WO2023191050A1 - Composition pharmaceutique destinée à être administrée pour un cancer, ou composition immunostimulante - Google Patents

Composition pharmaceutique destinée à être administrée pour un cancer, ou composition immunostimulante Download PDF

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WO2023191050A1
WO2023191050A1 PCT/JP2023/013553 JP2023013553W WO2023191050A1 WO 2023191050 A1 WO2023191050 A1 WO 2023191050A1 JP 2023013553 W JP2023013553 W JP 2023013553W WO 2023191050 A1 WO2023191050 A1 WO 2023191050A1
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lipid
lipid nanoparticles
cl15f
alkyl group
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Japanese (ja)
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彩夏 大津
佳己 前田
一毅 橋場
将光 田口
左知子 坂元
卓矢 宍戸
悠介 佐藤
秀吉 原島
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日東電工株式会社
国立大学法人北海道大学
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    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
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    • 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
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
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    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • 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
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers

Definitions

  • the present invention relates to lipid nanoparticles useful as pharmaceutical compositions for cancer delivery or immunostimulatory compositions.
  • Lipid nanoparticles are used as carriers to encapsulate fat-soluble drugs, siRNA (short interfering RNA), mRNA, and other nucleic acids and deliver them to target cells.
  • siRNA short interfering RNA
  • mRNA short interfering RNA
  • lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids such as siRNA into target cells they are electrically neutral at physiological pH and become cationic in weakly acidic pH environments such as endosomes.
  • Lipid nanoparticles containing changing pH-sensitive cationic lipids as constituent lipids have been reported (Patent Document 1 and Non-Patent Document 1).
  • Jayaraman et al. developed DLin-MC3-DMA and achieved an ED 50 of 0.005 mg siRNA/kg in factor 7 (F7) knockdown in mouse liver (non-patent Reference 2).
  • the present inventors have also developed original pH-sensitive cationic lipids YSK05 and YSK13-C3, and achieved ED50 of 0.06 and 0.015 mg siRNA/kg, respectively, in F7 knockdown ( Non-patent documents 3 to 5).
  • Non-patent Documents 6 to 8 L319, which is MC3-DMA with biodegradability, and reported that it has both an ED 50 of 0.01 mg siRNA/kg and high safety.
  • Non-patent Documents 6 to 8 it has been revealed that the endosomal escape efficiency of lipid nanoparticles containing these pH-sensitive cationic lipids is still only about a few percent (Non-Patent Document 9), and it is possible to further improve bioavailability.
  • the development of new technology is desired.
  • Non-Patent Document 10 a unique lipid-like substance cKK-E12 through high-throughput screening and achieved an ED 50 of 0.002 mg siRNA/kg in F7 knockdown.
  • An object of the present invention is to provide lipid nanoparticles useful as pharmaceutical compositions for cancer delivery or immunostimulatory compositions.
  • lipid nanoparticles containing pH-sensitive cationic lipids with branched hydrocarbon chains as constituent lipids are useful as pharmaceutical compositions for cancer delivery or immunostimulatory compositions. , completed the present invention.
  • the present invention provides the following lipid nanoparticles.
  • [1-2] The lipid nanoparticle of [1-1] above, further containing a sterol and polyalkylene glycol-modified lipid.
  • [1-3] The lipid nanoparticle of [1-1] or [1-2] above, which contains a nucleic acid.
  • the lipid nanoparticle of [1-3] above, wherein the nucleic acid is siRNA.
  • [1-5] The lipid nanoparticle according to [1-3] above, wherein the nucleic acid is mRNA or plasmid DNA.
  • [1-6] The lipid nanoparticle according to any one of [1-3] to [1-5], wherein the nucleic acid is a gene expressed in liver cells.
  • [1-7] A pharmaceutical composition containing the lipid nanoparticles according to any one of [1-1] to [1-6] above as an active ingredient.
  • [1-8] The pharmaceutical composition of [1-7] above, which is used for gene therapy.
  • the lipid nanoparticles according to any one of [1-1] to [1-6] above, which encapsulate a foreign gene that is intended to be expressed in liver cells, are administered to a test animal (however, , excluding humans), and the foreign gene is expressed in the liver of the test animal.
  • [5-3] The lipid nanoparticle preparation of [5-1] or [5-2], wherein the nucleic acid is mRNA.
  • [5-4] The lipid nanoparticle formulation according to any one of [5-1] to [5-3], in which the lipid nanoparticles are suspended in an aqueous solution.
  • [5-5] The lipid nanoparticle preparation according to [5-4], wherein the concentration of the disaccharide is 1% to 20% by weight.
  • [5-6] The lipid nanoparticle formulation according to either [5-4] or [5-5], which has a pH of 6.8 to 8.0 at 25°C.
  • [5-7] The lyophilized lipid nanoparticle formulation according to any one of [5-1] to [5-3].
  • [5-8] A resuspension formulation in which water or an aqueous solution is added to the lipid nanoparticle formulation of [5-7].
  • [6-2] The volume ratio of the tetrahydrofuran (THF) and the dimethylpropylene urea (DMPU) in the step is 10:1 to 1:1 (v/v).
  • [6-3] The method for producing [6-1] or [6-2], wherein the organic lithium is lithium diisopropylamide (LDA).
  • [6-4] The production method according to any one of [6-1] to [6-3], wherein the alkyl halide is iodoalkyl.
  • [6-5] The production method according to any one of [6-1] to [6-4], further comprising a step of purifying the branched fatty acid by reverse phase chromatography.
  • the chemical formula of the pH-sensitive cationic lipid is as follows:
  • a production method comprising at least the step of hydrolyzing and heating a reaction solution obtained by reacting a malonic acid ester and an alkyl halide in the presence of a base to obtain a branched fatty acid.
  • [7-2] (i) Sterol or sterol derivative, The pharmaceutical composition for cancer delivery according to [7-1], comprising (ii) a polyalkylene glycol-modified lipid and (iii) a buffer.
  • [7-3] The pharmaceutical composition for cancer delivery according to [7-2], wherein the polyalkylene glycol-modified lipid contains 1,2-distearoyl-rac-glycerolmethoxypolyethylene glycol (DSG-PEG).
  • [7-5] The pharmaceutical composition for cancer delivery according to any one of [7-1] to [7-4], further comprising a nucleic acid.
  • [7-6] The pharmaceutical composition for cancer delivery according to [7-5], wherein the nucleic acid is at least one selected from the group consisting of siRNA, mRNA, aptamer, and plasmid DNA.
  • [7-8] The pharmaceutical composition for cancer delivery according to [7-7], which has a pH of 6.8 to 8.0 at 25°C.
  • a pharmaceutical composition for cancer treatment for treating individuals with cancer comprising the composition for cancer delivery according to any one of [7-1] to [7-10].
  • a pharmaceutical composition for cancer treatment comprising:
  • the lipid nanoparticles according to the present invention can highly express the encapsulated genes in the liver or spleen. Therefore, the lipid nanoparticles are useful as liver-specific gene delivery carriers or spleen-specific gene delivery carriers used in gene therapy. Moreover, the lipid nanoparticles according to the present invention have excellent stability.
  • FIG. 2 is a diagram showing the results of measuring the pKa of each F7siRNA-loaded lipid nanoparticle in Example 1.
  • Figure 1 (A) shows the results for lipid nanoparticles prepared using CL4F6, CL4G6, or CL4H6, and
  • Figure 1 (B) shows the results for lipid nanoparticles prepared using CL15F6, CL15G6, or CL15H6. be.
  • FIG. 2 is a diagram showing the results of calculating the relative plasma F7 enzyme activity (%) of mice to which each F7 siRNA-loaded lipid nanoparticle was administered in Example 1.
  • FIG. 2 is a diagram showing the measurement results of Nluc activity (RLU/mg protein) in the liver and spleen of mice to which each Nluc mRNA-loaded lipid nanoparticle was administered in Example 2.
  • 3 is a diagram showing the measurement results of Fluc activity of HeLa-GFP cells into which each pFluc-loaded lipid nanoparticle was introduced in Example 3.
  • FIG. 3 is a diagram showing the measurement results of Fluc activity (RLU/mg protein) in the liver and spleen of mice to which each pFluc-loaded lipid nanoparticle was administered in Example 3.
  • X1 to X2 (X1 and X2 are real numbers satisfying X1 ⁇ X2)" means "more than or equal to X1 and less than or equal to X2.”
  • Lipid nanoparticles according to the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid represented by the following general formula (I) (hereinafter sometimes referred to as "pH-sensitive cationic lipid of the present invention"). It is.
  • pH-sensitive cationic lipid represented by general formula (I) as a constituent lipid of the lipid nanoparticles, the lipid nanoparticles according to the present invention have high selectivity to the liver or spleen.
  • a represents an integer of 3 to 5, preferably 4.
  • b indicates 0 or 1. When b is 0, it means that there is no -O-CO- group and it is a single bond.
  • R 1 and R 2 each independently represent a group represented by the following general formula (A).
  • R 11 and R 12 each independently represent a linear or branched C 1-15 alkyl group (alkyl group having 1 to 15 carbon atoms); c is 0 or 1 ; v represents an integer from 4 to 12.
  • R 11 and R 12 are each independently preferably a linear or branched C 1-12 alkyl group (an alkyl group having 1 to 12 carbon atoms); A linear or branched C 2-12 alkyl group (alkyl group having 2 to 12 carbon atoms) is more preferable ; It is even more preferably a straight-chain or branched C 5-10 alkyl group (an alkyl group having 5 to 10 carbon atoms); Most preferably, it is a C 6-9 alkyl group (alkyl group having 6 to 9 carbon atoms).
  • R 1 and R 2 may be groups represented by the general formula (A), and may be the same group or different groups. .
  • X represents a group represented by the following general formula (B) or a 5- to 7-membered non-aromatic heterocyclic group.
  • the 5- to 7-membered non-aromatic heterocyclic group represented by X is bonded to (O-CO)b- through a carbon atom.
