteral administration route.
  • a dose of about 0.001 mg/kg to about 10 mg/kg of the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject.
  • FIG. 1 Chemiluminescent imaging of mice under experimental conditions 1, 2, 3, 4, and 5 in Example 5.
  • the numerical range represented by "value A to value B” refers to a range including the endpoint values A and B.
  • any value or any sub-range falling within the range is indicated to be specifically disclosed.
  • each numerical range of a parameter disclosed herein should be understood to include each numerical value and sub-range therein.
  • C 1-4 should be understood to include any sub-range and each point value therein, such as C 2-4 , C 3-4 , C 1-2 , C 1-3 , C 1-4 , etc., as well as C 1 , C 2 , C 3 , C 4 , etc.
  • compositions, methods, or apparatus comprising a list of elements is not necessarily limited to only the elements expressly listed but may also include other elements not expressly listed or inherent to such composition, method, or apparatus.
  • references to “some specific/preferred embodiments”, “other specific/preferred embodiments”, “embodiments”, etc. mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments.
  • the elements may be combined in various embodiments in any suitable manner.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention that is substantially non-toxic to an organism.
  • Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting the compound of the present invention with a pharmaceutically acceptable inorganic/organic acid or inorganic/organic base, and such salts are also referred to as acid addition salts or base addition salts.
  • Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.
  • common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc.
  • common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutol, etc.
  • pharmaceutically acceptable carrier refers to an excipient that is administered together with the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation, and is suitable for contacting the tissues of humans and/or other animals without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit/risk ratio within the scope of reasonable medical judgment.
  • Pharmaceutically acceptable carriers that can be used in the present invention include, but are not limited to: a) diluents; b) lubricants; c) binders; d) disintegrants; e) absorbents, colorants, flavorings and/or sweeteners; f) emulsifiers or dispersants; and/or g) substances that enhance the absorption of compounds, etc.
  • substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl.
  • substituent Y may be hydrogen, halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X may be hydrogen, halogen, hydroxyl, cyano, alkyl or aryl.
  • alkyl refers to a linear or branched monovalent saturated aliphatic hydrocarbon group.
  • C 1-8 alkyl refers to a linear or branched monovalent saturated aliphatic hydrocarbon group containing 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.
  • alkylene refers to a straight or branched divalent saturated aliphatic hydrocarbon group, and the two groups (or fragments) connected thereto may be connected to the same carbon atom or to different carbon atoms.
  • C 1-8 alkylene used herein refers to an alkylene group having 1 to 8 carbon atoms (such as methylene, 1,1-ethylene, 1,2-ethylene, 1,2-propylene, 1,3-butylene, etc.).
  • alkenyl refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond.
  • C2-30 alkenyl refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond.
  • Non-limiting examples of alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.
  • branched alkyl refers to an alkyl group containing a branch, which is connected to the parent molecule and forms at least two branch structures.
  • branched alkenyl refers to an alkenyl group containing a branch, which is attached to the parent molecule and forms at least two branch structures.
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).
  • the reagents or instruments used in the examples are all conventional products that can be obtained commercially. If no specific conditions are specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer.
  • the term "room temperature” used in the present invention refers to 20°C ⁇ 5°C.
  • the term “about” used in the present invention refers to an acceptable error range for those skilled in the art including the value or numerical range and the value or numerical range, for example, the error range is ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, etc.
  • Flash silica gel chromatography was performed using a Biotage flash column chromatograph.
  • a mixture of raw material 2 (682.89 mg, 9.22 mmol, 741.47 ⁇ L, 0.5 eq) and THF (10 mL) was added to the reaction mixture, and the reaction mixture was stirred at 20° C. for 1 hour.
  • the raw material 3 (200 mg, 484.73 ⁇ mol, 1 eq) was dissolved in DCM (5 mL), and DIEA (313.24 mg, 2.42 mmol, 422.15 ⁇ L, 5 eq) and CDI (235.79 mg, 1.45 mmol, 3 eq) were added. The mixture was stirred at 50°C for 16 hours. The reaction mixture was diluted with 20 mL of H 2 O and extracted with DCM (40 mL, 20 mL*2). The combined organic layer was washed with brine (30 mL), dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to obtain a yellow jelly, which was the raw material 4.
  • the raw material 7 (100 mg, 266.98 ⁇ mol, 1 eq) and 2-hexyldecanoic acid (205.38 mg, 800.95 ⁇ mol, 3 eq) were dissolved in DCM (3 mL), and EDCI (102.36 mg, 533.96 ⁇ mol, 2 eq) and DMAP (3.26 mg, 26.70 ⁇ mol, 0.1 eq) were added.
  • the reaction was stirred at 15°C for 18 hours.
  • the reaction mixture was diluted with H 2 O (60 mL) and extracted with DCM (100 mL, 50 mL*2).
  • TEA was added to a solution of [7-benzyloxy-1-(6-benzyloxyhexyl)heptyl]imidazole-1-carboxylate and N'-ethyl-N'-methyl-ethane-1,2-diamine in ACN at 25° C. After the addition, the mixture was warmed to 50° C. and stirred at 50° C. for 16 hours.
  • the preparation of lipid nanoparticles includes mixing and dissolving the above-mentioned ionizable lipids, auxiliary lipids, structural lipids and polymer-bound lipids in anhydrous ethanol according to a certain molar ratio so that the total lipid phase concentration is 0.5-20 mg/mL.
  • the nucleic acid is dissolved in a buffer solution with a pH lower than 7 (e.g., 3-6.5) and is prepared by rapidly mixing with an ethanol solution of the lipid.
