WO2023054241A1 - 核酸を脳組織に送達するために用いられる脂質ナノ粒子 - Google Patents

核酸を脳組織に送達するために用いられる脂質ナノ粒子 Download PDF

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WO2023054241A1
WO2023054241A1 PCT/JP2022/035636 JP2022035636W WO2023054241A1 WO 2023054241 A1 WO2023054241 A1 WO 2023054241A1 JP 2022035636 W JP2022035636 W JP 2022035636W WO 2023054241 A1 WO2023054241 A1 WO 2023054241A1
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group
lipid
carbon atoms
formula
represented
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French (fr)
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耕太 丹下
宏樹 吉岡
悠太 中井
英万 秋田
遊 櫻井
浩揮 田中
翔也 藤田
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Chiba University NUC
NOF Corp
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Chiba University NUC
NOF Corp
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Priority to JP2023551462A priority Critical patent/JPWO2023054241A1/ja
Priority to US18/697,213 priority patent/US20240408230A1/en
Priority to CN202280066448.6A priority patent/CN118284407A/zh
Priority to CA3234320A priority patent/CA3234320A1/en
Priority to EP22876108.6A priority patent/EP4410275A4/en
Publication of WO2023054241A1 publication Critical patent/WO2023054241A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 present invention relates to lipid nanoparticles used for delivering nucleic acids to brain tissue, and methods for delivering nucleic acids to brain tissue using the same.
  • Viral vectors are nucleic acid delivery carriers with high expression efficiency, but have practical problems from the viewpoint of safety. Therefore, non-viral nucleic acid delivery carriers that can be used more safely are being developed. is a career.
  • Ionic lipids are roughly divided into amine and lipid moieties.
  • the amine moieties which are protonated under acidic conditions, and the nucleic acids, which are polyanions, interact electrostatically to form lipid nanoparticles. It promotes uptake into cells and delivers nucleic acids into cells.
  • DODAP 1,2-dioleoyl-3-dimethylammonium propane
  • Patent Document 1 describes an ionic lipid having a structure in which compounds consisting of one or two amine sites and one lipid site are linked by a biodegradable disulfide bond.
  • the ionic lipid can improve pharmacokinetics such as blood stability and tumor targetability, and by changing the structure around the amine site, the pKa of the lipid membrane structure can be increased to cells. It has been shown that it can be adjusted to a value that is advantageous for endosomal escape from the endosome, and that it has the effect of dissociating nucleic acids from the lipid membrane structure by utilizing intracellular cleavage of disulfide bonds.
  • DODAP which is a known ionic lipid
  • DODAP which is a known ionic lipid
  • Patent Document 2 in addition to the tertiary amine site and disulfide bond, the use of an ionic lipid with an aromatic ring introduced near the lipid site enhances the fusibility with the endosomal membrane and further enhances the efficiency of nucleic acid introduction into the cytoplasm. Elevated lipid membrane structures are shown.
  • ionic lipids with improved intracellular dynamics have been developed by increasing endosomal escape efficiency and membrane fusion ability.
  • directivity to target organs and cells is required.
  • DMG-PEG dimyristoylglycerol PEG
  • lipid nanoparticles using DMG-PEG with a PEG number average molecular weight of 2000 are administered into the blood, the PEG lipids gradually dissociate from the lipid nanoparticles in the blood, and apolipoprotein E present in the blood ( ApoE) is known to adhere to lipid nanoparticles and increase its accumulation in the liver, which expresses the ApoE receptor (Non-Patent Document 2).
  • lipid nanoparticles imparted with tropism to organs other than the liver for example, instead of DMG-PEG having a hydrophobic group derived from myristic acid as a PEG lipid, DMG-PEG having a hydrophobic group derived from stearic acid can be used.
  • DMG-PEG lipid nanoparticles obtained using stearoylglycerol PEG (DSG-PEG) (Non-Patent Document 3). Since DSG-PEG is more difficult to dissociate from lipid nanoparticles in blood than DMG-PEG, it avoids adhesion of ApoE in blood, suppresses accumulation in the liver, and has high blood retention. As a result, the accumulation in tumors increases.
  • lipid nanoparticles that have improved intracellular dynamics
  • lipid nanoparticles that control organ accumulation in intravenous injection by changing the PEG lipid, one of the constituents.
  • organs and cells that can be drug discovery targets, and there is a demand for the development of lipid nanoparticles that have directivity to various organs and cells.
  • the brain is an extremely difficult organ to deliver drugs to, because the blood-brain barrier that exists in the capillaries in the brain strictly restricts the transfer of substances from the blood to the brain tissue. For this reason, there are many intractable diseases for which there are no effective treatments for cranial nerve diseases, and there is a need for lipid nanoparticles for efficiently delivering therapeutic nucleic acids to the brain.
  • Non-Patent Document 4 shows an example of delivering nucleic acids to brain tissue by directly administering nucleic acid-encapsulated lipid nanoparticles into the ventricle.
  • direct administration of drugs into the ventricles of the brain imposes a heavy burden on the patient, so a minimally invasive method is desired.
  • An object of the present invention is to provide lipid nanoparticles that can efficiently deliver nucleic acids to brain tissue, and to provide a method for delivering nucleic acids to brain tissue using the lipid nanoparticles.
  • the present inventors have made intensive studies in view of the above problems, and found that an ionic lipid that has a pKa suitable for endosomal escape and specifically decomposes in an intracellular reductive environment and a specific ratio of the following formula (2 ) prepared using dimyristoylglycerol PEG can efficiently deliver nucleic acids to brain tissue.
  • the present invention based on this knowledge is as follows.
  • R 1a and R 1b each independently represent an alkylene group having 1 to 6 carbon atoms
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and represents a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups
  • R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having 8 or less carbon atoms
  • Y a and Y b each independently represent an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom.
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or a residue derived from a reaction product with glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.
  • Dimyristoylglycerol PEG represented by Lipid nanoparticles comprising The amount of dimyristoylglycerol PEG represented by formula (2) is 1 to 6 mol% with respect to the total amount of ionic lipid, phospholipid and cholesterol represented by formula (1), and the nucleic acid is transferred to brain tissue. Lipid nanoparticles used for delivery. [2] The above-mentioned [1], wherein the lipids constituting the lipid nanoparticles are composed of an ionic lipid represented by formula (1), a phospholipid, cholesterol, and dimyristoylglycerol PEG represented by formula (2). lipid nanoparticles. [3] The ionic lipid represented by formula (1) is represented by the following formula:
  • the amount of the ionic lipid represented by formula (1) is 15 to 70 mol% with respect to the total of the ionic lipid, phospholipid and cholesterol represented by formula (1), and the amount of phospholipid is 5 to 25 mol%, and the amount of cholesterol is 25 to 80 mol%.