  • d represents an integer of 0 to 3. When d is 0, it means that there is no --(CH 2 )- group and it is a single bond.
  • R 3 and R 4 each independently represent a C 1-4 alkyl group (alkyl group having 1 to 4 carbon atoms) or a C 2-4 alkenyl group (alkenyl group having 1 to 4 carbon atoms). show. In the C 1-4 alkyl group or C 2-4 alkenyl group represented by R 3 and R 4 , one or two hydrogen atoms may be substituted with a phenyl group.
  • R 3 and R 4 may be a C 1-4 alkyl group or a C 2-4 alkenyl group, and may be the same group or different groups.
  • Examples of the C 1-4 alkyl group include a methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, and tert-butyl group.
  • Examples of the C 2-4 alkenyl group include vinyl group, 1-propenyl group, 2-propenyl group, 1-methylvinyl group, 2-methyl-1-propenyl group, 1-butenyl group, 2-butenyl group, 3-butenyl group. Examples include groups.
  • R 3 and R 4 may be bonded to each other to form a 5- to 7-membered non-aromatic heterocycle.
  • Examples of the 5- to 7-membered non-aromatic heterocycle formed by R 3 and R 4 bonding to each other include 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, and 1-piperazinyl group. .
  • the 5- to 7-membered non-aromatic heterocycle formed by combining R 3 and R 4 with each other has one or two hydrogen atoms in the ring converted into a C 1-4 alkyl group or a C 2-4 alkenyl group. May be replaced. When two hydrogen atoms in the ring are substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group, they may be substituted with the same group, or may be substituted with different groups. Good too.
  • heteroatom contained in the heterocyclic group examples include a nitrogen atom, an oxygen atom, a sulfur atom, etc. .
  • the number of heteroatoms constituting the heterocycle in the heterocyclic group may be one, or two or more of the same or different heteroatoms.
  • the heterocycle in the heterocyclic group may be a saturated heterocycle and may contain one or more double bonds, but the heterocycle does not become an aromatic ring.
  • R 1 and R 2 are each independently, and in general formula (A), R 11 and R 12 are each independently, linear is a C 1-12 alkyl group in the form of a shaped or branched chain, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 1, X is a 5- to 7-membered non-aromatic heterocyclic group (bonded to (O-CO)b- via a carbon atom in the heterocyclic group), preferably 1-pyrrolidinyl group, 1-piperidinyl group, 1- Morpholinyl group or 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, one hydrogen atom is substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group) ) or in the general formula (I), R 1 and R 2 are each independently, and in the general formula (A), R 11
  • R 1 and R 2 are each independently, and in general formula (A), R 11 and R 12 are each independently linear or branched C 2-12 alkyl a group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 5- to 7-membered non-aromatic heterocycle group (bonded to (O-CO)b- through a carbon atom in the heterocyclic group), preferably a 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, or 1-piperazinyl group (bonded to (O-CO)b- through a carbon atom in the heterocyclic group) is bonded to (O-CO)b- through a carbon atom, and one hydrogen atom may be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group);
  • R 11 and R 12 are each independently linear or
  • R 1 and R 2 are each independently, and in general formula (A), R 11 and R 12 are each independently a linear or branched C 5- 12 alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, b is 1, and X is a 5- to 7-membered non-aromatic A heterocyclic group (bonded to (O-CO)b- through a carbon atom in the heterocyclic group), preferably a 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, or 1-piperazinyl group ( A compound which is bonded to (O-CO)b- through a carbon atom in the ring, and one hydrogen atom may be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group.
  • R 1 and R 2 are each independently, and in the general formula (A), R 11 and R 12 are each independently a linear or branched C 5- 12 alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 0, and X is a group of general formula (B) , d is 0, and R 3 and R 4 are each independently a C 1-4 alkyl group or a C 2-4 alkenyl group (a C 1-4 alkyl group or a C 2-4 alkenyl group represented by R 3 and R 4 is preferably a compound in which one or two hydrogen atoms may be substituted with a phenyl group.
  • the pH-sensitive cationic lipid of the present invention is such that in general formula (I), R 1 and R 2 are each independently, and in general formula (A), R 11 and R 12 are each independently , a linear or branched C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5, b is 1, and
  • the hydrogen atom may be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group.
  • R 11 and R 12 are each independently a linear or branched C 6-9 alkyl group, c is 1, and v is an integer from 6 to 10; and a is an integer of 3 to 5, b is 0, X is general formula (B), d is 0, and R 3 and R 4 are each independently C 1-4 Compounds that are alkyl groups are preferred.
  • R 1 and R 2 are each independently, and in general formula (A), R 11 and R 12 are each independently is a linear C 6-9 alkyl group, c is 1, v is an integer from 6 to 10, a is an integer from 3 to 5, and b is 1.
  • X is a 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, or 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, and one hydrogen atom is C (Optionally substituted with 1-4 alkyl group or C 2-4 alkenyl group);
  • R 1 and R 2 are each independently substituted with general formula (A).
  • R 11 and R 12 are each independently a branched C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is 3 to 5 is an integer of 1, b is 1, and and one hydrogen atom may be substituted with a C 1-4 alkyl group or a C 2-4 alkenyl group; in the general formula (I), R 1 and R 2 are each independently In the general formula (A), R 11 and R 12 are each independently a linear C 6-9 alkyl group, c is 1, and v is an integer from 6 to 10.
  • R 3 and R 4 are each independently C 1-4 A compound that is an alkyl group; in the general formula (I), R 1 and R 2 are each independently, and in the general formula (A), R 11 and R 12 are each independently a branched C 6-9 It is an alkyl group, c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 0, and X is of general formula (B).
  • compounds in which d is 0 and R 3 and R 4 are each independently a C 1-4 alkyl group;
  • R 1 and R 2 are the same group, and in general formula (A), R 11 and R 12 are each independently linear is a C 6-9 alkyl group in which c is 1, v is an integer of 6 to 10, a is an integer of 3 to 5, b is 1, and X is 1-pyrrolidinyl group, 1-piperidinyl group, 1-morpholinyl group, or 1-piperazinyl group (bonded to (O-CO)b- by a carbon atom in the ring, one hydrogen atom is C 1-4 (optionally substituted with an alkyl group or a C 2-4 alkenyl group); in the general formula (I), R 1 and R 2 are the same group, and in the general formula (A), R 11 and R 12 are each independently a branched C 6-9 alkyl group, c is 1, v is an integer of 6 to 10, and a is an integer of 3 to 5 , b is 1, and X is a 1-
  • the pH-sensitive cationic lipid of the present invention is, for example, a pH-sensitive cationic lipid having the following structure, a stereoisomer or a mixture of stereoisomers thereof:
  • the invention in one aspect, relates to the pH-sensitive cationic lipids of the invention.
  • the pKa of the pH-sensitive cationic lipid represented by general formula (I) is not particularly limited, but can be selected, for example, from about 4.0 to 9.0, preferably from about 4.5 to 8.5; It is preferred to select the type of each substituent to provide a range of pKas.
  • the pH-sensitive cationic lipid represented by general formula (I) can be easily produced, for example, by the method specifically shown in the Examples of this specification. By referring to this production method and appropriately selecting raw material compounds, reagents, reaction conditions, etc., those skilled in the art can easily produce any lipid falling within the scope of general formula (I).
  • the group represented by the general formula (A) is a group having a branched structure in which two hydrocarbon chains (R 11 and R 12 ) are connected to a -CO-O- group. That is, the pH-sensitive cationic lipid of the present invention has two branched hydrocarbon chains (R 1 and R 2 ), and these hydrocarbon chains are embedded in the lipid membrane of the lipid nanoparticle. becomes a hydrophobic scaffold.
  • the lipid nanoparticles according to the present invention have a characteristic of high selectivity to the liver or spleen by using the pH-sensitive cationic lipid of the present invention, which has a hydrophobic scaffold consisting of a branched chain structure, as a component of the lipid.
  • the number of pH-sensitive cationic lipids of the present invention constituting the lipid nanoparticles of the present invention may be one type or two or more types.
  • the amount of the pH-sensitive cationic lipids of the present invention is determined based on the amount of the pH-sensitive cationic lipids of the present invention among the lipid molecules constituting the lipid nanoparticles. It refers to the total amount of lipid molecules corresponding to the pH-sensitive cationic lipids of the invention.
  • the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles is preferably 20 mol% or more.
  • the proportion of pH-sensitive cationic lipids in the lipid molecules constituting the lipid nanoparticles is more preferably 30 mol% or more, even more preferably 30 to 70 mol%, even more preferably 40 to 60 mol%.
  • lipids other than the pH-sensitive cationic lipid of the present invention lipids generally used when forming liposomes can be used.
  • examples of such lipids include phospholipids, sterols or sterol derivatives, glycolipids, and saturated or unsaturated fatty acids. These can be used alone or in combination of two or more.
  • phospholipids examples include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phospharidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, and phosphatidic acid; sphingomyelin, ceramide phosphorylglycerol, and ceramide. Examples include sphingophospholipids such as phosphorylethanolamine; and the like. Furthermore, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.
  • glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phospharidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglyce
  • the fatty acid residues in glycerophospholipids and sphingophospholipids are not particularly limited, but examples include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms, and saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms. Fatty acid residues are preferred. Specifically, mention may be made of acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, and lignoceric acid. I can do it. When these glycerolipids or sphingolipids have two or more fatty acid residues, all the fatty acid residues may be the same group or may be mutually different groups.
  • sterols or sterol derivatives examples include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant sterols such as stigmasterol, sitosterol, ⁇ -sitosterol, campesterol, and brassicasterol; Sterols derived from microorganisms (phytosterols); sterols derived from microorganisms such as thymosterol and ergosterol.
  • animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol
  • plant sterols such as stigmasterol, sitosterol, ⁇ -sitosterol, campesterol, and brassicasterol
  • Sterols derived from microorganisms sterols
  • sterols derived from microorganisms such as thymosterol and
  • glycolipids examples include glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; glycosphingolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside; and the like. It will be done.
  • saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms, such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.