  • ionizable lipids MC3, compound 1, compound 14, compound 15
  • auxiliary lipids DSPC, DOPE
  • structural lipids cholesterol
  • polymer-bound lipids DMG-PEG2000
  • the aqueous buffer was 50 mM citric acid buffer at pH 4.0 to dissolve the corresponding concentration of Luc-eGFP mRNA.
  • the mass ratio of mRNA to total lipids was 1:30.
  • LNP was prepared using the Myana prescription screening chip, with a total flow rate of 12 ml/min and a lipid phase to water phase flow rate ratio of 1:3.
  • the collected lipid nanoparticles were dialyzed with PBS at 4°C overnight to remove ethanol and acidic buffer salts.
  • the dialysate volume was more than 400 times the sample volume.
  • the dialyzed sample was filtered using a 0.2 micron filter membrane.
  • lipid nanoparticles can also be prepared using other methods such as injection mixing. After dialysis filtration, the sample was concentrated using a 10kDa cellulose ultrafiltration membrane.
  • the centrifugation parameters were 1000g to 2000g, and the centrifugation time was 30 minutes to 1 hour.
  • the centrifugation temperature was 4°C.
  • Particle size determination The particle size and polydispersity index (PDI) of the prepared lipid nanoparticles were measured using a Malvern particle size analyzer.
  • Quant-iT TM RNA kit detection method For lipid nanoparticles encapsulating mRNA, Quant-iT TM RNA kit detection method. The specific operation is as follows: Use TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.5) to dilute the sample to 10-20 times. Add an equal volume of demulsifier (2% Triton X-100) to the sample to be tested and dilute it again 10-20 times. The reagent was diluted 100 times in TE buffer and 100 ⁇ l was added to a 96-well plate. 100 ⁇ l of the sample to be tested was added to the corresponding 96-well plate. A standard curve was prepared using mRNA standards at the same time.
  • TE buffer 10mM Tris-HCl, 1mM EDTA, pH 7.5
  • demulsifier 2% Triton X-100
  • the fluorescence value at 520 nm excited by 480 nm excitation light was read in an ELISA reader.
  • the concentration of the encapsulated and free mRNA was calculated using the standard curve.
  • the encapsulation efficiency was calculated by the ratio of the concentration of the encapsulated mRNA to the concentration of the total mRNA.
  • composition and characterization data of lipid nanoparticles are shown in Table 1.
  • the nucleic acid delivery efficiency of lipid nanoparticles was characterized by observing the fluorescence expression in mice.
  • Lipid nanoparticles encapsulating Luc-eGFP mRNA were injected into mice via the tail vein at a certain dose.