  • a method of delivering a nucleic acid to brain tissue comprising transnasally administering to a subject the lipid nanoparticles of any one of [1] to [5] encapsulating the nucleic acid.
  • the lipid nanoparticles of the present invention can efficiently deliver nucleic acids to brain tissue.
  • FIG. 4 shows the effect of molecular weight of PEG chains of PEG-lipid (DMG-PEG) and dosing method on brain localization of LNP.
  • FIG. 3 shows the effect of the composition ratio of phospholipid (DOPC) on the brain penetration of LNP in vivo administration.
  • FIG. 4 shows the effect of phospholipid type on the brain localization of LNP upon in vivo administration.
  • FIG. 4 shows the composition ratio effect of cholesterol on the brain localization of LNPs upon in vivo administration.
  • FIG. 10 is a diagram showing the effect of the number of administrations (administering once to each of the left and right nasal cavities as one set; the same applies to the following figures) on the brain penetration of mRNA-encapsulated LNP in in vivo administration.
  • FIG. 2 shows the effects of the number of administrations on the brain localization of mRNA-encapsulated LNP (left) and the expression of the mRNA in the brain (right) upon in vivo administration.
  • FIG. 2 shows that the optimized lipid composition and dosage regimen of the present invention is significantly superior to the lipid composition and dosage regimen for liver localization with respect to brain localization of mRNA-encapsulated LNP upon in vivo administration.
  • the optimized lipid composition and dosage for liver migration are Fig. 10 shows that it is significantly superior in
  • the present invention provides an ionic lipid represented by formula (1) (that is, an ionic lipid having a tertiary amino group, a lipid moiety, and a disulfide bond that is a biodegradable group), a phospholipid, cholesterol, and formula (
  • the present invention relates to lipid nanoparticles containing dimyristoylglycerol PEG represented by 2) and a method for delivering nucleic acids to brain tissue using the same.
  • lipid nanoparticles are hydrophilic groups of amphiphilic lipids on the aqueous phase side of the interface. Particles having a oriented membrane structure and a particle size of less than 1 ⁇ m are meant, and by “amphiphilic lipid” is meant a lipid having both hydrophilic and hydrophobic groups.
  • the particle size of the lipid nanoparticles of the present invention is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm.
  • the particle size can be measured using a particle size distribution analyzer such as Zetasizer Nano (Malvern).
  • the particle size of the lipid nanoparticles can be appropriately adjusted by the method for producing the lipid nanoparticles.
  • particle size means an average particle size (number average) measured by a dynamic light scattering method.
  • amphipathic lipids examples include ionic lipids, phospholipids, PEG lipids, and the like.
  • PEG means polyethylene glycol
  • PEG lipid means a lipid modified with PEG
  • Y modified with X e.g., X: PEG, Y: lipid
  • PEG lipid means a lipid with PEG attached.
  • the lipid nanoparticles of the present invention are lipids other than ionic lipids, phospholipids, cholesterol represented by formula (1), and dimyristoylglycerol PEG represented by formula (2) (hereinafter referred to as "other lipids"). may include).
  • Other lipids include, for example, sterols other than cholesterol and PEG lipids other than dimyristoylglycerol PEG represented by formula (2).
  • the amount of other lipids in the lipid nanoparticles of the present invention is preferably 0 to 50 mol%, more preferably 0 to 30 mol%, still more preferably 0 to 10 mol% relative to the total amount of lipids in the lipid nanoparticles.
  • the "total amount of lipids in the lipid nanoparticles” means, for example, that the lipid nanoparticles are composed of ionic lipids, phospholipids, cholesterol represented by formula (1), and divalent lipids represented by formula (2).
  • ionic lipids, phospholipids, cholesterol represented by formula (1), and dimyristoylglycerol PEG and other lipids represented by formula (2) means the total amount of Further, in the present specification, “amount of B relative to A (mol%)” means “100 ⁇ amount of B (mol)/amount of A (mol)". For example, “the amount of other lipids (mol%) relative to the total amount of lipids” means “100 x the amount of other lipids (mol)/total amount of lipids (mol)".
  • lipids constituting the lipid nanoparticles of the present invention are ionic lipids, phospholipids, cholesterol represented by formula (1), and represented by formula (2). Most preferably, it consists of dimyristoylglycerol PEG.
  • Ionic lipid used in the present invention is an ionic lipid represented by the following formula (1) (hereinafter sometimes abbreviated as "ionic lipid (1)").
  • the ionic lipid (1) may be used alone or in combination of two or more.
  • R 1a and R 1b each independently represent an alkylene group having 1 to 6 carbon atoms
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and represents a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups
  • R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having 8 or less carbon atoms
  • Y a and Y b each independently represent an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom.
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or a residue derived from a reaction product with glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.
  • Each of R 1a and R 1b independently represents an alkylene group having 1 to 6 carbon atoms, which may be linear or branched, but is preferably linear.
  • the alkylene group preferably has 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms.
  • Specific examples of the alkylene group having 1 to 6 carbon atoms include methylene group, ethylene group, trimethylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group and neopentylene group.
  • R 1a and R 1b are preferably each independently methylene, ethylene, trimethylene, isopropylene or tetramethylene, most preferably each ethylene.
  • R 1a may be the same as or different from R 1b , but preferably R 1a is the same group as R 1b .
  • X a and X b are each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and represents a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups, preferably each independently having 2 to 5 carbon atoms and having 1 to 2 tertiary amino groups; is an alkylene tertiary amino group.
  • the alkyl group having 1 to 6 carbon atoms in the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group may be linear or branched. or cyclic.
  • the number of carbon atoms in the alkyl group is preferably 1-3.
  • Specific examples of alkyl groups having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group and isopentyl group.
  • a cyclohexyl group and the like can be mentioned, preferably a methyl group, an ethyl group, a propyl group or an isopropyl group, most preferably a methyl group.
  • a preferred specific structure of the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is represented by X 1 .
  • R 5 of X 1 represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched or cyclic.
  • the number of carbon atoms in the alkyl group is preferably 1-3.
  • Specific examples of alkyl groups having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group and isopentyl group.
  • a cyclohexyl group and the like can be mentioned, preferably a methyl group, an ethyl group, a propyl group or an isopropyl group, most preferably a methyl group.
  • the number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups is preferably 4 to 5.
  • Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include an aziridylene group, an azetidylene group, a pyrrolidylene group, a piperidylene group, an imidazolidylene group, It is a piperazylene group, preferably a pyrrolidylene group, a piperidylene group or a piperazylene group, most preferably a piperidylene group.
  • a preferred specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group is represented by X2 .
  • p of X2 is 1 or 2; When p is 1, X2 is a pyrrolidylene group, and when p is 2, X2 is a piperidylene group. Preferably p is two.
  • a preferred specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is represented by X3 .