  • the constituent lipids of the lipid nanoparticles according to the present invention preferably include a neutral lipid, more preferably a phospholipid or a sterol, and preferably a sterol. It is more preferable, and even more preferable that it contains cholesterol.
  • the lipid nanoparticles according to the present invention preferably contain a polyalkylene glycol-modified lipid as a lipid component.
  • Polyalkylene glycol is a hydrophilic polymer, and by constructing lipid nanoparticles using polyalkylene glycol-modified lipids as lipid membrane-constituting lipids, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may improve the stability of lipid nanoparticles, such as their retention in blood.
  • polyethylene glycol for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc.
  • the molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.
  • stearylated polyethylene glycol eg, stearic acid PEG45 (STR-PEG45), etc.
  • stearylated polyethylene glycol can be used to modify lipids with polyethylene glycol.
  • the ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles of the present invention determines the liver selectivity or spleen selectivity of the pH-sensitive cationic lipid of the present invention, specifically, The amount is not particularly limited as long as it does not impair liver-specific gene expression activity or spleen-specific gene expression activity when lipid nanoparticles are used as gene carriers.
  • the ratio of polyalkylene glycol-modified lipids to the total amount of lipids constituting lipid nanoparticles is preferably 0.5 to 3 mol%.
  • the lipid nanoparticles according to the present invention can be subjected to appropriate surface modification, etc., if necessary.
  • the lipid nanoparticles according to the present invention can have improved blood retention by modifying the surface with a hydrophilic polymer or the like.
  • surface modification can be performed by using lipids modified with these modifying groups as constituent lipids of lipid nanoparticles.
  • examples of lipid derivatives for improving blood retention include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerol phospholipid derivatives, etc.
  • You can also use In addition to polyalkylene glycol, we also use dextran, pullulan, Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, and divinyl ether-maleic anhydride alternating copolymer as hydrophilic polymers to increase blood retention.
  • amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc. can also be used for surface modification.
  • the lipid nanoparticles can be surface-modified with an oligosaccharide compound having three or more saccharides.
  • the type of oligosaccharide compound having three or more saccharides is not particularly limited, but for example, an oligosaccharide compound having about 3 to 10 sugar units bonded to it can be used, preferably about 3 to 6 sugar units bonded to it. Oligosaccharide compounds can be used. Among these, oligosaccharide compounds that are glucose trimers or hexamers can be preferably used, and oligosaccharide compounds that are glucose trimers or tetramers can be used more preferably.
  • isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, maltohexaose, etc. can be suitably used. More preferred are triose, maltotetraose, maltopentaose, or maltohexaose. Particularly preferred is maltotriose or maltotetraose, most preferred is maltotriose.
  • the amount of surface modification of lipid nanoparticles with oligosaccharide compounds is not particularly limited, but for example, it is about 1 to 30 mol%, preferably about 2 to 20 mol%, more preferably about 5 to 10 mol%, based on the total lipid amount. It is.
  • the method of surface-modifying lipid nanoparticles with oligosaccharide compounds is not particularly limited, but for example, liposomes (International Publication No. 2007/102481) in which lipid nanoparticles are surface-modified with monosaccharides such as galactose or mannose are known. Therefore, the surface modification method described in this publication can be adopted.
  • liposomes International Publication No. 2007/102481
  • monosaccharides such as galactose or mannose
  • the lipid nanoparticles according to the present invention can be provided with any one or more functions such as, for example, a temperature change sensitive function, a membrane permeation function, a gene expression function, and a pH sensitive function.
  • a temperature change sensitive function e.g., a thermosensitive function
  • a membrane permeation function e.g., a membrane permeation function
  • a gene expression function e.g., a gene expression function
  • a pH sensitive function e.g., a pH sensitive function
  • the lipid nanoparticles according to the present invention include one or more kinds selected from the group consisting of an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, a charged substance, and a membrane polypeptide. It may contain substances such as Examples of charged substances that impart a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and examples of charged substances that impart a negative charge include dicetyl phosphate and cholesteryl hemis. Cusinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid and the like can be mentioned. Examples of membrane polypeptides include membrane superficial polypeptides and integral membrane polypeptides. The blending amount of these substances is not particularly limited and can be appropriately selected depending on the purpose.
  • the average particle size is preferably 400 nm or less, and the average particle size is 300 nm or less, since high delivery efficiency to in vivo liver cells or spleen cells can be easily obtained.
  • the average particle diameter is more preferably 200 nm or less, even more preferably 150 nm or less.
  • the average particle diameter of lipid nanoparticles means the number average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be carried out in a conventional manner using a commercially available DLS device or the like.
  • the polydispersity index (PDI) of the lipid nanoparticles according to the present invention is about 0.01 to 0.7, preferably about 0.01 to 0.6, and more preferably about 0.03 to 0.3.
  • the zeta potential at pH 7.4 can range from -50 mV to 5 mV, preferably from -45 mV to 5 mV.
  • the form of the lipid nanoparticles according to the present invention is not particularly limited, but examples of forms dispersed in an aqueous solvent include unilamellar liposomes, multilamellar liposomes, spherical micelles, and amorphous layered structures.
  • the lipid nanoparticles according to the present invention are preferably unilamellar liposomes or multilamellar liposomes.
  • the lipid nanoparticles according to the present invention preferably contain components to be delivered into target cells inside the particles covered with a lipid membrane.
  • the components that the lipid nanoparticles according to the present invention encapsulate inside the particles are not particularly limited as long as they have a size that can be encapsulated.
  • the lipid nanoparticles according to the present invention can encapsulate arbitrary substances such as nucleic acids, saccharides, peptides, low molecular compounds, and metal compounds.
  • Nucleic acids are preferred as components to be included in the lipid nanoparticles according to the present invention.
  • the nucleic acid may be DNA, RNA, or an analog or derivative thereof (eg, peptide nucleic acid (PNA), phosphorothioate DNA, etc.).
  • the nucleic acid to be encapsulated in the lipid nanoparticle according to the present invention may be a single-stranded nucleic acid, a double-stranded nucleic acid, a linear shape, or a circular shape.
  • lipid nanoparticles according to the invention comprise a pH-sensitive cationic lipid of the invention, a stereoisomer or mixture of stereoisomers thereof, and a nucleic acid.
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention preferably contains a foreign gene for expression within the target cell, and is a nucleic acid that functions to express the foreign gene within the cell by being taken into the cell. It is more preferable that there be.
  • the foreign gene may be a gene originally contained in the genomic DNA of the target cells (preferably liver cells and spleen cells), or may be a gene not contained in the genomic DNA.
  • Examples of such nucleic acids include gene expression vectors containing a nucleic acid consisting of a base sequence encoding a gene of interest to be expressed.
  • the gene expression vector may exist as an extrachromosomal gene in the introduced cell, or may be incorporated into genomic DNA by homologous recombination.
  • the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is not particularly limited, and vectors commonly used in gene therapy and the like can be used.
  • the gene expression vector to be encapsulated in the lipid nanoparticles according to the present invention is preferably a nucleic acid vector such as a plasmid vector.
  • the plasmid vector may remain circular or may be previously cut into linear forms and encapsulated in the lipid nanoparticles of the present invention.
  • Gene expression vectors can be designed by conventional methods using commonly used molecular biological tools based on the base sequence information of the gene to be expressed, and can be manufactured by various known methods. .
  • the nucleic acid encapsulated in the lipid nanoparticle according to the present invention is a functional nucleic acid that controls the expression of a target gene present in a target cell.
  • the functional nucleic acids include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, mRNA, aptamers, plasmid DNA, and the like.
  • it may be plasmid DNA (pDNA) that serves as an siRNA expression vector for expressing siRNA in cells.
  • the siRNA expression vector can be prepared from a commercially available siRNA expression vector, and may be modified as appropriate.
  • the nucleic acid to be encapsulated in the lipid nanoparticles according to the present invention is preferably mRNA or pDNA, since it has particularly good selectivity to the liver or spleen.
  • the lipid nanoparticles of the invention comprise a pH-sensitive cationic lipid of the invention, a stereoisomer or mixture of stereoisomers thereof, and a nucleic acid, wherein said nucleic acid is an mRNA or a plasmid. It is DNA.
  • the method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be adopted.
  • all lipid components are dissolved in an organic solvent such as chloroform, and a lipid film is formed by drying under reduced pressure using an evaporator or spray drying using a spray dryer, and then the components are encapsulated in the lipid nanoparticles.
  • it can be produced by adding an aqueous solvent containing a nucleic acid or the like to the dried above-mentioned mixture, and further emulsifying it using an emulsifying machine such as a homogenizer, an ultrasonic emulsifying machine, a high-pressure injection emulsifying machine, etc.
  • liposomes can also be produced by well-known methods such as reverse phase evaporation. If it is desired to control the size of lipid nanoparticles, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter with uniform pore sizes.
  • composition of the aqueous solvent is not particularly limited, but examples include buffer solutions such as phosphate buffer, citrate buffer, and phosphate buffered saline, physiological saline, and medium for cell culture. I can do it.
  • aqueous solvents can stably disperse lipid nanoparticles, but they also contain monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, xylose, lactose, sucrose, cellobiose, trehalose, Disaccharides such as maltose, trisaccharides such as raffinose and melesinose, polysaccharides such as cyclodextrin, sugars (aqueous solutions) such as sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol, as well as glycerin, diglycerin, and polyglycerin.
  • monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, xylose, lactose, sucrose, cellobios
  • polyhydric alcohol such as propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol, etc. .
  • aqueous solution such as propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol, etc.
  • the pH of the aqueous solvent should be set from weakly acidic to around neutral (about pH 3.0 to 8.0), and/or dissolved oxygen should be removed by nitrogen bubbling, etc. is desirable.
  • the lipid nanoparticles according to the present invention can also be produced by an alcohol dilution method using a channel.
  • a solution in which a lipid component is dissolved in an alcohol solvent and a solution in which a water-soluble component to be included in lipid nanoparticles is dissolved in an aqueous solvent are introduced through separate flow channels, and the two are combined.