  • Female C57/BL6 mice weighing 18-22g were randomly divided into groups, with 2 mice in each group.
  • LNPs encapsulating Luc-eGFP mRNA (in sterile PBS solution) were injected into the tail vein at a dose of 0.5mg/kg (0.5mg here refers to the dose of mRNA).
  • the luciferase substrate was dissolved in a sterile PBS solution to prepare a solution with a concentration of 30mg/ml.
  • each mouse was intraperitoneally injected with 150ul of luciferase substrate solution.
  • the mice were left alone for 5 minutes, then anesthetized in a carbon dioxide chamber for 3 minutes. After anesthesia, they were placed in a small animal in vivo imaging device for imaging.
  • mice in Figure 2 are from left to right the mice in experimental conditions 1, 2, 3, 4, and 5.
  • the lipid nanoparticles formed by the compounds of the present invention can significantly improve the delivery efficiency of nucleic acids in mice, have good liver targeting and high transfection potential in the liver, confirming the feasibility of in vivo application.

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Abstract

本发明涉及可电离脂质化合物、包含其的脂质载体及应用。本发明提供了一系列式(1)所示的化合物、包含其作为可电离脂质的脂质载体、核酸脂质纳米颗粒组合物及其制剂，由该可电离脂质形成的脂质纳米颗粒能将核酸分子递送至体内，提高核酸分子的转运率，从而提高核酸药物的治疗效果。

Description

可电离脂质化合物、包含其的脂质载体及应用 技术领域
本发明属于生物医药领域，具体涉及一种可电离脂质化合物及包含其的脂质载体、核酸脂质纳米颗粒组合物和药物制剂。
背景技术
脂质纳米颗粒被广泛用于药物输送领域，而其中，可电离脂质不但是优良的蛋白/多肽抗原载体，还是一种新型的免疫佐剂，可直接活化抗原呈递细胞，增强疫苗诱导的免疫反应，因此可电离脂质在疫苗领域广泛地用于封装、转运核酸分子。可电离脂质是脂质纳米颗粒产生靶向性和递送性最关键的一环，其与负电荷性的核酸结合，有助于内涵体逃逸、核酸分子体内转染，具有pH敏感性等多种特点。可电离脂质决定了脂质纳米颗粒递送系统的递送效率和转染效率，因此设计靶向性和递送性良好的可电离脂质，是脂质纳米颗粒必不可少的关键要素。
目前，市面上最常用的，DLin-MC3-DMA(以下简称MC3)作为新型可电离脂质，具有“低毒高效”的优势，是全球首个应用于siRNA脂质体产品(Onpattro)的可电离脂质。而随着Onpattro在美国上市，MC3以及新型可电离脂质，得到了更多科学家的关注。相比于常见的DOTAP、DOTMA、DC-CHOL等可电离脂质，MC3这类新型可电离脂质的毒性更低，载药量和安全性均有显著提高。
目前，全球有4款(包含紧急使用授权)siRNA和mRNA药物得到FDA的认证，均使用了新型的可电离脂质。因此，新型的可电离脂质在核酸药物递送中具有广泛的应用前景。
发明内容
本发明提供了一系列式(1)所示的化合物、包含其作为可电离脂质的脂质载体、核酸脂质纳米颗粒组合物及其制剂，由该可电离脂质形成的脂质纳米颗粒能将核酸分子递送至体内，提高核酸分子的转运率，从而提高核酸分子的治疗效果。
第一方面，本发明提供了式(1)所示的化合物或其药学上可接受的盐：
其中，
R1和R2独立地选自C1-8烷基，所述烷基任选地被一个或多个OH、NH2或卤素取代；
A为C1-8亚烷基；
R3和R4独立地选自-R6、-G3-O-C(＝O)-R5、-G3-C(＝O)-O-R5、-G3-NH-C(＝O)-O-R5、-G3-NH-O-C(＝O)-R5、-G3-NH-C(＝O)-R5或-G3-O-C(＝O)-NH-R5；
G3独立地为C2-10亚烷基；
R5独立地选自C2-30支链烷基或C2-30支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-30烷基、-C(＝O)-O-C1-30烷基、-O-C(＝O)-C1-30烷基、-O-C2-30烯基、-C(＝O)-O-C2-30烯基或-O-C(＝O)-C2-30烯基取代；
R6为C2-30烯基。
在一些实施方案中，R1和R2独立地选自C1-6烷基，所述烷基任选地被一个或多个OH、NH2或卤素取代。
在一些实施方案中，R1和R2独立地选自C1-4烷基，所述烷基任选地被一个或多个OH、NH2或卤素取代。
在一些实施方案中，R1和R2独立地选自-CH3、-CH2CH3、-CH2CH2OH或-CH2CH2CH3；或者，R1和R2独立地选自-CH2CH3或-CH2CH2OH。