  • w of X3 is 1 or 2; When w is 1, X3 is an imidazolidylene group, and when w is 2, X3 is a piperazylene group.
  • Xa may be the same as or different from Xb , but preferably Xa is the same group as Xb .
  • R 2a and R 2b each independently represent an alkylene group or oxydialkylene group having 8 or less carbon atoms, preferably each independently an alkylene group having 8 or less carbon atoms.
  • the alkylene group having 8 or less carbon atoms may be linear or branched, but is preferably linear.
  • the number of carbon atoms contained in the alkylene group is preferably 6 or less, most preferably 4 or less.
  • Examples of the alkylene group having 8 or less carbon atoms include methylene group, ethylene group, trimethylene group, isopropylene group, tetramethylene group, isobutylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group and the like. are preferably methylene group, ethylene group, trimethylene group and tetramethylene group, and most preferably ethylene group.
  • oxydialkylene group having 8 or less carbon atoms means an alkylene group via an ether bond (alkylene-O-alkylene, in other words, “alkyleneoxyalkylene group”), and there are two It means a group in which the total number of carbon atoms in the alkylene group is 8 or less.
  • alkylenes present may be the same or different, but are preferably the same.
  • Examples of the oxydialkylene group having 8 or less carbon atoms include, specifically, an oxydimethylene group, an oxydiethylene group, an oxydi(trimethylene) group (i.e., trimethyleneoxytrimethylene group), and an oxydi(tetramethylene) group (i.e., tetramethyleneoxytetramethylene group) and the like.
  • an oxydimethylene group an oxydiethylene group, an oxydi(trimethylene) group (i.e., trimethyleneoxytrimethylene group), and an oxydi(tetramethylene) group (i.e., tetramethyleneoxytetramethylene group) and the like.
  • Preferred are oxydimethylene, oxydiethylene and oxydi(trimethylene) groups, and most preferred is oxydiethylene.
  • R 2a may be the same as or different from R 2b , but preferably R 2a is the same group as R 2b .
  • Y a and Y b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, preferably each independently an ester bond, an amide bond or a carbamate bond, more preferably each It is independently an ester bond or an amide bond, most preferably each an ester bond.
  • the orientation of the bonds of Y a and Y b is not limited, when Y a and Y b are ester bonds, they are preferably -Z a -CO-OR 2a - and -Z b -CO-OR 2b -.
  • Y a may be the same as or different from Y b , but Y a is preferably the same group as Y b .
  • Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom. represents The number of carbon atoms contained in the aromatic compound is preferably 6-12, most preferably 6-7. Also, the number of aromatic rings contained in the aromatic compound is preferably one.
  • Examples of types of aromatic rings contained in aromatic compounds having 3 to 16 carbon atoms include benzene ring, naphthalene ring and anthracene ring for aromatic hydrocarbon rings, imidazole ring, pyrazole ring and oxazole ring for aromatic hetero rings, isoxazole ring, thiazole ring, isothiazole ring, triazine ring, pyrrole ring, furan ring, thiophene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, purine ring, pteridine ring, benzimidazole ring, indole ring, benzofuran ring , quinazoline ring, phthalazine ring, quinoline ring, isoquinoline ring, coumarin ring, chromone ring, benzodiazepine ring, phenoxazine ring, pheno
  • the aromatic ring may have a substituent, and examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, an alkylcarbamoyl group having 2 to 4 carbon atoms, and an alkylcarbamoyl group having 2 to 4 carbon atoms.
  • acyloxy groups acylamino groups with 2 to 4 carbon atoms, alkoxycarbonylamino groups with 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylsulfanyl groups with 1 to 4 carbon atoms, carbon atoms 1-4 alkylsulfonyl group, 6-10 carbon arylsulfonyl group, nitro group, trifluoromethyl group, cyano group, 1-4 carbon alkyl group, ureido group, 2-4 carbon alkylureido group , an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aryloxy group having 6 to 10 carbon atoms, and the like.
  • Preferred examples are acetyl group, methoxycarbonyl group, methylcarbamoyl group, acetoxy group, acetamide group, methoxycarbonylamino group, fluorine atom, chlorine atom, bromine atom, iodine atom, methylsulfanyl group, phenylsulfonyl group, nitro group, trifluoromethyl group, cyano group, methyl group, ethyl group, propyl group, Examples include isopropyl group, tert-butyl group, ureido group, methoxy group, ethoxy group, propoxy group, isopropoxy group, tert-butoxy group, phenyl group and phenoxy group.
  • Preferred specific structures of Z a and Z b include Z 1 .
  • s represents an integer of 0 to 3
  • t represents an integer of 0 to 3
  • u represents an integer of 0 to 4
  • u R 4 each independently represents a substituent.
  • s of Z 1 is preferably an integer of 0 to 1, more preferably 0.
  • t of Z 1 is preferably an integer of 0 to 2, more preferably 1.
  • u of Z 1 is preferably an integer of 0-2, more preferably an integer of 0-1.
  • R 4 of Z 1 is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not interfere with the reaction in the process of synthesizing the ionic lipid.
  • substituents include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, an alkylcarbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, and an acyl group having 2 to 4 carbon atoms.
  • acylamino group alkoxycarbonylamino group having 2 to 4 carbon atoms, fluorine atom, chlorine atom, bromine atom, iodine atom, alkylsulfanyl group having 1 to 4 carbon atoms, alkylsulfonyl group having 1 to 4 carbon atoms, 6 to 6 carbon atoms 10 arylsulfonyl group, nitro group, trifluoromethyl group, cyano group, alkyl group having 1 to 4 carbon atoms, ureido group, alkylureido group having 2 to 4 carbon atoms, alkoxy group having 1 to 4 carbon atoms, carbon 6 to 10 aryl groups, 6 to 10 carbon atoms aryloxy groups, etc.
  • Preferred examples include acetyl group, methoxycarbonyl group, methylcarbamoyl group, acetoxy group, acetamido group, methoxycarbonylamino group, fluorine atom. , chlorine atom, bromine atom, iodine atom, methylsulfanyl group, phenylsulfonyl group, nitro group, trifluoromethyl group, cyano group, methyl group, ethyl group, propyl group, isopropyl group, tert-butyl group, ureido group, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups, phenyl groups and phenoxy groups.
  • each R 4 may be the same or different.
  • Z a may be the same as or different from Z b , but Z a is preferably the same group as Z b .
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or glutaric acid
  • Fat-soluble vitamins having a hydroxyl group include, for example, retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol, and tocotrienol. Fat-soluble vitamins having hydroxyl groups are preferably tocopherols.
  • sterol derivatives having a hydroxyl group examples include cholesterol, cholestanol, stigmasterol, ⁇ -sitosterol, lanosterol, and ergosterol, preferably cholesterol or cholestanol.