  • This is a method for producing lipid nanoparticles.
  • baffles baffles having a constant width relative to the channel width are alternately arranged from both sides.
  • aqueous solvent used in the alcohol dilution method those mentioned above can be used.
  • aqueous dispersion of lipid nanoparticles When the obtained aqueous dispersion of lipid nanoparticles is freeze-dried or spray-dried, for example, monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar, lactose, sucrose, cellobiose, trehalose, Stability can be improved by using sugars (aqueous solutions) such as disaccharides such as maltose, trisaccharides such as raffinose and melesinose, polysaccharides such as cyclodextrin, and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol.
  • sugars aqueous solutions
  • sugars such as disaccharides such as maltose, trisaccharides such as raffinose and melesinose, poly
  • the above-mentioned saccharides, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether Stability may be improved by using polyhydric alcohols (aqueous solutions) such as , diethylene glycol monoalkyl ether, and 1,3-butylene glycol.
  • the lipid nanoparticles according to the invention are lyophilized.
  • the invention relates in one aspect to lipid nanoparticle formulations comprising the pH-sensitive cationic lipids of the invention, stereoisomers or mixtures of stereoisomers thereof.
  • the present invention provides (i) a sterol or sterol derivative, (ii) a polyalkylene glycol-modified lipid, (iii) a nucleic acid, (iv) a buffer, (v) a disaccharide, and (vi) a pH of the present invention.
  • the present invention relates to lipid nanoparticle formulations containing sensitive cationic lipids, stereoisomers or mixtures of stereoisomers thereof.
  • Examples of sterols or sterol derivatives include cholesterol and sitosterol, with cholesterol being preferred.
  • polyalkylene glycol-modified lipids examples include polyethylene glycol-modified lipids, polypropylene glycol-modified lipids, etc., and polyethylene glycol-modified lipids are preferred.
  • the nucleic acid examples include siRNA, pDNA, mRNA, guide RNA (gRNA), aptamer, and plasmid, with mRNA being preferred.
  • the buffer examples include HEPES buffer, phosphate buffer, Tris buffer, and the like.
  • the disaccharide examples include lactose, sucrose, cellobiose, trehalose, and maltose, with sucrose being preferred.
  • the concentration of disaccharide in the lipid nanoparticle formulation is, for example, 1% to 20% by weight, preferably 5% to 15% by weight.
  • the molar ratio of sterol or sterol derivative to pH-sensitive cationic lipid, stereoisomer or mixture of stereoisomers thereof is, for example, from 68.5:20 to 28.5:60.
  • lipid nanoparticle formulations may be prepared by suspending lipid nanoparticles in an aqueous solution.
  • the pH of the lipid nanoparticle formulation of the present invention at 25° C. is, for example, 5.5 to 8.5, preferably 6.8 to 8.0.
  • the present invention relates to a resuspension formulation in which a lipid nanoparticle formulation is resuspended by the addition of water or an aqueous solution.
  • the lipid nanoparticles of the present invention have excellent stability.
  • the lipid nanoparticles of the present invention are, for example, stable for one week or more than one week when stored at -80°C, and/or for one week, two weeks, three weeks, four weeks, five weeks when stored at 5°C. or is stable for 5 weeks or more, and/or is stable for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 5 weeks or more when stored at 25°C, and/or is stable for 3 days when stored at 40°C. , stable for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or more than 5 weeks.
  • the average particle diameter, PDI, and nucleic acid encapsulation rate are compared with the values immediately after preparation after leaving the lipid nanoparticles at a predetermined temperature and storing them for a predetermined period of time.
  • Particles may be defined as lipid nanoparticles of good quality.
  • lipid nanoparticles that maintain good quality for more than a week when left standing at 5°C or lipid nanoparticles that maintain good quality for more than one week when left standing at 40°C are considered to be stable. It may be evaluated that the lipid nanoparticles are excellent.
  • the gene expression vector encapsulated in the lipid nanoparticles is selectively expressed in the liver or spleen rather than in other organs.
  • the lipid nanoparticles of the present invention encapsulating siRNA expression vectors are administered to individual animals, the siRNA expression vectors encapsulated in the lipid nanoparticles are selectively expressed in the liver or spleen than in other organs. , the expression of the gene targeted by the expression vector is suppressed.
  • the lipid nanoparticles according to the present invention encapsulating a foreign gene to be expressed in liver cells or spleen cells are administered to a test animal, the foreign gene is expressed in the liver or spleen of the test animal. I can do it.
  • the lipid nanoparticles according to the present invention function as a gene expression carrier targeting the liver or spleen.
  • the lipid nanoparticles according to the present invention are useful as active ingredients in pharmaceutical compositions used in gene therapy, and are particularly effective in pharmaceutical compositions used in gene therapy that targets the liver or spleen. Useful as an ingredient.
  • the invention relates in one aspect to a pharmaceutical composition for liver delivery containing a pH-sensitive cationic lipid of the invention, a stereoisomer or a mixture of stereoisomers thereof.
  • the invention in another aspect, relates to a pharmaceutical composition for splenic delivery containing a pH-sensitive cationic lipid of the invention, a stereoisomer or a mixture of stereoisomers thereof.
  • the animal to which the lipid nanoparticles according to the present invention are administered is not particularly limited, and may be a human or a non-human animal.
  • non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quail, and ducks.
  • a pharmaceutical composition for cancer delivery comprises a pH-sensitive cationic lipid of the invention.
  • pH-sensitive cationic lipids include, but are not limited to, CL15F 7-3, CL15F 7-5, CL15F 8-6, CL15F 9-3, CL15F 9-5, CL15F 9-7, CL15F 10-4, CL15F 10-5, CL15F6, CL15F 10-8, CL15F 11-5, CL15F 11-6, CL15F 11-7, CL15F 11-9, CL15F 12-4, CL15F 13-3, CL15F 13-11 , CL15F 14-2, CL15F 14-12, CL15F 16-1, etc.
  • a pharmaceutical composition for cancer delivery comprises, in addition to the pH-sensitive cationic lipids of the invention, a sterol or sterol derivative, a polyalkylene glycol-modified lipid, and a buffer.
  • sterols or sterol derivatives include, but are not limited to, cholesterol, sitosterol, etc., and are preferably cholesterol.
  • polyalkylene glycol-modified lipids include, but are not limited to, polyethylene glycol-modified lipids, polypropylene glycol-modified lipids, etc., and preferably polyethylene glycol-modified lipids.
  • polyethylene glycol-modified lipids include, but are not limited to, 1,2-distearoyl-rac-glycerolmethoxypolyethylene glycol (DSG-PEG), preferably 1,2-distearoyl-rac - Glycerol methoxypolyethylene glycol (DSG-PEG).
  • the lipid nanoparticles When the molecular weight of the polyethylene glycol of DSG-PEG is 500 to 10,000, the lipid nanoparticles accumulate better in cancer tissues, and when it is 1000 to 3000, the lipid particles accumulate better in cancer tissues. , 2000, the accumulation in cancer tissue is even better.
  • buffers include, but are not limited to, HEPES buffer, phosphate buffer, Tris buffer, and the like.
  • the pharmaceutical composition for cancer delivery further comprises a nucleic acid.
  • nucleic acids include, but are not limited to, siRNA, mRNA, guide RNA (gRNA), aptamer, and plasmid DNA.
  • the pharmaceutical composition for cancer delivery is a suspension of lipid nanoparticles.
  • the pharmaceutical composition for cancer delivery has a pH of 6.8 to 8.0 at 25°C.
  • the pharmaceutical composition for cancer delivery comprises a disaccharide.
  • disaccharides include, but are not limited to, lactose, sucrose, cellobiose, trehalose, maltose, etc., and preferably sucrose.
  • the concentration of disaccharide is, for example, 1% to 20% by weight, preferably 5% to 15% by weight.
  • the pharmaceutical composition for cancer delivery is a lyophilized formulation of lipid nanoparticles.
  • the present invention in one aspect, relates to a pharmaceutical composition for treating cancer for treating an individual with cancer.
  • a pharmaceutical composition for treating cancer comprises a composition for cancer delivery of the invention.
  • cancer includes, but is not limited to, malignant tumors that originate from epithelial cells such as the skin, gastric and intestinal mucosa, and non-epithelial tumors that originate from non-epithelial cells such as muscle, fiber, bone, fat, blood vessels, and nerves. These include malignant tumors that have developed, leukemia and malignant lymphoma that have developed from blood-forming organs.
  • the individual to whom the pharmaceutical composition for cancer treatment according to the present invention is administered is not particularly limited, and may be a human or a non-human animal.
  • non-human animals examples include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quail, and ducks.
  • the pharmaceutical composition for cancer treatment may further contain a pharmaceutically acceptable carrier.
  • carriers include, but are not limited to, excipients, fillers, fillers, binders, wetting agents, disintegrants, surfactants, coloring agents, flavoring agents, preservatives, and stabilizers. , buffering agents, suspending agents, tonicity agents, lubricants, fluidity promoters, etc.
  • the present invention relates to a method of treating cancer, which method comprises administering the pharmaceutical composition for treating cancer according to the present invention to an individual in need thereof.
  • the invention relates to the use of the lipid nanoparticles according to the invention for the production of a pharmaceutical composition for cancer treatment.
  • the present invention in one aspect, relates to immunostimulatory compositions.
  • immunostimulatory composition refers to a composition for producing an immunostimulatory effect on an individual.
  • the immunostimulatory composition comprises the pH-sensitive cationic lipid of the invention.
  • pH-sensitive cationic lipids include, but are not limited to, CL4F 6-4, CL4F 7-3, CL4F7-4, CL4F 7-5, CL4F 8-4, CL4F 8-5, CL4F 8 -6, CL4F 9-3, CL4F 9-4, CL4F 9-5, CL4F 9-6, CL4F 9-7, CL4F 10-2, CL4F 10-4, CL4F6, CL4F 10-8, CL4F 11-5, CL4F 12-4, CL4F 12-6, CL15F 7-5, CL15F 8-6, CL15F 9-3, CL15F 9-5, CL15F 9-7, CL15F 10-4, CLF 10-5, CL15F6, CL15F 10- 8, CL15F 11-5, CL15F 11-6, CL15F 11-7, CL15F 11-9, CL15F 12-4, CL15F 13-3, CL15F 14-2, etc.