在一些实施方案中，A为C1-6亚烷基。
在一些实施方案中，A为C1-4亚烷基。
在一些实施方案中，A为C2-4亚烷基。
在一些实施方案中，A选自-CH2-、-CH2CH2-或-CH2CH2CH2-；或者，A为-CH2CH2-。
在一些实施方案中，R3和R4独立地选自-R6、-G3-O-C(＝O)-R5、-G3-C(＝O)-O-R5、-G3-NH-C(＝O)-O-R5或-G3-NH-O-C(＝O)-R5。
在一些实施方案中，R3和R4独立地选自-R6、-G3-O-C(＝O)-R5或-G3-C(＝O)-O-R5，或者，R3和R4独立地选自-R6或-G3-O-C(＝O)-R5；或者，R3和R4独立地选自-G3-O-C(＝O)-R5。
在一些实施方案中，G3独立地为C2-8亚烷基。
在一些实施方案中，G3独立地为C2-6亚烷基。
在一些实施方案中，G3独立地选自 或者，G3独立地选自
在一些实施方案中，R5选自C2-24支链烷基或C2-24支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-24烷基、-C(＝O)-O-C1-24烷基、-O-C(＝O)-C1-24烷基、-O-C2-24烯基、-C(＝O)-O-C2-24烯基或-O-C(＝O)-C2-24烯基取代。
在一些实施方案中，R5选自C4-24支链烷基或C4-24支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-24烷基、-C(＝O)-O-C1-24烷基、-O-C(＝O)-C1-24烷基、-O-C2-24烯基、-C(＝O)-O-C2-24烯基或-O-C(＝O)-C2-24烯基取代。
在一些实施方案中，R5选自C6-24支链烷基或C6-24支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-24烷基、-C(＝O)-O-C1-24烷基、-O-C(＝O)-C1-24烷基、-O-C2-24烯基、-C(＝O)-O-C2-24烯基或-O-C(＝O)-C2-24烯基取代。
在一些实施方案中，R5选自C8-24支链烷基或C8-24支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-24烷基、-C(＝O)-O-C1-24烷基、-O-C(＝O)-C1-24烷基、-O-C2-24烯基、-C(＝O)-O-C2-24烯基或-O-C(＝O)-C2-24烯基取代。
在一些实施方案中，R5选自C10-20支链烷基或C10-20支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-20烷基、-C(＝O)-O-C1-20烷基、-O-C(＝O)-C1-20烷基、-O-C2-20烯基、-C(＝O)-O-C2-20烯基或-O-C(＝O)-C2-20烯基取代。
在一些实施方案中，R5选自
在一些实施方案中，R5选自
在一些实施方案中，R5选自
在一些实施方案中，R6为C2-24烯基；或者，R6为C6-24烯基；或者，R6为C8-24烯基；或者，R6为C10-20烯基。
在一些实施方案中，R6选自
在一些实施方案中，R3和R4独立地选自
在一些实施方案中，R3和R4独立地选自
在一些实施方案中，R3和R4独立地选自
本发明还提供如下化合物或其药学上可接受的盐：


第二方面，本发明提供了一种脂质载体，其包含式(1)所示的化合物或其药学上可接受的盐作为可电离脂质。
在一些实施方案中，所述脂质载体包含式(1)所示的化合物或其药学上可接受的盐、辅助脂质、结构脂质和聚合物结合的脂质。
在一些实施方案中，所述辅助脂质为选自1,2-二油酰-sn-甘油-3-磷脂酰乙醇胺DOPE、1,2-二硬脂酰-sn-甘油-3-磷脂酰胆碱DSPC、二油酰基磷脂酰丝氨酸DOPS、二硬脂酰磷脂酰丝氨酸DSPS、1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺DSPE、二棕榈酰磷脂酰丝氨酸DPPS、1,2-二棕榈酰-sn-甘油-3-磷脂酰胆碱DPPC、1,2-二油酰-sn-甘油-3-磷脂酰胆碱DOPC、二棕榈酰磷酯酰甘油DPPG、油酰磷脂酰胆碱POPC、1-棕榈酰基-2-油酰基磷脂酰乙醇胺POPE、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺DPPE、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺DMPE、二硬脂酰磷脂酰乙醇胺DSPE和1-硬脂酰基-2-油酰基磷脂酰乙醇胺SOPE中的至少一种。
在一些实施方案中，所述辅助脂质为选自1,2-二油酰-sn-甘油-3-磷脂酰乙醇胺DOPE、1,2-二硬脂酰-sn-甘油-3-磷脂酰胆碱DSPC中的至少一种。
在一些实施方案中，所述结构脂质为选自胆固醇、非甾醇、谷固醇、麦角固醇、菜油甾醇、豆甾醇、芸苔甾醇、番茄碱、熊果酸、α-生育酚、粪固醇和皮质类固醇中的至少一种。
在一些实施方案中，所述结构脂质为胆固醇。
在一些实施方案中，所述聚合物结合的脂质为选自1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)、DMG-PEG2000-甘露糖、胆固醇-PEG2000、1,2-二肉豆蔻酰基-sn-甘油甲氧基-聚乙二醇PEG-DMG、二肉豆蔻酰甘油-聚乙二醇PEG-c-DMG、聚乙二醇-二肉豆蔻酰基甘油PEG-C14、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺PEG-c-DMA、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)]PEG-DSPE、聚乙二醇化磷脂酰乙醇胺PEG-PE、PEG修饰的神经酰胺、PEG修饰的二烷基胺、PEG修饰的二酰基甘油、吐温-20、吐温-80、1,2-二棕榈基-sn-甘油-甲氧基聚乙二醇PEG-DPG、4-O-(2’，3’-二(十四烷酰氧基)丙基-1-O-(ω-甲氧基(聚乙氧基)乙基)丁二酸酯PEG-s-DMG、PEG-二烷氧基丙基PEG-DAA、mPEG2000-1,2-二-O-烷基-sn3-氨基甲酰基甘油酯PEG-c-DOMG和N-乙酰半乳糖胺((R)-2,3-双(十八烷氧基)丙基-1-(甲氧基聚(乙二醇)2000)丙基氨基甲酸酯))GalNAc-PEG-DSG中的至少一种。