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched.
  • the aliphatic hydrocarbon group may be saturated or unsaturated.
  • the number of unsaturated bonds contained in the aliphatic hydrocarbon group is generally 1-6, preferably 1-3, more preferably 1-2.
  • Unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds, preferably carbon-carbon double bonds.
  • the number of carbon atoms contained in the aliphatic hydrocarbon group is preferably 13-19, most preferably 13-17.
  • Aliphatic hydrocarbon groups include alkyl groups, alkenyl groups, alkynyl groups and the like, and preferably include alkyl groups or alkenyl groups.
  • aliphatic hydrocarbon groups having 12 to 22 carbon atoms include dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, henicosyl group and docosyl group.
  • dodecenyl group tridecenyl group, tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenyl group, octadecenyl group, nonadecenyl group, icosenyl group, henicosenyl group, docosenyl group, dodecadienyl group, tridecadienyl group, tetradecadienyl group, pentadecadienyl group, hexadecadienyl group, heptadecadienyl group, octadecadienyl group, nonadecadienyl group, icosadienyl group, henicosadienyl group, docosadienyl group, octadecatrienyl group, icosatrienyl group, icosatetraenyl group,
  • the aliphatic hydrocarbon group having 12 to 22 carbon atoms is preferably tridecyl group, pentadecyl group, heptadecyl group, nonadecyl group, heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group, particularly preferably tridecyl group, They are a heptadecyl group, a heptadecenyl group and a heptadecadienyl group.
  • the C 12-22 aliphatic hydrocarbon groups represented by R 3a and R 3b are derived from fatty acids.
  • the carbonyl carbon derived from the fatty acid is included in -CO-O- in formula (1).
  • a specific example of the aliphatic hydrocarbon group is a heptadecadienyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.
  • R 3a may be the same as or different from R 3b , but preferably R 3a is the same group as R 3b .
  • R 1a is the same as R 1b
  • X a is the same as Xb
  • R 2a is the same as R 2b
  • Y a is the same as Yb
  • Z a is is the same as Zb
  • R3a is the same as R3b .
  • ionic lipid (1) include the following ionic lipids.
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic having 12 to 22 carbon atoms a hydrocarbon group (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group); Ionic lipids (1).
  • a fat-soluble vitamin having a hydroxyl group e.g., tocopherol
  • succinic anhydride or glutaric anhydride or an aliphatic having 12 to 22 carbon atoms a hydrocarbon group (e.g., heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group)
  • Ionic lipids (1).
  • each of R 1a and R 1b is independently an alkylene group having 1 to 4 carbon atoms (e.g., methylene group, ethylene group); each of X a and X b is independently an acyclic alkyl tertiary amino group having 1 to 3 carbon atoms and having 1 tertiary amino group (eg, —N(CH 3 )—); or a cyclic alkylene tertiary amino group (e.g., piperidylene group) having 2 to 5 carbon atoms and one tertiary amino group; R 2a and R 2b are each independently an alkylene group having 6 or less carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Y a and Y b are each independently an ester bond or an amide bond; Z a and Z b are each independently a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, having one aromatic ring, and
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g. , heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group); Ionic lipids (1).
  • a fat-soluble vitamin having a hydroxyl group e.g., tocopherol
  • succinic anhydride e.g., an aliphatic hydrocarbon group having 13 to 19 carbon atoms
  • each of R 1a and R 1b is independently an alkylene group having 1 to 2 carbon atoms (that is, a methylene group or an ethylene group);
  • X a and X b are each independently X 1 :
  • R 5 is an alkyl group having 1 to 3 carbon atoms (eg, methyl group)
  • X 2 is an alkyl group having 1 to 3 carbon atoms (eg, methyl group)
  • R 2a and R 2b are each independently an alkylene group having 4 or less carbon atoms (e.g., methylene group, ethylene group, trimethylene group); Y a and Y b are each independently an ester bond or an amide bond; Z a and Z b are each independently Z 1 :
  • R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g. , heptadecenyl group, heptadecadienyl group, 1-hexylnonyl group); Ionic lipids (1).
  • ionic lipid (1) examples include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD -Ph-P4C2, O-Ph-amide-P4C2, O-Ph-C3M.
  • the ionic lipid (1) is preferably an ionic lipid represented by the following formula.
  • the amount of ionic lipid (1) in the lipid nanoparticles of the present invention is, from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency in cells, and stability of lipid nanoparticles, ionic lipid (1), It is preferably 15 to 70 mol %, more preferably 15 to 55 mol %, still more preferably 15 to 35 mol % relative to the total of phospholipid and cholesterol.
  • the ionic lipid (1) can be produced by a known method (eg, the method described in WO 2019/188867 A1).
  • the lipid nanoparticles of the present invention comprise phospholipids. Only one type of phospholipid may be used, or two or more types may be used in combination.
  • phospholipids examples include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn -glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), their lyso forms etc.
  • PC 1,2-diacyl-sn-glycero-3-phosphocholine
  • PE 1,2-diacyl-sn-glycero-3-phosphoethanolamine
  • PS 1,2-diacyl-sn -glycero-3-phosphoserine
  • PG 1,2-diacyl-sn-glycero-3-phosphoglycerol
  • PA 1,2-diacyl-sn-glycero-3-phosphatidic acid
  • 1,2-diacyl-sn-glycero-3-phosphocholine are 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-dierucyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-myristoy
  • phospholipids are sometimes referred to by their abbreviations.
  • 1,2-diacyl-sn-glycero-3-phosphocholine is sometimes described as PC and 1,2-didecanoyl-sn-glycero-3-phosphocholine is sometimes described as DDPC.
  • 1,2-diacyl-sn-glycero-3-phosphoethanolamine are 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (DDPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1,2-dierucyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine (MPPE), 1-myristoy
  • 1,2-diacyl-sn-glycero-3-phosphoserine PS
  • PS 1,2-didecanoyl-sn-glycero-3-phosphoserine
  • DLPS 1,2-dilauroyl-sn-glycero-3-phosphoserine
  • DPPS 1,2-dipalmitoyl-sn-glycero-3-phosphoserine
  • DSPS 1,2-distearoyl-sn-glycero-3-phosphoserine
  • DOPS 1,2-dioleoyl-sn-glycero-3-phosphoserine
  • DOPS 1,2-dilinoleoyl-sn-glycero-3-phosphoserine
  • DEPS 1,2-dierucyl-sn-glycero-3-phosphoserine
  • DEPS 1,2-dierucyl-sn-glycero-3-phosphoserine
  • 1,2-diacyl-sn-glycero-3-phosphoglycerol are 1,2-didecanoyl-sn-glycero-3-phosphoglycerol (DDPG), 1,2-dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol (DLoPG), 1,2-dierucyl-sn-glycero-3-phosphoglycerol (DEPG), 1-myristoyl-2-palmito
  • 1,2-diacyl-sn-glycero-3-phosphatidic acid PA
  • 1,2-didecanoyl-sn-glycero-3-phosphatidic acid DDPA
  • 1,2-dilauroyl-sn-glycero-3-phosphatidic acid DLPA
  • 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid DMPA
  • 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid DPPA
  • 1,2-distearoyl-sn-glycero-3-phosphatidic acid DSPA
  • 1,2-dioleoyl-sn-glycero-3-phosphatidic acid DOPA
  • 1,2-dilinoleoyl-sn-glycero-3-phosphatidic acid DLoPA
  • 1,2-dierucyl-sn-glycero-3-phosphatidic acid DEPA
  • the phospholipid is preferably PC and/or PE.