  • the immunostimulatory composition further comprises a nucleic acid.
  • nucleic acids include, but are not limited to, siRNA, mRNA, guide RNA (gRNA), aptamer, and plasmid DNA.
  • the immunostimulatory composition is a suspension of lipid nanoparticles.
  • the immunostimulatory composition has a pH of 6.8 to pH 8.0 at 25°C.
  • the individual to whom the immunostimulatory composition according to the present invention is administered is not particularly limited, and may be a human or a non-human animal.
  • non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quail, and ducks.
  • the immunostimulatory composition may further contain a pharmaceutically acceptable carrier.
  • carriers include, but are not limited to, excipients, fillers, fillers, binders, wetting agents, disintegrants, surfactants, coloring agents, flavoring agents, preservatives, and stabilizers. , buffering agents, suspending agents, tonicity agents, lubricants, fluidity promoters, etc.
  • the immunostimulatory composition comprises a disaccharide.
  • disaccharides include, but are not limited to, lactose, sucrose, cellobiose, trehalose, maltose, etc., and preferably sucrose.
  • the concentration of disaccharide is, for example, 1% to 20% by weight, preferably 5% to 15% by weight.
  • the immunostimulatory composition is a lyophilized formulation of lipid nanoparticles.
  • the present invention relates to a method of activating immunity in an individual, the method comprising administering to the individual an immunostimulating composition according to the present invention.
  • the invention relates to the use of lipid nanoparticles according to the invention for the production of immunostimulatory compositions.
  • the pH-sensitive cationic lipids, stereoisomers or mixtures of stereoisomers thereof of the present invention can be prepared, for example, from the basic skeleton 7-(4-(dipropylamino)butyl)tridecane-1,7,13-triol or It is synthesized by condensing 5,11-dihydroxy 5-(6-hydroxyhexyl)undecyl 1-methylpiperidine-4-carboxylate and a branched fatty acid.
  • the present invention relates in one aspect to a method for producing the pH-sensitive cationic lipids, stereoisomers or mixtures of stereoisomers thereof of the present invention.
  • the method for producing a pH-sensitive cationic lipid of the present invention comprises reacting an alkyl carboxylic acid and an alkyl halide in the presence of organolithium, dimethylpropylene urea (DMPU), and tetrahydrofuran (THF).
  • the method includes at least a step (step A) of obtaining a branched fatty acid.
  • the alkyl carboxylic acid is, for example, octanoic acid, decanoic acid, tridecanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, It is hexadecenoic acid.
  • the halogenated alkyl is, for example, 1-iodohexane, 1-iodobutane, 2-iodohexane, 1-bromohexane, iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1- Iodohexane, 1-iodoheptane, 1-iodooctane, 1-iodononane, 1-iododecane, 1-iodoundecane, 1-iodododecane, 1-iodotridecane, 1-iodotetradecane, 1-iodopentadecane, 1- It is iodohexadecane.
  • the organolithium is, for example, lithium diisopropylamide lithium (LDA), t-butyllithium, or n-
  • the method for producing a pH-sensitive cationic lipid of the present invention comprises hydrolyzing and heating a reaction solution obtained by reacting a malonic acid ester and an alkyl halide in the presence of a base. It includes at least a step (step B) of obtaining a branched fatty acid.
  • the malonate ester is, for example, dimethyl malonate, diethyl malonate, diisopropyl malonate, and preferably dimethyl malonate.
  • the halogenated alkyl is, for example, iodoalkyl
  • the iodoalkyl is, for example, 1-iodohexane, 1-iodopropane, 2-iodohexane.
  • the base is, for example, sodium hydride, calcium hydride, sodium ethoxide and lithium bis(trismethylsilyl)amide, preferably sodium hydride.
  • the hydrolysis treatment is performed using, for example, any one of sodium hydroxide, calcium hydroxide, and lithium hydroxide.
  • the heat treatment is preferably carried out at 120°C to 170°C, more preferably at 150°C to 170°C, simultaneously and/or after the hydrolysis treatment.
  • the method further includes purifying the branched fatty acids by reverse phase chromatography.
  • branched fatty acids used in the synthesis of , branched fatty acids can be obtained in higher yield by performing step A than by step B.
  • branched fatty acids can be obtained in higher yield by performing step B than by step A.
  • the branched fatty acid used in the synthesis of the pH-sensitive cationic lipid of the present invention may be obtained, for example, by the method described in Japanese Patent No. 2756756.
  • the crude product was purified by subjecting it to silica gel chromatography [eluent: dichloromethane:methanol (continuous gradient)] to give 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13- Diyl bis(2-hexyldecanoate) (CL4F6) was obtained.
  • the filtrate was separated and washed with 0.5N aqueous sodium oxide solution and saturated brine.
  • the organic layer was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product.
  • the crude product was purified by subjecting it to silica gel chromatography [eluent; dichloromethane:methanol (continuous gradient)] to give 7-hydroxy-7-(4-((1-methylpiperidine)-4-carbonyl)oxy) Butyl]tridecane-1,13-diyl bis(2-hexyldecanoate) (CL15F6) was obtained.
  • lipid nanoparticles were prepared by an alcohol dilution method using a channel.
  • a microfluidic device "iLiNP" manufactured by Lilac Pharma
  • a built-in mixer was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and an acetate buffer (25 mM, pH 4.0) adjusted to an siRNA concentration of 71.1 ⁇ g/mL were added at 0.375 mL/min and 1.125 mL/min, respectively.
  • a liquid was sent into the microchannel, and the lipid nanoparticle solution excreted from the channel was collected.
  • the lipid nanoparticle solution was placed in a dialysis membrane (MWCO 12,000-14,000), and the external aqueous phase was dialyzed against 20mM MES buffer (pH 6.0) at 4°C for 2 hours or more. Thereafter, the external aqueous phase was replaced with PBS(-) (pH 7.4), and after further dialysis at 4°C for 2 hours or more, the lipid nanoparticle solution was collected from the dialysis membrane.
  • CL4F6, CL4G6, CL15F6, and CL15G6 were those synthesized in Synthesis Examples 1 to 4.
  • 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1,13-diyl dioleate (CL4H6) and 7-hydroxy-7-(4-((1-methylpiperidine-4-carbonyl)oxy) ) butyl) tridecane-1,13-diyl dioleate (CL15H6) was synthesized by the method described in Patent Document 1.
  • cholesterol chol
  • PEG-DMG polyethylene glycol 2000 modified dimyristoylglycerol
  • ⁇ Measurement of pKa of lipid nanoparticles The pKa of lipid nanoparticles was measured using p-toluidino-2-naphthalenesulfonic acid (TNS). First, TNS (final concentration: 0.75 ⁇ M) and lipid nanoparticles (final concentration: 30 mM) were mixed in a buffer solution adjusted to each pH. The fluorescence intensity of the prepared mixture was measured using a microplate reader. Among the measured values, the highest and lowest values were taken as 100% and 0% charge rate, respectively, and the pH showing 50% charge rate was calculated as pKa.
  • Example 1 Using pH-sensitive cationic lipid, cholesterol, and PEG-DMG in a molar ratio of 50:50:1, lipid nanoparticles loaded with siRNA against F7 (F7 siRNA-loaded lipid nanoparticles) were created by alcohol dilution method. did. CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 was used as the pH-sensitive cationic lipid.
  • lipid nanoparticles produced using pH-sensitive cationic lipid X will be referred to as X-LNP.
  • lipid nanoparticles prepared using the pH-sensitive cationic lipid CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 can be prepared using CL4F-LNP, CL4G6-LNP, CL4H6-LNP, CL15F-LNP, CL15G6- It is called LNP or CL15H6-LNP.
  • Table 1 shows the base sequence of siRNA for F7. In the table, uppercase letters represent natural RNA (T only is natural DNA), lowercase letters represent 2'-fluoro modified products, and * represents a phosphorothioate bond.
  • Each of the prepared lipid nanoparticles had an average particle diameter of 80 to 120 nm, and the siRNA encapsulation rate was 90% or more.
  • the results of measuring the pKa of each lipid nanoparticle are shown in FIGS. 1(A) and 1(B).
  • lipid nanoparticles manufactured using CL4F6, CL4G6, CL15F6, or CL15G6, which have a branched scaffold structure are different from lipid nanoparticles manufactured using CL4H6 or CL15H6, which have a linear scaffold structure. showed a lower pKa compared to other lipid nanoparticles.
  • each of the prepared F7 siRNA-loaded lipid nanoparticles was administered to ICR mice (4 weeks old, female), and in vivo F7 knockdown activity was examined. Specifically, each F7 siRNA-loaded lipid nanoparticle was intravenously administered to ICR mice at 0.003 to 0.1 mg siRNA/kg, and plasma F7 enzyme activity was measured 24 hours later. The relative plasma F7 enzyme activity (%) of the mice to which each F7 siRNA-loaded lipid nanoparticle was administered was calculated, taking the plasma F7 enzyme activity of untreated mice as 100%. The results are shown in FIGS. 2(A) and 2(B). FIG. 2(B) shows the results of intravenous administration of each F7 siRNA-loaded lipid nanoparticle at 0.1 mg siRNA/kg.
  • lipid nanoparticles manufactured using CL4F6, CL4G6, CL15F6, or CL15G6, which have a branched scaffold structure are different from lipid nanoparticles manufactured using CL4H6 or CL15H6, which have a linear scaffold structure.
  • the lipid nanoparticles exhibited comparable in vivo F7 knockdown activity.
  • Example 2 Lipid nanoparticles loaded with mRNA instead of siRNA were prepared, and in vivo gene expression activity was examined.