在一些实施方案中，所述聚合物结合的脂质为1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)。
在一些实施方案中，在脂质载体中，可电离脂质、辅助脂质、结构脂质和聚合物结合的脂质的摩尔比为(20～75):(2～25):(15～55):(0～15)；示例性地，可电离脂质、辅助脂质、结构脂质和聚合物结合的脂质的摩尔比可以为45:10:42:3、30:25:30:10、46:15:40:3、50:10:38.5:1.5、50:10:37:3、50:9:38:3、60:5:34.5:0.5、75:5:19.5:0.5、65:5:29.5:0.5、55:8:36.5:0.5、50:8:41.5:0.5、50:10:39.5:0.5、50:9.5:40:0.5等。
第三方面，本发明提供了一种核酸脂质纳米颗粒组合物，其包括式(1)所示的化合物或其药学上可接受的盐或上述脂质载体、以及核酸。
在一些实施方案中，所述核酸为选自DNA、RNA、含有DNA或RNA的复合物(如DNA和RNA的复合物、DNA和多肽/蛋白的复合物、RNA和多肽/蛋白的复合物)、修饰后的DNA、修饰后的RNA以及修饰后的含有DNA或RNA的复合物中的至少一种。
在一些实施方案中，所述核酸为RNA。
在一些实施方案中，所述RNA选自mRNA、siRNA、dsRNA、rRNA、circRNA、saRNA、tRNA、snRNA或shRNA，优选地为mRNA。
在一些实施方案中，上述核酸与上述任一种化合物或其药学上可接受的盐的质量比为1:(3～40)。
在一些实施方案中，上述核酸与上述脂质载体的质量比为1:(3～40)。
示例性地，上述质量比为1:3、1:5、1:10、1:15、1:20、1:30等。
第四方面，本发明提供了一种药物制剂，其包含上述任一种化合物或其药学上可接受的盐、或上述脂质载体、或上述核酸脂质纳米颗粒组合物，以及药学上可接受的载体。
在一些实施方案中，药物制剂的粒径为30～500nm，示例性地，粒径可以为30nm、40nm、50nm、60nm、70nm、80nm、90nm、100nm、110nm、120nm、130nm、140nm、150nm、200nm、250nm、300nm、350nm、500nm等。
在一些实施方案中，核酸在药物制剂中的包封率大于50％。示例性地，包封率可以为55％、60％、65％、70％、75％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％等。
第五方面，本发明还提供上述化合物或其药学上可接受的盐或上述脂质载体或上述核酸脂质纳米颗粒组合物或上述药物制剂在制备核酸药物或基因疫苗中的用途。
本发明还提供用于体内递送核酸药物或基因疫苗的方法，所述方法包括向有需要的受试者施用上述核酸脂质纳米颗粒组合物或上述药物制剂。
本发明还提供一种治疗或预防肝部疾病的方法，所述方法包括向有需要的受试者施用上述核酸脂质纳米颗粒组合物或上述药物制剂，所述肝部疾病选自肝炎、脂肪肝、肝纤维化、肝硬化、酒精性/非酒精性肝损伤或肝细胞癌。
本发明还提供上述化合物或其药学上可接受的盐或上述脂质载体或上述核酸脂质纳米颗粒组合物在制备用于治疗或预防肝部疾病的药物中的用途。
在一些实施方案中，上述核酸脂质纳米颗粒组合物或上述药物制剂通过以下施用途径之一施用：口服、鼻内、静脉内、腹膜内、肌肉内、关节内、病灶内、气管内、皮下以及皮内。在一些实施方案中，上述核酸脂质纳米颗粒组合物或上述药物制剂例如经由肠内或肠胃外施用途径施用。在一些实施方案中，将约0.001mg/kg至约10mg/kg剂量的所述核酸脂质纳米颗粒组合物或药物制剂施用给所述受试者。
附图说明
图1：化合物1的核磁共振氢谱图。
图2：实施例5中实验条件1、2、3、4、5的小鼠的化学发光成像图。
图3：化合物14的核磁共振氢谱图。
图4：化合物15的核磁共振氢谱图。
具体实施方式
为更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本说明书中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。当数值范围的下限和上限被公开时，落入该范围中的任何数值或任何亚范围都表示被具体公开。特别地，本文中所公开的参数的每一个数值范围(例如，以“约a至b”，或同等的“大约a至b”，或同等的“约a-b”的形式)均应理解为涵盖其中的每一个数值和亚范围。例如，“C1-4”应理解为涵盖其中的任意亚范围以及每一个点值，如C2-4、C3-4、C1-2、C1-3、C1-4等，以及C1、C2、C3、C4等。
术语“包含”、“包括”、“具有”或“含有”或其任何其它变体旨在涵盖非排他性或开放式的包含内容。例如，包含一系列元素的组合物、方法或装置不一定仅限于已明确列出的元素，而是可能还包含其它未明确列出的元素或上述组合物、方法或装置所固有的元素。
本说明书中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。
本说明书中，所提及的“一些具体/优选的实施方案”、“另一些具体/优选的实施方案”、“实施方案”等是指所描述的与该实施方案有关的特定要素(例如，特征、结构、性质和/或特性)包括在此处所述的至少一种实施方案中，并且可存在于其它实施方案中或者可不存在于其它实施方案中。另外，应理解，所述要素可以任何合适的方式组合在各种实施方案中。
在进一步描述本发明之前，应当理解，本发明不限于本文中所述的特定实施方案；还应该理解，本文中所使用的术语仅用于描述而非限定特定实施方案。