  • the phospholipid is more preferably at least one selected from the group consisting of DOPC, POPC, DOPE and POPE.
  • the phospholipid is more preferably at least one selected from the group consisting of DOPC, SOPC, POPC, DPPC and DEPC.
  • the phospholipid is more preferably DOPC.
  • the amount of phospholipids in the lipid nanoparticles of the present invention is ionic lipid (1), phospholipid and cholesterol from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency in cells and stability of lipid nanoparticles is preferably 5 to 25 mol%, more preferably 5 to 20 mol%, and still more preferably 10 to 20 mol% of the total of
  • the lipid nanoparticles of the present invention contain cholesterol.
  • the amount of cholesterol in the lipid nanoparticles of the present invention is determined from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency in cells, and lipid nanoparticle stability. It is preferably 25 to 80 mol %, more preferably 50 to 70 mol %, still more preferably 55 to 65 mol % of the total.
  • Dimyristoylglycerol PEG The lipid nanoparticles of the present invention have the formula (2): CH2 ( OR6 )-CH( OR7 ) -CH2 ( OR8 ) (2) (In formula (2), Two of R 6 , R 7 and R 8 represent a myristoyl group, and the remaining one has 1 to 6 carbon atoms and is linked via a polyethylene glycol (PEG) chain having a number average molecular weight of 4,000 to 6,000. represents an alkyl group of ) Dimyristoylglycerol PEG represented by (this specification may be abbreviated as “dimyristoylglycerol PEG (2)”).
  • the number average molecular weight of the PEG chain in formula (2) is 4,000-6,000, preferably 4,500-5,500.
  • the number average molecular weight of the PEG used to form this PEG chain can be measured by gel permeation chromatography (GPC).
  • the alkyl group having 1 to 6 carbon atoms may be linear, branched or cyclic.
  • the number of carbon atoms in the alkyl group is preferably 1-3.
  • Specific examples of alkyl groups having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentyl group and isopentyl group.
  • a methyl group is preferred.
  • the amount of dimyristoylglycerol PEG (2) in the lipid nanoparticles of the present invention is ionic lipid (1) from the viewpoint of nucleic acid encapsulation efficiency, nucleic acid release efficiency in cells, and lipid nanoparticle stability. , preferably 1 to 6 mol %, more preferably 2 to 5 mol %, still more preferably 2 to 3 mol %, relative to the total of phospholipid and cholesterol.
  • Optimal Composition The optimal molar ratio of ionic lipid (1):phospholipid:cholesterol:dimyristoylglycerol PEG (2) in the lipid nanoparticles of the present invention is 25:15:60:3.
  • the lipid nanoparticles of the present invention are prepared by mixing lipid raw materials containing ionic lipid (1), phospholipid, cholesterol, and dimyristoylglycerol PEG (2) in an appropriate dispersion medium (e.g., aqueous dispersion medium, alcohol It can be produced by dispersing in an organic dispersion medium) and, if necessary, performing an operation to induce organization.
  • an appropriate dispersion medium e.g., aqueous dispersion medium, alcohol It can be produced by dispersing in an organic dispersion medium) and, if necessary, performing an operation to induce organization.
  • Examples of the "organization-inducing operation" for producing the lipid nanoparticles of the present invention include ethanol dilution using a microchannel or vortex, simple hydration, ultrasonication, heating, vortex, ether Methods known per se such as injection method, French press method, cholic acid method, Ca 2+ fusion method, freeze-thaw method, reverse phase evaporation method, etc., preferably ethanol dilution method using microchannel or vortex. More preferably, it is an ethanol dilution method using a microchannel.
  • an acidic buffer solution containing nucleic acid and an ethanol solution of lipid are mixed to form a dispersion containing lipid nanoparticles.
  • liquid can be produced.
  • the dispersion produced by this method contains lipid nanoparticles and a dispersion medium (acidic buffer and ethanol). (especially buffer solution) can be exchanged.
  • the present invention also provides a method for delivering nucleic acid to brain tissue, comprising administering to a subject the lipid nanoparticles of the present invention encapsulating nucleic acid.
  • the lipid nanoparticles are nasally administered to the subject.
  • nucleic acids examples include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, DNA/RNA hybrids, and the like.
  • the nucleic acid may be either single-stranded or triple-stranded, preferably single-stranded or double-stranded.
  • Nucleic acids can be, for example, nucleotides with N-glycosides of purine or pyrimidine bases, oligomers with non-nucleotide backbones (such as commercially available peptide nucleic acids (PNA), etc.), or oligomers with special linkages (wherein said oligomers are DNA or contain nucleotides having a configuration that allows base pairing or base attachment as found in RNA).
  • PNA commercially available peptide nucleic acids
  • nucleic acids include, for example, nucleic acids with known modifications, labeled nucleic acids known in the art, capped nucleic acids, methylated nucleic acids, one or more natural nucleotides replaced with analogues.
  • Nucleic acids containing intercurrent compounds e.g., acridine, psoralen, etc.
  • nucleic acids containing chelate compounds e.g., metals, radioactive metals, boron, oxidizing metals, etc.
  • alkylating agents and nucleic acids having modified bonds eg, ⁇ -anomeric nucleic acids, etc.
  • DNA that can be used in the present invention is not particularly limited, and can be appropriately selected according to the purpose of use.
  • Examples of DNA include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo and the like, preferably plasmid DNA, cDNA and antisense DNA, more preferably plasmid DNA.
  • Circular DNA such as plasmid DNA can be appropriately digested with a restriction enzyme or the like and used as linear DNA.
  • RNA that can be used in the present invention is not particularly limited, and can be appropriately selected according to the purpose of use.
  • examples of RNA include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., preferably siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon.
  • nucleic acids used in the present invention are preferably purified by methods commonly used by those skilled in the art.
  • the nucleic acid used in the present invention preferably has preventive and/or therapeutic activity (prophylactic/therapeutic nucleic acid) for a given disease.
  • Such nucleic acids include, for example, nucleic acids used for so-called gene therapy.