  • the mRNA used was one prepared by performing an in vitro transcription reaction on pDNA encoding NanoLuc (registered trademark) luciferase (Nluc) (manufactured by Promega) (Nluc mRNA).
  • a pH-sensitive cationic lipid a pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG were used in a molar ratio of 60:10:40:1 and diluted with alcohol.
  • Lipid nanoparticles loaded with Nluc mRNA lipid nanoparticles loaded with Nluc mRNA
  • CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 was used as the pH-sensitive cationic lipid.
  • the average particle diameter, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were examined. The measurement results are shown in Table 2.
  • "CL” means cationic lipid.
  • the average particle diameters calculated by dynamic light scattering were all 70 to 130 nm (Table 2).
  • the prepared lipid nanoparticles only CL15H6-LNP had a large PDI and formed particles with low uniformity (Table 2).
  • the mRNA encapsulation rate was less than 80% for CL4H6-LNP, while it was more than 90% for all LNPs containing other cationic lipids.
  • each of the prepared Nluc mRNA-loaded lipid nanoparticles was administered to ICR mice (4 weeks old, female), and in vivo gene expression activity was examined. Specifically, each Nluc mRNA-loaded lipid nanoparticle was intravenously administered to ICR mice at 0.04 mg mRNA/kg, and Nluc activity in the liver and spleen was measured 24 hours later. Nluc activity was measured using a luminometer (RLU) and corrected by the amount of protein quantified by the BCA method.
  • RLU luminometer
  • FIG. 3 shows the measurement results of Nluc activity (RLU/mg protein) in the liver and spleen of mice administered with each Nluc mRNA-loaded lipid nanoparticle.
  • FIG. 3(A) shows the measurement results of Nluc activity in the liver
  • FIG. 3(B) shows the measurement results of Nluc activity in the spleen.
  • liver selectivity of gene expression was calculated by dividing the gene expression activity in the liver by the gene expression activity in the spleen.
  • FIG. 3(C) is a diagram showing the calculation results of [Nluc activity in the liver]/[Nluc activity in the spleen] of mice to which each Nluc mRNA-loaded lipid nanoparticle was administered. As shown in FIG.
  • mice administered with CL4F6-LNP, CL15F6-LNP, and CL15G6-LNP showed Nluc activity equivalent to that of mice administered with CL4H6-LNP in the liver. Furthermore, as shown in FIG. 3(C), mice administered with CL4F6-LNP and CL4G6-LNP showed higher liver selectivity compared to mice administered with CL4H6-LNP. Similarly, mice treated with CL15F6-LNP and CL15G6-LNP showed increased liver selectivity compared to mice treated with CL15H6-LNP.
  • lipid nanoparticles containing a pH-sensitive cationic lipid with a branched scaffold structure are different from lipid nanoparticles containing a pH-sensitive cationic lipid with a linear scaffold structure when encapsulating mRNA. It was found that the selectivity to the liver was higher than that of the conventional one, and it was found to be useful as a delivery carrier that specifically delivers to the liver.
  • Example 3 Lipid nanoparticles loaded with pDNA instead of siRNA were prepared, and in vivo gene expression activity was examined.
  • pDNA a plasmid (pFluc) expressing firefly luciferase (Fluc) under the CMV promoter was used.
  • lipid nanoparticles loaded with pFluc were prepared.
  • CL4F6, CL4G6, CL4H6, CL15F6, CL15G6, or CL15H6 was used as the pH-sensitive cationic lipid. Further, the N/P ratio in the microchannel was set to 9.
  • the average particle diameter, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were examined. The measurement results are shown in Table 3.
  • "CL” means cationic lipid.
  • the average particle diameters calculated by dynamic light scattering were all 90 to 150 nm (Table 3).
  • Regarding the pDNA encapsulation rate, CL4H6-LNP and CL15H6-LNP were 70% and 82%, respectively.
  • lipid nanoparticles containing other cationic lipids showed a good pDNA encapsulation rate of 90% or more.
  • pFluc-loaded lipid nanoparticles were introduced into cultured cells, and in vitro gene expression activity was examined. Specifically, HeLa-GFP cells cultured in a 96-well plate were transfected with pFluc-loaded lipid nanoparticles at 0.0625 ⁇ g pDNA/well, and Fluc activity was measured 24 hours later. As a positive control, pFluc was introduced into HeLa-GFP cells using the introduction reagent "Lipofectamine 3000" (manufactured by Thermo Fisher Scientific). Fluc activity was measured using a luminometer (RLU) and corrected by the amount of protein quantified by the BCA method.
  • RLU luminometer
  • Figure 4 shows the measurement results of Fluc activity of HeLa-GFP cells into which each pFluc-loaded lipid nanoparticle was introduced.
  • "Lipo3K” means a positive control in which the gene was introduced using Lipofectamine 3000.
  • cells introduced with CL15F6-LNP showed higher activity than cells introduced with CL4H6-LNP, CL15H6-LNP, and the positive control.
  • pFluc-loaded lipid nanoparticles were prepared in the same manner as described above, except that the N/P ratio in the microchannel was 6.
  • the average particle diameter, PDI, zeta potential, and mRNA encapsulation rate of the prepared lipid nanoparticles were examined. The measurement results are shown in Table 4.
  • "CL” means cationic lipid.
  • the average particle diameters calculated by dynamic light scattering were all 70 to 125 nm (Table 4).
  • each of the prepared pFluc-loaded lipid nanoparticles was administered to ICR mice (4 weeks old, female), and in vivo gene expression activity was examined. Specifically, each pFluc-loaded lipid nanoparticle was intravenously administered to ICR mice at 0.5 mg mRNA/kg, and Fluc activity in the liver and spleen was measured 6 hours later. Fluc activity was measured using a luminometer (RLU) and corrected by the amount of protein quantified by the BCA method.
  • RLU luminometer
  • FIG. 5 shows the measurement results of Fluc activity (RLU/mg protein) in the liver and spleen of mice to which each pFluc-loaded lipid nanoparticle was administered.
  • FIG. 5(A) shows the measurement results of Fluc activity in the liver
  • FIG. 5(B) shows the measurement results of Fluc activity in the spleen.
  • liver selectivity of gene expression was calculated by dividing the gene expression activity in the liver by the gene expression activity in the spleen.
  • FIG. 5(C) is a diagram showing the calculation results of [Fluc activity in the liver]/[Fluc activity in the spleen] of mice to which each pFluc-loaded lipid nanoparticle was administered. As shown in FIG.
  • mice administered with CL4F6-LNP and CL15F6-LNP Fluc activity in the liver was better in mice administered with CL4F6-LNP and CL15F6-LNP than in mice administered with CL4H6-LNP and CL15H6-LNP.
  • mice administered with CL4F6-LNP and CL4G6-LNP showed higher liver selectivity compared to mice administered with CL4H6-LNP.
  • mice treated with CL15F6-LNP and CL15G6-LNP showed increased liver selectivity compared to mice treated with CL15H6-LNP.
  • lipid nanoparticles containing pH-sensitive cationic lipids with a branched scaffold structure have a straight scaffold structure. It was found that the lipid nanoparticles have higher selectivity to the liver than lipid nanoparticles containing chain-type pH-sensitive cationic lipids, and are useful as delivery carriers that specifically deliver to the liver.
  • the above branched fatty acids were synthesized as follows using straight chain fatty acids or dimethyl malonate as raw materials. ⁇ Synthesis of branched fatty acids using straight chain fatty acids as raw materials> Straight chain fatty acid (10.28 mmol) was dissolved in 36 mL of THF, and then lithium diisopropylamide (24 mmol) was added dropwise at -20°C or lower, followed by stirring at 0°C for 30 minutes. Subsequently, DMPU (18 mL) was added, and the mixture was stirred at 0°C for 60 minutes. Subsequently, iodoalkane (23.2 mmol) was added, and the mixture was reacted overnight at 10°C.
  • the mixture was diluted with ethyl acetate and washed with saturated brine.
  • the organic layer was dehydrated by adding anhydrous sodium sulfate. After filtering this, the solvent was distilled off using a rotary evaporator. The components from which the solvent was removed were dissolved in 16 mL of ethanol, 5 mL of 8N aqueous sodium hydroxide solution was added, and the mixture was reacted at 60° C. overnight. After neutralizing with 6N hydrochloric acid, the mixture was diluted with ethyl acetate and washed with saturated brine. The organic layer was dehydrated by adding anhydrous sodium sulfate.
  • lipid nanoparticles using CL4F6 derivatives and CL15F6 derivatives 1.
  • Preparation and evaluation of mRNA-loaded lipid nanoparticles ⁇ Preparation of mRNA-loaded lipid nanoparticles (mRNA-LNP)> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A microfluidic device "NanoAssemblr" (manufactured by Precision NanoSystems) with a built-in mixer was used as the flow path.
  • an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 ⁇ g/mL were added to the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution that was excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.
  • lipid nanoparticles of lipid nanoparticles > pH-sensitive cationic lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:
  • lipid nanoparticles loaded with FlucmRNA lipid nanoparticles loaded with FlucmRNA
  • FlucmRNA lipid nanoparticles loaded with FlucmRNA
  • CleanCap registered trademark
  • FLuc mRNA 5 moU
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • pKa of lipid nanoparticles was measured using p-toluidino-2-naphthalenesulfonic acid (TNS).
  • TNS p-toluidino-2-naphthalenesulfonic acid
  • lipid nanoparticles final concentration: 60 ⁇ M
  • the fluorescence intensity of the prepared mixture was measured using a microplate reader.
  • the excitation wavelength was calculated by setting the measured value at pH 3.5 as 100% charge rate, the measured value at pH 9.5 as 0% charge rate, and the pH showing 50% charge rate as pKa.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of siRNA and mRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • the mRNA encapsulation rate was less than 80% for CL4F 6-2-LNP and CL4F 16-1-LNP, while it was more than 80% for all LNPs containing other cationic lipids.
  • the average particle diameter of the CL15F6 derivative nanoparticles in Table 7 was over 300 nm for CL15F 6-4-LNP and CL15F 7-3-LNP, but was 90-200 nm for all LNPs containing other cationic lipids.