术语“药学上可接受的盐”是指对生物体基本上无毒性的本发明的化合物的盐。药学上可接受的盐通常包括(但不限于)本发明的化合物与药学上可接受的无机/有机酸或无机/有机碱反应而形成的盐，此类盐又被称为酸加成盐或碱加成盐。常见的无机酸包括(但不限于)盐酸、氢溴酸、硫酸、磷酸等，常见的有机酸包括(但不限于)三氟乙酸、柠檬酸、马来酸、富马酸、琥珀酸、酒石酸、乳酸、丙酮酸、草酸、甲酸、乙酸、苯甲酸、甲磺酸、苯磺酸、对甲苯磺酸等，常见的无机碱包括(但不限于)氢氧化钠、氢氧化钾、氢氧化钙、氢氧化钡等，常见的有机碱包括(但不限于)二乙胺、三乙胺、乙胺丁醇等。
术语“药学上可接受的载体”是指与上述核酸脂质纳米颗粒组合物或上述药物制剂一同给药的辅料，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激性、过敏反应或与合理的益处/风险比相应的其它问题或并发症。在本发明中可使用的药学上可接受的载体包括但不限于：a)稀释剂；b)润滑剂；c)粘合剂；d)崩解剂；e)吸收剂、着色剂、调味剂和/或甜味剂；f)乳化剂或分散剂；和/或g)增强化合物的吸收的物质等。
术语“独立地”是指结构中存在的取值范围相同或相近的至少两个基团(或环系)可以在特定情形下具有相同或不同的含义。例如，取代基X和取代基Y各自独立地为氢、卤素、羟基、氰基、烷基或芳基，则当取代基X为氢时，取代基Y既可以为氢，也可以为卤素、羟基、氰基、烷基或芳基；同理，当取代基Y为氢时，取代基X既可以为氢，也可以为卤素、羟基、氰基、烷基或芳基。
术语“烷基”是指直链或支链的一价饱和脂肪族烃基。例如，“C1-8烷基”指包含1至8个碳原子的直链或支链的一价饱和脂肪族烃基，例如为甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基或叔丁基等。
术语“亚烷基”是指直链或支链的二价饱和脂肪族烃基，其所连接的两个基团(或片段)既可以连接同一个碳原子，又可以连接不同的碳原子。例如，本文中所使用的术语“C1-8亚烷基”是指具有1-8个碳原子的亚烷基(如亚甲基、1,1-亚乙基、1,2-亚乙基、1,2-亚丙基、1,3-亚丁基等)。
术语“烯基”是指由碳原子和氢原子组成的直链或支链的具有至少一个双键的不饱和脂肪族烃基。例如“C2-30烯基”指包含2至30个碳原子并且具有至少1个碳碳双键的一价直链或支链烃基。烯基的非限制性实例包括但不限于乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、异丁烯基、1,3-丁二烯基等。
术语“支链烷基”即含支链的烷基，是指与母体分子相连并自身形成至少两个分支结构的烷基。例如
术语“支链烯基”即含支链的烯基，是与母体分子相连并自身形成至少两个分支结构的烯基。例如
术语“卤素”是指氟(F)、氯(Cl)、溴(Br)和碘(I)。
为了使本发明的目的和技术方案更加清楚，以下结合实施例对本发明的实施方案进行详细描述。但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。
实施例中所使用的试剂或仪器均为可以通过市购获得的常规产品。未注明具体条件者，均按照常规条件或制造商建议的条件进行。本发明中所使用的术语“室温”是指20℃±5℃。在用于修饰某一数值或数值范围时，本发明中所使用的术语“约”是指包括该数值或数值范围以及该数值或数值范围的本领域技术人员可接受的误差范围，例如该误差范围为±10％、±5％、±4％、±3％、±2％、±1％、±0.5％等。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
在下述的实施例中，所用溶剂和药品均为分析纯或化学纯；溶剂在使用前均经过重新蒸馏；无水溶剂均按照标准方法或文献方法进行处理。
本文中所用的缩写具有以下含义：

1H NMR波谱法采用Bruker超导核磁共振波谱仪(型号AVACE III HD 400MHz)。
快速硅胶色谱法使用Biotage快速柱色谱仪。
实施例1：化合物1的制备方法
将原料1(5g，18.44mmol，1eq)和THF(20mL)的混合物添加至Mg(672.17mg，27.66mmol，1.5eq)和TMSCl(200.30mg，1.84mmol，234.00μL，0.1eq)在THF(4mL)中的混合物中，并且将混合物在20℃搅拌1小时。向反应混合物中加入原料2(682.89mg，9.22mmol，741.47μL，0.5eq)和THF(10mL)的混合物，并将反应混合物在20℃下搅拌1小时。将反应混合物倒入用冰冷却的10％的硫酸水溶液(30mL)中，在其中加入乙酸乙酯(60mL)，分离有机层并用无水硫酸镁干燥，然后减压蒸去溶剂。将THF(20mL)、EtOH(10mL)和KOH(10M，5mL，2.71eq)加入到获得的残余物中，并将混合物在40℃下搅拌1小时。向反应混合物中加入乙酸乙酯(60mL)和水(50mL)，分离有机层，然后用无水硫酸镁干燥，减压蒸去溶剂。残余物通过快速硅胶色谱纯化，得到的白色固体即为原料3(1.37g)。
将原料3(200mg，484.73μmol，1eq)溶解于DCM(5mL)中，加入DIEA(313.24mg，2.42mmol，422.15μL，5eq)和CDI(235.79mg，1.45mmol，3eq)。将混合物在50℃下搅拌16小时。反应混合物用20mL的H2O稀释，并用DCM(40mL，20mL*2)萃取。合并的有机层用盐水(30mL)洗涤，用无水MgSO4干燥，过滤和减压浓缩，得到的黄色胶状物即为原料4。
向原料4的乙腈溶液(5mL)中加入TEA(419.40mg，4.14mmol，576.88μL，10当量)、DMAP(50.63mg，414.47μmol，1eq)和原料5(481.63mg，4.14mmol，582.38μL，10当量)。将混合物在50℃下搅拌16小时。残余物经快速硅胶色谱纯化，得到的无色油状物即为原料6。