  • the particle size of the nucleic acid-encapsulated lipid nanoparticles is not particularly limited, but is preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm.
  • the particle size can be measured using a particle size distribution analyzer such as Zetasizer Nano (Malvern).
  • the particle size of the nucleic acid-encapsulated lipid nanoparticles can be appropriately adjusted depending on the production method.
  • the surface potential (zeta potential) of the nucleic acid-encapsulated lipid nanoparticles is not particularly limited, but is preferably -15 to +15 mV, more preferably -10 to +10 mV.
  • particles with positively charged surfaces have mainly been used. This is useful as a method for promoting electrostatic interaction with negatively charged cell surface heparin sulfate and promoting cell uptake.
  • the positive surface potential can cause (a) suppression of nucleic acid release from the carrier due to interaction with delivery nucleic acid in cells, and (b) suppression of protein synthesis due to interaction of mRNA with delivery nucleic acid. have a nature. This problem can be solved by adjusting the surface potential (zeta potential) within the above range.
  • the surface potential (zeta potential) can be measured using a zeta potential measuring device such as Zetasizer Nano.
  • the surface potential (zeta potential) of lipid nanoparticles can be adjusted by the composition of the constituent components of the lipid nanoparticles.
  • the lipid nanoparticles By administering the nucleic acid-encapsulated lipid nanoparticles of the present invention to a subject, the lipid nanoparticles reach and come into contact with the brain tissue, and the nucleic acid encapsulated in the lipid nanoparticles is delivered to the brain tissue in vivo. be done.
  • Subjects to which the lipid nanoparticles can be administered are not particularly limited. , frogs, etc.), fish (eg, zebrafish, killifish, etc.), and the like.
  • the subject of administration of the lipid nanoparticles is preferably humans or other mammals.
  • the method of administering nucleic acid-encapsulated lipid nanoparticles to a subject is not particularly limited as long as the lipid nanoparticles can deliver nucleic acids to brain tissue, and per se known administration methods (e.g., oral administration, parenteral administration (e.g., , nasal administration, intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.) can be selected as appropriate.
  • parenteral administration e.g., nasal administration, intravenous administration, intramuscular administration, topical administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.
  • nasal administration is preferred.
  • the dose of the lipid nanoparticles can be appropriately selected in consideration of the type of administration subject, administration method, and the like.
  • the lipid nanoparticles of the present invention can be used as they are or mixed with a pharmaceutically acceptable carrier, and can be used in oral formulations (eg, tablets, capsules, etc.) or parenteral formulations (eg, intranasal formulations, injections, inhalation). agent, etc.), preferably as a parenteral agent (more preferably, as an intranasal agent).
  • the pharmaceutically acceptable carrier those commonly used as pharmaceutical materials are used.
  • excipients such as lubricants, binders, disintegrants, etc.
  • liquid formulations such as solvents, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like are used.
  • formulation additives such as preservatives, antioxidants, coloring agents, and sweetening agents can be used as necessary.
  • nasal formulations are used in the form of nasal drops or sprays.
  • ionic lipid (1) is indicated by the names described in the table above. Abbreviations used in the following examples and the like have the following meanings.
  • Chol Cholesterol
  • DDW Deionized distilled water
  • DEPC 1,2-dierucyl-sn-glycero-3-phosphocholine
  • DiR 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine Iodide
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (PEG number average molecular weight (Mn): 2000)
  • DMG-PEG5000 1,2-dimyristoyl-rac-glycerol, methoxypolyethylene glycol (PEG number average molecular weight (Mn): 5000)
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • EPC egg phosphatidy
  • Example 1 Preparation of Lipid Nanoparticles (LNP) As lipids, SS-OP, DOPC and Chol, and DMG-PEG5000 or DMG-PEG2000 (comparative examples) were used.
  • the molar ratio of SS-OP:DOPC:Chol used was 52.5:7.5:40 and 3 mol% of DMG-PEG5000 or DMG-PEG2000 was used for the sum of SS-OP, DOPC and Chol.
  • 0.5 mol % of the fluorescent dye DiR with respect to the sum of SS-OP, DOPC and Chol was added to the lipid ethanol solution.
  • MES buffer pH 6.5
  • the resulting mixture was transferred to Amicon Ultra 15 and subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to obtain approximately 1500 ⁇ L.
  • concentrated to The resulting concentrate was diluted to 15 mL with MES buffer (pH 6.5), and again subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to concentrate to about 1500 ⁇ L.
  • the obtained concentrate was diluted to 15 mL with PBS and concentrated to about 1500 ⁇ L by ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes).
  • the obtained concentrate was diluted to 15 mL with DDW, subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes), and concentrated to about 250 ⁇ L.
  • the resulting concentrate was transferred to Amicon Ultra 4, diluted to 4 mL using DDW, and again subjected to ultrafiltration under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to approximately 100 ⁇ L.
  • the resulting concentrate was diluted with DDW to a lipid concentration of 40 mM to obtain a dispersion containing LNP.
  • antisedan 100 ⁇ L/20 g mouse
  • mice were returned to their cages.
  • antisedan 100 ⁇ L/20 g mouse
  • the mouse head was excised and left on ice. After 5 minutes, the whole brain was excised using dissecting scissors.
  • IVIS Lumina II (Caliper) was used (measurement conditions: Imaging Mode: Fluorescent, Exposure Time: 10 sec, Binning: Medium, F/Stop: 2, Lamp Level: High, Excitation Filter: 745 nm, Emission Filter: ICG), imaging was performed.
  • DMG-PEG5000 tended to result in better translocation of LNP to the brain than the use of DMG-PEG2000.
  • LNP using DMG-PEG5000 was administered by the LHB method, a tendency toward better penetration into the brain was observed (Fig. 1).
  • DMG2k conventional method or “DMG5k conventional method” in FIG. 1 indicates the results of the conventional method using DMG-PEG2000 or DMG-PEG5000, respectively
  • DMG2k LHB method or “DMG2k method” in FIG. LHB method” indicates the results of the LHB method using DMG-PEG2000 or DMG-PEG5000, respectively.
  • Example 2 Preparation of various LNPs with different molar ratios of ionic lipid (1) and phospholipids SS-OP, DOPC, Chol and DMG-PEG5000 were used as lipids.
  • the molar ratios of SS-OP:DOPC:Chol used were "52.5:7.5:40", “45:15:40", or "30:30:40” and SS-OP, DOPC 3 mol % of DMG-PEG5000 was used with respect to the sum of C and Chol.
  • lipid nanoparticles containing egg phosphatidylcholine EPC
  • EPC egg phosphatidylcholine
  • EPC/Chol 70/30
  • 3 mol % of DMG-PEG5000 was added to the total of EPC and Chol.