  • Ta For PDI, small and highly uniform particles were formed except for CL15F 6-4-LNP, CL15F 7-3-LNP, CL15F 16-0-LNP, and CL15H6-LNP.
  • the mRNA encapsulation rate was less than 80% for CL15F 6-4-LNP and CL15F 7-3-LNP, while it was higher than 80% for all LNPs containing other cationic lipids.
  • each of the prepared FlucmRNA-loaded lipid nanoparticles was administered to Balb/c mice (Charles River Japan, 7 weeks old, female), and in vivo gene expression activity was examined. Specifically, each Fluc mRNA-loaded lipid nanoparticle was intravenously administered to Balb/c mice at 0.1 mg mRNA/kg, and Fluc activity in the liver and spleen was measured 6 hours later. Fluc activity was determined by administering VivoGlo Luciferin, In Vivo Grade (Promega, P1041) dissolved at 15 mg/mL in PBS through the tail vein at a dose of 1.5 mg per mouse, using an in vivo imaging system (Perkin Elmer, IVIS200). ). The unit of Fluc activity is the emission intensity per unit area (Avg Radiance [p/s/cm2/sr]) at a maximum emission wavelength of about 560 nm.
  • Tables 5 to 7 show the measurement results of Fluc activity (Avg Radiance [p/s/cm2/sr]) in the liver and spleen of mice to which each Fluc mRNA-loaded lipid nanoparticle was administered.
  • the Fluc activity in the liver of the CL4F6 derivative nanoparticles in Tables 5 and 6 was determined by the Fluc activity in the liver of CL4F6-containing Fluc mRNA lipid nanoparticles in mice administered with CL4F 8-6, CL4F 9-7, or CL4F 11-6-containing Flucm RNA-loaded lipid nanoparticles. showed higher Fluc activity than mice administered particles.
  • the Fluc activity of CL4F6 derivative nanoparticles in the spleen was CL4F 7-5, CL4F 8-4, CL4F 9-3, CL4F 10-2, CL4F 8-6, CL4F 10-4, CL4F 10-5, CL4F 12 -4, CL4F 13-3, CL4F 14-2, CL4F 7-4, CL4F 8-5, CL4F 9-4, or CL4F 9-5 containing Flucm RNA lipid nanoparticles were administered to mice. They showed higher Fluc activity than mice administered nanoparticles.
  • the Fluc activity in the liver of CL15F6 derivative nanoparticles in Table 7 is CL15F 9-7, CL15F 11-5, CL15F 11-6, CL15F 10-8, CL15F 11-7, CL15F 11-9, CL15F 12-10, or Mice administered with CL15F 14-2-containing FlucmRNA-loaded lipid nanoparticles showed higher Fluc activity than mice administered with CL15F6-containing FlucmRNA-loaded lipid nanoparticles.
  • Fluc activity in the spleen of CL15F6 in Table 7 is as follows: CL15F 6-4, CL15F 7-3, CL15F 7-5, CL15F 9-3, CL15F 9-5, CL15F 10-5, CL15F 13-3, CL15F 11-6 , CL15F 10-8, CL15F 11-7, CL15F 11-9, or CL15F 14-2-containing FlucmRNA lipid nanoparticles had higher Fluc activity in mice than in mice receiving CL15F6-containing FlucmRNA lipid nanoparticles. Indicated.
  • siRNA-loaded lipid nanoparticles Lipid nanoparticles loaded with siRNA instead of mRNA were prepared, and in vivo F7 knockdown activity was examined. siRNA-loaded lipid nanoparticles were prepared in the same manner as described above, except that the N/P ratio in the microchannel was 6.
  • Table 8 shows the base sequence of siRNA for F7. In the table, uppercase letters represent natural RNA (T is only natural DNA), lowercase letters represent 2'-fluoro modified products, and * represents a phosphorothioate bond.
  • lipid nanoparticles loaded with siRNA for F7 were produced by an alcohol dilution method.
  • the average particle diameter, PDI, siRNA encapsulation rate, and pKa of the prepared lipid nanoparticles were examined.
  • the measurement results for CL4F6 derivative nanoparticles are shown in Tables 9 and 10, and the measurement results for CL15F6 derivative nanoparticles are shown in Table 11.
  • the average particle diameter of the CL4F6 derivative nanoparticles is 60 to 280 nm, and the siRNA encapsulation rate was less than 90% for CL4F 6-2-LNP, while it was more than 90% for all LNPs containing other cationic lipids. showed that. Except for CL4F 16-2-LNP, particles with small PDI and high uniformity were formed.
  • the average particle diameter of the CL15F6 derivative nanoparticles was over 300 nm for CL15F 6-2-LNP, but was 85 to 290 nm for all other LNPs containing cationic lipids.
  • the siRNA encapsulation rate was 90% or more for all LNPs. Except for CL15F 6-2-LNP, CL15F 6-4-LNP, CL15F 7-3-LNP, and CL15F 16-0-LNP, highly uniform particles with small PDI were formed.
  • each of the prepared F7 siRNA-loaded lipid nanoparticles was administered to Balb/c mice (Charles River Japan, 5 weeks old, female), and in vivo F7 knockdown activity was examined.
  • mice were given 0.025 mg siRNA/kg of F7siRNA-loaded lipid nanoparticles containing the lipids listed in Table 9, and F7siRNA-loaded lipid nanoparticles containing the lipids listed in Tables 10 and 11. was administered intravenously at 0.025 mg siRNA/kg, and plasma F7 enzyme activity was measured 24 hours later using BIOPHEN FVII (Biophen, A221304). The relative plasma F7 enzyme activity (%) of the mice to which each F7 siRNA-loaded lipid nanoparticle was administered was calculated, taking the plasma F7 enzyme activity of the untreated group mice as 100%.
  • the relative plasma F7 enzyme activity (%) of the mouse to which the MC3-containing F7siRNA-loaded lipid nanoparticles were administered was set as 1, and the ratio was calculated.
  • the measurement results for CL4F6 derivative nanoparticles are shown in Tables 9 and 10, and the measurement results for CL15F6 derivative nanoparticles are shown in Table 11.
  • the F7siRNA-loaded lipid nanoparticles containing CL4F 10-4, CL4F 8-5, CL4F 9-5, or CL4F 12-6 in Tables 9 and 10 exhibited higher knockdown activity than the F7siRNA-loaded lipid nanoparticles containing MC3. Ta.
  • F7siRNA-loaded lipid nanoparticles containing CL15F 9-7, CL15F 11-5, CL15F 12-4, CL15F 11-6, CL15F 10-8, CL15F 11-7, CL15F 11-9, or CL15F 12-10 in Table 11 showed higher knockdown activity than MC3-containing F7siRNA-loaded lipid nanoparticles.
  • pDNA-loaded lipid nanoparticles Lipid nanoparticles loaded with pDNA encoding eGFP instead of mRNA were prepared, and the luminescence of eGFP in vitro was investigated.
  • pDNA encoding eGFP was prepared by inserting the gene encoding eGFP into pCMV-LacI from LacSwitch II Mammalian Expression System (Agilent). The pDNA-loaded lipid nanoparticles were prepared in the same manner as the mRNA-loaded lipid nanoparticles.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The entrapment rate of pDNA in lipid nanoparticles was measured using Picogreen (registered trademark) reagent (Quant-iTTM dsDNA Assay Kit, broad range, manufactured by Thermofisher Scientific).
  • Picogreen registered trademark
  • Quant-iTTM dsDNA Assay Kit broad range, manufactured by Thermofisher Scientific.
  • the nucleic acid concentration of the nanoparticle surface nucleic acid concentration measurement solution was 1.6 ⁇ g/mL
  • the nucleic acid concentration of the total nucleic acid concentration measurement solution was 24 ng/mL
  • the nucleic acid concentration used for the calibration curve was 0 to 0.
  • the method was the same as the method for measuring the encapsulation rate of siRNA and mRNA, except that the concentration was 5 ⁇ g/mL and Picogreen (registered trademark) reagent (Quant-iT TM dsDNA Assay Kit, broad range, manufactured by Thermofisher Scientific) was used as the measurement reagent.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g) /mL)) ⁇ 100
  • Lipid nanoparticles were prepared by an alcohol dilution method using a channel.
  • a microfluidic device "NanoAssemblr" manufactured by Precision NanoSystems
  • a citrate buffer 50 mM, pH 3.5
  • an mRNA concentration of 46.1 ⁇ g/mL were added to the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution that was excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.
  • lipid nanoparticles Using a pH-sensitive cationic lipid, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG in a molar ratio of 50:10:38.5:1.5, FlucmRNA-loaded lipid nanoparticles (FlucmRNA-loaded lipid nanoparticles) were produced by an alcohol dilution method. As Fluc mRNA, CleanCap (registered trademark) FLuc mRNA (5 moU) from TriLink Biotechnologies was used.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles > The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses dynamic light scattering.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The mRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (manufactured by Life Technologies). ⁇ Storage stability evaluation> The lipid nanoparticles were left standing at each temperature of -80°C, 5°C, 25°C, and 40°C, and after each period of storage, the average particle diameter, PDI, and nucleic acid encapsulation rate were measured. Lipid nano that satisfies all three conditions: the average particle diameter is maintained within ⁇ 20 nm based on the LNP preparation date, the PDI is 0.2 or less and highly uniform, and the nucleic acid encapsulation rate is maintained at 80% or more. It was judged that the quality of the particles was kept good.
  • Lipid nanoparticles were prepared by an alcohol dilution method using a channel.
  • a microfluidic device "NanoAssemblr" manufactured by Precision NanoSystems
  • a citrate buffer 50mM, pH 3.5
  • a nucleic acid concentration 46.1 ⁇ g/mL
  • the lipid nanoparticle solution that was excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.
  • Lipids were prepared with the compositions listed in Tables 15-18.
  • CL4F 10-5, CL4F 9-7, CL4F 8-4, CL4F6, CL15 10-5 and CL15F6 were used as pH-sensitive cationic lipids.