向原料6(190mg，308.22μmol，1eq)的MeOH(5mL)溶液中加入Pd/C(32.80mg，30.82μmol，10％纯度，0.1eq)。将悬浮液在真空下脱气并用氢气吹扫数次。将混合物在氢气(50psi)和50℃下搅拌16小时。将混合物在50℃下搅拌16小时。将混合物减压浓缩，得到的无色油状物即为原料7。
将原料7(100mg，266.98μmol，1eq)和2-己基癸酸(205.38mg，800.95μmol，3eq)溶解于DCM(3mL)中，加入EDCI(102.36mg，533.96μmol，2当量)和DMAP(3.26mg，26.70μmol，0.1当量)。反应在15℃下搅拌18小时。反应混合物用H2O(60mL)稀释，并用DCM(100mL，50mL*2)萃取。合并的有机层用盐水(100mL)洗涤，用无水MgSO4干燥，过滤并在减压下浓缩，得到残余物。残余物通过快速硅胶色谱纯化，得到的无色液体即为化合物1。化合物1的核磁共振氢谱数据如图1所示。
实施例2：化合物14的制备方法
在25℃下，向[7-苄氧基-1-(6-苄氧基己基)庚基]咪唑-1-羧酸酯和N’-乙基-N’-甲基-乙烷-1,2-二胺的ACN溶液中加入TEA。加入后，将混合物升温至50℃并在50℃下搅拌16小时。
在25℃和Ar2气氛下，向[7-苄氧基-1-(6-苄氧基己基)庚基]-N-[2-[乙基(甲基)氨基]乙基]氨基甲酸酯的MeOH溶液中加入Pd/C。加入后，将混合物升温至50℃并在50℃下搅拌16小时。
在25℃下，向[7-羟基-1-(6-羟基己基)庚基]-N-[2-[乙基(甲基)氨基]乙基]氨基甲酸酯和2-己基癸酸在DCM中的溶液中加入DMAP。将混合物冷却至0℃，并在0℃下在N2气氛下加入EDCI。加入后，将混合物升温至25℃并在25℃搅拌16小时。反应经过后处理得到化合物14(产率：95％)。化合物14的核磁共振氢谱数据如图3所示。
实施例3：化合物15的制备方法
向250mL圆底烧瓶中加入Mg和I2，加入THF，并滴加1-溴-3-甲基丁烷。该反应在N2下于20℃搅拌2小时。然后迅速加入DMAP和溴化亚铜-二甲基硫醚。将烧瓶冷却至-70℃，滴加丙-2-烯酸叔丁酯、TMSCl和THF。混合物在N2下于20℃搅拌12小时。
在N2下将LiAlH4在THF中的溶液冷却至0℃，然后在0℃下加入6-甲基庚酸在THF中的溶液。在N2下将混合物在25℃下搅拌2小时。
在N2下将PPh3和咪唑在MeCN中的溶液冷却至0℃，然后在0℃下加入6-甲基庚-1-醇在MeCN中的溶液。将混合物在0℃下搅拌15分钟。然后分批加入I2，混合物在N2下于20℃搅拌1.5小时。
在N2下，将N-异丙基丙-2-胺在THF中的溶液冷却至-70℃，然后加入n-BuLi。在N2下，将混合物在-70℃搅拌0.5小时。将反应升温至0℃，将6-甲基庚酸叔丁酯在THF中的溶液冷却至0℃，然后加入LDA溶液，将混合物在0℃搅拌30分钟。在N2下于0℃滴加1-碘-6-甲基庚烷和HMPA。将混合物在N2下于20℃搅拌12小时。将反应混合物进行后处理和纯化，得到淡黄色油状2-异己基-8-甲基壬酸叔丁酯。
向2-异己基-8-甲基壬酸叔丁酯在CH2Cl2(2ml)中的溶液中加入HCl/二噁烷。将混合物在20℃搅拌24小时。
向2-异己基-8-甲基壬酸和[7-羟基-1-(6-羟基己基)庚基]-N-[2-(二乙基氨基)乙基]氨基甲酸酯的CH2Cl2(20mL)溶液中加入DMAP并在20℃搅拌0.5小时。然后加入EDCI，将反应混合物在20℃搅拌12小时。化合物15的核磁共振氢谱数据如图4所示。
实施例4：脂质纳米颗粒的制备和表征
1、脂质纳米颗粒(LNP)的制备
脂质纳米颗粒的制备包括将如上提到的可电离脂质、辅助脂质、结构脂质和聚合物结合的脂质等按照一定摩尔比混合溶解在无水乙醇中，以使总脂质相浓度为0.5-20mg/mL。将核酸溶解在pH低于7(例如3-6.5)的缓冲液中，并通过与脂质的乙醇溶液快速混合达到制备的目的。
在该实施例中，将可电离脂质(MC3、化合物1、化合物14、化合物15)、辅助脂质(DSPC、DOPE)、结构脂质(胆固醇)和聚合物结合的脂质(DMG-PEG2000)按照表1所示的摩尔比溶解在无水乙醇中，配制的总脂质相浓度为1mg/mL。
用水相缓冲液为pH4.0的50mM柠檬酸缓冲液溶解相应浓度的Luc-eGFP mRNA。mRNA与总脂质的质量比为1:30。
利用迈安纳处方筛选芯片制备LNP，总流速12ml/min，脂质相与水相流速比为1：3。将收集得到的脂质纳米颗粒用PBS在4℃透析过夜以除去乙醇和酸性缓冲盐。透析液体积为样品体积的400倍以上。利用0.2微米的滤膜对透析后的样品进行过滤。除了利用微流控芯片，还可以利用其他方式比如注射混合等方式来制备脂质纳米颗粒。样品在透析过滤后，利用10kDa的纤维素超滤膜对样品进行浓缩。离心参数为1000g到2000g，离心时间在30分钟至1小时。离心温度为4℃。
2、粒径测定:利用马尔文粒度仪测量制备的脂质纳米颗粒的粒径、多分散指数(PDI)。
3、mRNA包封率测定：
对于包裹了mRNA的脂质纳米颗粒，可以利用Quant-iTTM RNA试剂盒的检测方法。具体操作如下：利用TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 7.5)将样品稀释至10-20倍。向待测样品加入等体积的破乳剂(2％ Triton X-100)后再稀释10-20倍。将试剂在TE缓冲液中稀释100倍，并将100微升加入96孔板中。在相应的96孔板中加入100微升待测样品。同时利用mRNA标准品制备标准曲线。在酶标仪中读取利用480nm激发光激发的520nm的荧光值。通过标准曲线计算包裹的和游离的mRNA的浓度。通过包裹的mRNA的浓度和总mRNA的浓度的比值来计算包封率。
脂质纳米颗粒的组成成分和表征数据如表1所示。
表1：脂质纳米颗粒粒径大小和核酸包封率
实施例5：荧光素酶mRNA动物体内递送实验