  • 0.5 mol % of the fluorescent dye DiR relative to the sum of SS-OP, DOPC and Chol or the sum of EPC and Chol was added to ethanol solutions of lipids.
  • MES buffer pH 6.5
  • the resulting mixture was transferred to Amicon Ultra 15 and subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to obtain approximately 1500 ⁇ L.
  • concentrated to The resulting concentrate was diluted to 15 mL with MES buffer (pH 6.5), and again subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to concentrate to about 1500 ⁇ L.
  • the obtained concentrate was diluted to 15 mL with PBS and concentrated to about 1500 ⁇ L by ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes).
  • the obtained concentrate was diluted to 15 mL with DDW, subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes), and concentrated to about 250 ⁇ L.
  • the resulting concentrate was transferred to Amicon Ultra 4, diluted to 4 mL using DDW, and again subjected to ultrafiltration under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 50 ⁇ L.
  • the resulting concentrate was diluted with DDW to a lipid concentration of 40 mM to obtain a dispersion containing each LNP.
  • the resulting dispersion was used in Test Example 2 below.
  • Test Example 2 Effect of Composition Ratio of Phospholipids on Brain Translocation of LNP by In Vivo Administration Mice were intraperitoneally administered three kinds of mixed anesthesia (100 ⁇ L/20 g mouse), and then placed on a particle administration platform for LHB method. . Thirty minutes after administration of the triple anesthesia, a 10 ⁇ L pipette was used to administer the LNP-containing dispersion at 5 ⁇ L/20 g mouse to the left and right mouse nasal cavities at 3-minute intervals, for a total of 10 ⁇ L/20 g mouse.
  • Example 3 Preparation of various LNPs with different phospholipids SS-OP, phospholipids (DOPC, SOPC, POPC, DPPC or DEPC), Chol, and DMG-PEG5000 were used as lipids.
  • the SS-OP:Phospholipid:Chol molar ratio used was 45:15:40 and 3 mol % DMG-PEG5000 was used for the sum of SS-OP, phospholipid and Chol.
  • 0.5 mol % of the fluorescent dye DiR with respect to the sum of SS-OP, phospholipid and Chol was added to the lipid ethanol solution.
  • MES buffer pH 6.5
  • the resulting mixture was transferred to Amicon Ultra 15 and subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to obtain approximately 1500 ⁇ L.
  • concentrated to The resulting concentrate was diluted to 15 mL with MES buffer (pH 6.5), and again subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to concentrate to about 1500 ⁇ L.
  • the obtained concentrate was diluted to 15 mL with PBS and concentrated to about 1500 ⁇ L by ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes).
  • the obtained concentrate was diluted to 15 mL with DDW, subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes), and concentrated to about 250 ⁇ L.
  • the resulting concentrate was transferred to Amicon Ultra 4, diluted to 4 mL using DDW, and again subjected to ultrafiltration under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to about 50 ⁇ L.
  • the resulting concentrate was diluted with DDW to a lipid concentration of 40 mM to obtain a dispersion containing each LNP.
  • the resulting dispersion was used in Test Example 3 below.
  • Test Example 3 Effects of Types of Phospholipids on Brain Translocation of LNP by In Vivo Administration Mice were intraperitoneally administered three kinds of mixed anesthesia (100 ⁇ L/20 g mouse), and then placed on a particle administration table for the LHB method. Thirty minutes after administration of the triple anesthesia, a 10 ⁇ L pipette was used to administer the LNP-containing dispersion at 5 ⁇ L/20 g mouse to the left and right mouse nasal cavities at 3-minute intervals, for a total of 10 ⁇ L/20 g mouse.
  • Example 4 Preparation of various LNPs with different molar ratios of ionic lipid (1) and cholesterol SS-OP, DOPC, Chol and DMG-PEG5000 were used as lipids.
  • the molar ratios of SS-OP:DOPC:Chol used were "85:15:0", “65:15:20", “45:15:40", or "25:15:60” and SS -3 mol% of DMG-PEG5000 was used with respect to the sum of OP, DOPC and Chol.
  • the obtained concentrate was diluted to 15 mL with PBS and concentrated to about 1500 ⁇ L by ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes). Then, the obtained concentrate was diluted to 15 mL with DDW, subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes), and concentrated to about 250 ⁇ L. The resulting concentrate was transferred to Amicon Ultra 4, diluted to 4 mL using DDW, and concentrated again to approximately 50 ⁇ L under centrifugal conditions (25° C., 1000 g, 5 minutes). The resulting concentrate was diluted with DDW to a lipid concentration of 40 mM to obtain a dispersion containing each LNP. The resulting dispersion was used in Test Example 4 below.
  • Test Example 4 Effect of Cholesterol Composition Ratio on LNP Translocation to Brain by In Vivo Administration Mice were intraperitoneally administered triple anesthesia (100 ⁇ L/20 g mouse), and then placed on a particle administration table for the LHB method. Thirty minutes after administration of the triple anesthesia, a 10 ⁇ L pipette was used to administer the LNP-containing dispersion at 5 ⁇ L/20 g mouse to the left and right mouse nasal cavities at 3-minute intervals, for a total of 10 ⁇ L/20 g mouse.
  • antisedan 100 ⁇ L/20 g mouse
  • mice were returned to their cages.
  • Eighteen hours after administration the mouse head was excised and left on ice. Five minutes later, the brain was excised using dissecting scissors. After extracting the brain, it was placed in a crushing tube and frozen in liquid nitrogen.
  • In vivo Lysis Buffer 800 ⁇ L was added to the crushing tube and homogenized twice under the conditions of 4800 rpm and 30 seconds. The supernatant was collected and centrifuged at 13000 rpm, 4°C and 10 minutes.
  • Example 5 Preparation of mRNA-encapsulated LNP with optimized lipid composition SS-OP, DOPC, Chol, and DMG-PEG5000 were used as lipids, and were estimated to have the highest brain penetration from the above test examples. Ethanol solutions of lipids were analyzed by lipid composition (molar ratio of SS-OP:DOPC:Chol was 25:15:60, using 3 mol% DMG-PEG5000 for the sum of SS-OP, DOPC and Chol). prepared. For particle labeling, 0.5 mol % of the fluorescent dye DiR with respect to the sum of SS-OP, DOPC and Chol was added to the lipid ethanol solution.
  • Luciferase-encoding mRNA (CleanCap (registered trademark) FLuc mRNA-(L-7602)) at a final concentration of 0.0067 ⁇ g/ ⁇ L and acidic malic acid buffer (20 mM, pH 3.0) containing NaCl at a final concentration of 30 mM, respectively. 2100 ⁇ L (for 2 administrations), 3750 ⁇ L (for 4 administrations) or 7200 ⁇ L (for 8 administrations), and 800 ⁇ L (for 2 administrations) and 1350 ⁇ L (for 8 administrations) of ethanol solution of lipids, respectively. 4 doses) or 2500 ⁇ L (8 doses) was weighed into a syringe.