通过观察小鼠体内荧光表达的方式来表征脂质纳米颗粒的核酸递送效率。将实施例4中制备的包 裹Luc-eGFP mRNA的脂质纳米颗粒按照一定剂量通过尾静脉注射进老鼠体内。取体重在18-22g的雌性C57/BL6小鼠，随机分组，每组2只。适应性饲养完毕，按照0.5mg/kg的剂量(此处的0.5mg，指的是mRNA的剂量)，尾静脉注射包裹Luc-eGFP mRNA的LNP(无菌PBS溶液中)。为了观察荧光素酶的表达，将荧光素酶底物溶解在无菌PBS溶液中，制备成浓度为30mg/ml的溶液。
注射LNP 6小时后，每只小鼠腹腔注射150ul荧光素酶底物溶液。放置5分钟，然后在二氧化碳箱中麻醉3分钟，麻醉完毕放入小动物活体成像仪中，进行成像。
表2：脂质纳米颗粒递送Luc-eGFP mRNA的化学发光强度检测
图2中的小鼠从左到右依次为实验条件1、2、3、4、5的小鼠。
从表2和图2可以看出，与目前常用的可电离脂质MC3相比，本发明的化合物形成的脂质纳米颗粒在小鼠体内能够显著提高核酸的递送效率，具有良好的肝靶向以及在肝部高转染的潜力，证实了体内应用的可行性。

Claims (13)

  1. 一种式(1)所示的化合物或其药学上可接受的盐：
    其中，
    R1和R2独立地选自C1-8烷基，所述烷基任选地被一个或多个OH、NH2或卤素取代；
    A为C1-8亚烷基；
    R3和R4独立地选自-R6、-G3-O-C(＝O)-R5、-G3-C(＝O)-O-R5、-G3-NH-C(＝O)-O-R5、-G3-NH-O-C(＝O)-R5、-G3-NH-C(＝O)-R5或-G3-O-C(＝O)-NH-R5；
    G3独立地为C2-10亚烷基；
    R5独立地选自C2-30支链烷基或C2-30支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-30烷基、-C(＝O)-O-C1-30烷基、-O-C(＝O)-C1-30烷基、-O-C2-30烯基、-C(＝O)-O-C2-30烯基或-O-C(＝O)-C2-30烯基取代；
    R6为C2-30烯基。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，其中，
    R1和R2独立地选自C1-6烷基，所述烷基任选地被一个或多个OH、NH2或卤素取代；
    或者，R1和R2独立地选自C1-4烷基，所述烷基任选地被一个或多个OH、NH2或卤素取代；
    或者，R1和R2独立地选自-CH3、-CH2CH3、-CH2CH2OH或-CH2CH2CH3；
    或者，R1和R2独立地选自-CH2CH3或-CH2CH2OH。
  3. 根据权利要求1或2所述的化合物或其药学上可接受的盐，其中，
    A为C1-6亚烷基；
    或者，A为C1-4亚烷基；
    或者，A选自-CH2-、-CH2CH2-或-CH2CH2CH2-；
    或者，A为-CH2CH2-。
  4. 根据权利要求1-3中任一项所述的化合物或其药学上可接受的盐，其中，
    R3和R4独立地选自-R6、-G3-O-C(＝O)-R5、-G3-C(＝O)-O-R5、-G3-NH-C(＝O)-O-R5或-G3-NH-O-C(＝O)-R5；
    或者，R3和R4独立地选自-R6、-G3-O-C(＝O)-R5或-G3-C(＝O)-O-R5；
    或者，R3和R4独立地选自-R6或-G3-O-C(＝O)-R5；
    或者，R3和R4独立地选自-G3-O-C(＝O)-R5；
    或者，R3和R4独立地选自
    或者，R3和R4独立地选自
  5. 根据权利要求1-4中任一项所述的化合物或其药学上可接受的盐，其中，
    G3独立地为C2-8亚烷基；
    或者，G3独立地为C2-6亚烷基；
    或者，G3独立地选自
    或者，G3独立地选自
  6. 根据权利要求1-5中任一项所述的化合物或其药学上可接受的盐，其中，
    R5选自C2-24支链烷基或C2-24支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-24烷基、-C(＝O)-O-C1-24烷基、-O-C(＝O)-C1-24烷基、-O-C2-24烯基、-C(＝O)-O-C2-24烯基或-O-C(＝O)-C2-24烯基取代；
    或者，R5选自C8-24支链烷基或C8-24支链烯基，所述支链烷基或支链烯基任选地被一个或多个-O-C1-24烷基、-C(＝O)-O-C1-24烷基、-O-C(＝O)-C1-24烷基、-O-C2-24烯基、-C(＝O)-O-C2-24烯基或-O-C(＝O)-C2-24烯基取代；
    或者，R5选自
    或者，R5选自
  7. 一种化合物或其药学上可接受的盐，所述化合物选自如下化合物：


  8. 一种脂质载体，其包含权利要求1-7中任一项所述的化合物或其药学上可接受的盐作为可电离脂质。
  9. 根据权利要求8所述的脂质载体，其进一步包含辅助脂质、结构脂质和聚合物结合的脂质。
  10. 一种核酸脂质纳米颗粒组合物，其包括权利要求1-7中任一项所述的化合物或其药学上可接受的盐或者权利要求8或9所述的脂质载体、以及核酸。
  11. 根据权利要求10所述的核酸脂质纳米颗粒组合物，其中，所述核酸为选自DNA、RNA、含有DNA或RNA的复合物、修饰后的DNA、修饰后的RNA以及修饰后的含有DNA或RNA的复合物中的至少一种。
  12. 一种药物制剂，其包含权利要求1-7中任一项所述的化合物或其药学上可接受的盐、权利要求8或9所述的脂质载体或者权利要求10或11所述的核酸脂质纳米颗粒组合物，以及药学上可接受的载体。
  13. 权利要求1-7中任一项所述的化合物或其药学上可接受的盐、权利要求8或9所述的脂质载体、权利要求10或11所述的核酸脂质纳米颗粒组合物或者权利要求12所述的药物制剂在制备核酸药物或基因疫苗中的用途。
PCT/CN2024/097253 2023-06-07 2024-06-04 可电离脂质化合物、包含其的脂质载体及应用 Ceased WO2024251108A1 (zh)

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