  • NanoAssmblr registered trademark ultra-high-speed nanopharmaceutical preparation device (manufactured by Precision NanoSystems), addition rate of acidic buffer solution: 3 mL/min, addition rate of lipid ethanol solution: 1 mL/min, and syringe holder temperature: 25 ° C.
  • LNP was prepared under the conditions of and collected in a 15 mL tube. After adding 3000 ⁇ L of MES buffer (pH 6.5) to a 15 mL tube, the resulting mixture was transferred to Amicon Ultra 15 and subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to obtain approximately 1500 ⁇ L.
  • the resulting concentrate was diluted to 15 mL with MES buffer (pH 6.5), and again subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes) to concentrate to about 1500 ⁇ L. Also, the obtained concentrate was diluted to 15 mL with PBS and concentrated to about 1500 ⁇ L by ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes). Then, the obtained concentrate was diluted to 15 mL with DDW, subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes), and concentrated to about 250 ⁇ L.
  • MES buffer pH 6.5
  • the obtained concentrate was transferred to Amicon Ultra 4, diluted to 4 mL using DDW, and ultrafiltrated again under centrifugal conditions (25°C, 1000 g, 5 minutes) to about 50 ⁇ L (for 2 administrations). , 100 ⁇ L (for 4 doses), or 200 ⁇ L (for 8 doses).
  • the resulting concentrate was diluted with DDW to a lipid concentration of 40 mM to obtain a dispersion containing LNP.
  • the resulting dispersion was used in Test Example 5 below.
  • Test Example 5 Effect of Number of Administrations on LNP Translocation to Brain by In Vivo Administration Mice were intraperitoneally administered three kinds of mixed anesthesia (100 ⁇ L/20 g mouse), and then placed on a particle administration platform for LHB method. Three types of mixed anesthesia (30 minutes after administration, using a 10 ⁇ L pipette, 5 ⁇ L / 20 g mouse each of the dispersion containing LNP into the left and right mouse nasal cavities at intervals of 3 minutes, total 10 ⁇ L / 20 g mouse (2 administrations (1 administration set)), 20 ⁇ L/20 g mouse (4 administrations (2 administration sets)), or 40 ⁇ L/20 g mouse (8 administrations (4 administration sets)).
  • mice received antisedan (100 ⁇ L/20 g mouse) was administered intraperitoneally and the mice were returned to their cages. Eighteen hours after administration, the mouse head was excised and left on ice. After 5 minutes, the whole brain was excised using dissecting scissors. After excising the brain, IVIS Lumina II (Caliper) was used (measurement conditions: Imaging Mode: Fluorescent, Exposure Time: 10 sec, Binning: Medium, F/Stop: 2, Lamp Level: High, Excitation Filter: 745 nm, Emission Filter: ICG), imaging was performed. The brains were then placed in crushing tubes and frozen in liquid nitrogen.
  • In vivo Lysis Buffer (800 ⁇ L) was added to the crushing tube and homogenized twice (4800 rpm, 30 seconds). The supernatant was collected and centrifuged at 13000 rpm, 4°C and 10 minutes. Furthermore, the supernatant was recovered, 50 ⁇ L of luciferin was added to 20 ⁇ L of the supernatant, and luminescence was measured with a luminometer. As a result, dose-dependent brain migration (FIGS. 5-1 and 5-2, left) and luciferase intracerebral expression (FIG. 5-2, right) were observed.
  • Example 6 Preparation of mRNA-encapsulated LNP with lipid composition optimized for brain penetration
  • the lipid composition (SS-OP: DOPC :Chol molar ratio was 25:15:60 and 3 mol% DMG-PEG5000 was used for the sum of SS-OP, DOPC and Chol) to prepare an ethanol solution of lipids.
  • SS-OP, DOPC, Chol and DMG-PEG2000 were used as lipids, and the lipid composition known to have excellent transferability to the liver (the molar ratio of SS-OP:DOPC:Chol was 52.5:7.5:40 using 3 mol % DMG-PEG2000 for the sum of SS-OP, DOPC and Chol).
  • the obtained concentrate was diluted to 15 mL with PBS and concentrated to about 1500 ⁇ L by ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes). Then, the obtained concentrate was diluted to 15 mL with DDW, subjected to ultrafiltration under centrifugal conditions (25° C., 1000 g, 5 minutes), and concentrated to about 250 ⁇ L. The resulting concentrate was transferred to Amicon Ultra 4, diluted to 4 mL using DDW, and again subjected to ultrafiltration under centrifugal conditions (25°C, 1000 g, 5 minutes) to concentrate to approximately 250 ⁇ L. The resulting concentrate was diluted with DDW to a lipid concentration of 40 mM to obtain a dispersion containing each LNP. The resulting dispersion was used in Test Example 6 below.
  • Test Example 6 Verification of Optimization of Conditions for LNP Translocation to the Brain by In Vivo Administration
  • Mice were intraperitoneally administered three kinds of mixed anesthesia (100 ⁇ L/20 g mouse), and then placed on a particle administration table for LHB method. 30 minutes after administration of the three kinds of mixed anesthesia, a 10 ⁇ L pipette was used to administer a dispersion containing LNP at 5 ⁇ L/20 g mouse to the left and right mouse nasal cavities at intervals of 3 minutes, for a total of 40 ⁇ L/20 g mouse (8 administrations). (4 administration sets)).
  • antisedan 100 ⁇ L/20 g mouse
  • mice were returned to their cages.
  • the mouse head was excised and left on ice. After 5 minutes, the whole brain was excised using dissecting scissors.
  • IVIS Lumina II (Caliper) was used (measurement conditions: Imaging Mode: Fluorescent, Exposure Time: 10 sec, Binning: Medium, F/Stop: 2, Lamp Level: High, Excitation Filter: 745 nm, Emission Filter: ICG), imaging was performed. The brains were then placed in crushing tubes and frozen in liquid nitrogen.
  • In vivo Lysis Buffer (800 ⁇ L) was added to the crushing tube and homogenized twice under the conditions of 4800 rpm and 30 seconds. The supernatant was collected and centrifuged at 13000 rpm, 4°C and 10 minutes. Furthermore, the supernatant was recovered, 50 ⁇ L of luciferin was added to 20 ⁇ L of the supernatant, and luminescence was measured with a luminometer.
  • the composition selected for brain migration (NtB composition) and dosage and administration showed significantly more transferability to the brain (Fig. 6 -1 and FIG. 6-2 left) and luciferase expression levels in the brain (FIG. 6-2 right) were confirmed to be high, suggesting that the LNP conditions for brain transfer could be optimized.
  • the lipid nanoparticles of the present invention are useful for delivering nucleic acids to brain tissue.

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