  • lipids include cholesterol (Nacalai Tesque), ⁇ -sitosterol (22,23-Dihydrostigmasterol, beta-Sitosterol, 5-Stigmasten-3 ⁇ -ol, ⁇ -Dihydrofucosterol, 24 ⁇ -Ethylcholesterol, Sigma-Aldrich), DSPC (1,2- Distearoyl-sn-glycero-3-phosphocholine, COATSOME MC-8080, Yuka Sangyo Co., Ltd.), DMG-PEG2K (1,2-Dimyristoyl-rac-glycero-3-methylpolyoxyethylen, SUNBRIGHT GM-020, Yuka Sangyo Co., Ltd.) DOPC ( 1,2-Dioleoyl-sn-glycero-3-phosphocholine, COATSOME MC-8181, Yuka Sangyo), DOPE (1,2-Dioleoyl-sn-glycero-3-phospho
  • mRNA Fluc expression mRNA (CleanCap Fluc mRNA (5moU), TriLink Biotechnologies) and mCherry expression mRNA (CleanCap mCherry mRNA (5moU), TriLink Biotechnologies) were used.
  • ⁇ Storage stability evaluation> The prepared lipid nanoparticles were left standing at 40°C, and after each period of storage, the average particle diameter, PDI, and nucleic acid encapsulation rate were measured. Good quality was evaluated according to the following criteria: Good ( ⁇ ): Particle diameter is within ⁇ 20 nm of the particle diameter immediately after creation, PDI is 0.2 or less, and encapsulation rate is 80% or more; Not good ( ⁇ ): The particle diameter is more than ⁇ 20 nm of the particle diameter immediately after creation, or the encapsulation rate is less than 80%. Lipid nanoparticles that maintain good quality for one week or more when left standing at 40°C can be evaluated as having excellent stability.
  • E-MEM medium (contains L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, 1,500 mg/L sodium bicarbonate) (product code 055-08975, Wako Pure Chemical Industries) as a cell culture medium 10% serum (Fetal Bovine Serum Characterized, Corning) and 1% antibiotic (Penicillin-Streptomycin (10,000 U/mL), Thermo Fisher) were used.
  • Human embryonic kidney cells 293 (HEK293 cells) were added to a 96-well white plate (SIGMA) at 2.0 ⁇ 10 4 cells/well, and cultured overnight at 37°C in a 5% CO 2 atmosphere.
  • Particles were added at 100 ng/well in terms of mRNA content. After culturing under the same conditions for 24 hours, 100 ⁇ g/well of 300 ⁇ g/mL luciferin solution (Beetle luciferin, Promega) was added, and the luminescence intensity was measured using a multiplate reader (EnSight multimode plate reader, Perkin Elmer). At that time, the above-mentioned luciferin solution was added to the wells to which only the medium was added and to the cell wells to which no lipid nanoparticles were added, measurements were performed in the same manner, and the value obtained by subtracting the luminescence intensity of the medium wells as the background was used. Note that the luminescence intensity of cell wells to which lipid nanoparticles were not added was below background.
  • E-MEM medium containing L-glutamine, phenol red, sodium pyruvate, non-essential amino acids, 1,500 mg/L sodium bicarbonate (product code 055-08975, Wako Pure Chemical Industries, Ltd.) 10% fetal serum (Fetal Bovine Serum Characterized, Corning Inc.) and 1% antibiotic (Penicillin-Streptomycin (10,000 U/mL), Thermo Fisher Inc.) were added.
  • HEK293 cells Human embryonic kidney cells 293 (HEK293 cells) were added to a 96-well black plate (SIGMA) at 2.0 ⁇ 10 4 cells/well, and cultured overnight at 37°C in a 5% CO 2 atmosphere. Particles were added at an amount of 1000 ng/well in terms of mRNA content. After culturing under the same conditions for 24 hours, the fluorescence intensity (excitation wavelength 587 nm/fluorescence wavelength 610 nm) was measured using a multiplate reader (Ensight multimode plate reader, Perkin Elmer).
  • SIGMA 96-well black plate
  • an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an siRNA concentration of 151 ⁇ g/mL were introduced into the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution was pumped, and the lipid nanoparticle solution excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the hPLK-1siRNA-loaded lipid nanoparticle solution.
  • Table 24 shows the base sequence of the lipid carrying hPLK-1siRNA. In the table, lowercase letters indicate RNA bases, and * indicates 2'-OMe modification.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of siRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • mice 2 mg siRNA/kg of each hPLK-1 siRNA-loaded lipid nanoparticle prepared or pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) was administered intravenously to mice divided into groups. 24 hours after administration, the mice were euthanized by exsanguination from the posterior vena cava under isoflurane anesthesia, and the subcutaneous tumors were removed. The excised tumor was immersed in Allprotect Tissue Reagent (manufactured by QIAGEN) and allowed to stand overnight at 4°C, followed by RNA extraction (RNeasy Plus Universal Mini Kit, manufactured by QIAGEN).
  • Allprotect Tissue Reagent manufactured by QIAGEN
  • RNA was subjected to reverse transcription reaction using SuperScript VILO cDNA Synthesis Kit (manufactured by Invitrogen). Thereafter, the amount of hPLK-1 mRNA was measured by qPCR using Power SYBR TM Green PCR Master Mix (manufactured by Thermo).
  • the endogenous control gene was human GAPDH (hGAPDH).
  • the primers used are shown in Tables 25 and 26.
  • the amount of hPLK-1 mRNA in the tumor (%) is calculated based on the amount of hPLK-1 mRNA in the tumor of a mouse treated with pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) as 100%, and the amount of hPLK-1 siRNA-loaded lipid nano The amount (%) of hPLK-1 mRNA in the tumor of mice administered with the particles was calculated.
  • the average particle diameter, PDI, siRNA encapsulation rate, and amount (%) of hPLK-1 mRNA in the tumor of the prepared lipid nanoparticles loaded with hPLK-1 siRNA are shown in Tables 27 to 29.
  • the average particle diameter of the hPLK-1siRNA-loaded lipid nanoparticles was 74.5 to 177.2 nm, and the siRNA encapsulation rate was 95% or higher in all cases. Furthermore, particles with a small PDI and high uniformity were formed.
  • the expression level of hPLK-1 in tumors of mice administered with hPLK-1siRNA-loaded lipid nanoparticles was 20.8 to 66.9% of that of tumors of mice treated with HEPES buffer.
  • hGAPDHsiRNA The base sequence of hGAPDHsiRNA is shown in Table 30. In the table, uppercase letters indicate DNA bases, and lowercase letters indicate RNA bases.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of siRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • mice 2 mg siRNA/kg of each hGAPDH siRNA-loaded lipid nanoparticle prepared or pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) was administered intravenously to mice divided into groups. 24 hours after administration, the mice were euthanized by exsanguination from the posterior vena cava under isoflurane anesthesia, and the subcutaneous tumors were removed. The excised tumor was soaked in Allprotect Tissue Reagent (manufactured by QIAGEN) and allowed to stand overnight at 4°C, followed by RNA extraction (Rneasy Plus Universal Mini Kit, manufactured by QIAGEN). The obtained total RNA was subjected to reverse transcription reaction using SuperScript VILO cDNA Synthesis Kit (Invitrogen).
  • the amount of hGAPDH mRNA was measured by qPCR using Power SYBR TM Green PCR Master Mix (Thermo).
  • the endogenous control gene was human ⁇ -actin (h ⁇ -actin).
  • the primers used are shown in Tables 31 and 32.
  • the amount of hGAPDH mRNA in the tumor (%) is calculated based on the amount of hGAPDH mRNA in the tumor of the mouse treated with pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) as 100%, and the amount of hGAPDH mRNA in the mouse treated with each hGAPDH siRNA-loaded lipid nanoparticle.
  • the amount (%) of hGAPDH mRNA in the tumor was calculated.
  • Table 33 shows the average particle diameter, PDI, siRNA encapsulation rate, and amount (%) of hGAPDH mRNA in the tumor of the prepared lipid nanoparticles loaded with hGAPDH siRNA.
  • the average particle diameter of the hGAPDH siRNA-loaded lipid nanoparticles was 95.9 to 110.9 nm, and the siRNA encapsulation rate was 95% or higher in all cases. Furthermore, particles with a small PDI and high uniformity were formed.
  • the expression level of hGAPDH in the tumors of mice administered with hGAPDH siRNA-loaded lipid nanoparticles was 45.4 to 58.3% of that in tumors of mice treated with HEPES buffer.
  • Lipid nanoparticles loaded with hPLK-1siRNA were administered to mice in which subcutaneous tumors were formed by BT-474 cells, and in vivo knockdown was performed in the tumors. The activity was examined.
  • an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an siRNA concentration of 151 ⁇ g/mL were introduced into the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution was pumped, and the lipid nanoparticle solution excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the hPLK-1siRNA-loaded lipid nanoparticle solution.
  • Table 34 shows the base sequence of the lipid carrying hPLK-1siRNA. In the table, lowercase letters indicate RNA bases, and * indicates 2'-OMe modification.
  • CL15F 9-5, CL15F 11-9 and CL15F6 were used as pH-sensitive cationic lipids.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of siRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • mice were subcutaneously transplanted into BALB/c-nu/nu mice. After subcutaneous tumors were formed, each of the prepared PLK-1 siRNA-loaded lipid nanoparticles was administered, and the in vivo hPLK-1 (human PLK-1) knockdown activity in the tumors was examined.
  • BALB/c-nu/nu mice Charles River Japan, 5 weeks old, female
  • mice were subcutaneously injected into the right flank of 1.5 g/L Sodium bicarbonate (Sigma-Aldrich, 5 x 106 BT-474 cells (human breast cancer cell line, ATCC, HTB-20) was transplanted. After subcutaneous tumor formation, mice were divided into groups based on tumor volume.
  • mice mean tumor volume was 141-143 mm3 .
  • Allprotect Tissue Reagent QIAGEN
  • RNA was subjected to reverse transcription reaction using SuperScript VILO cDNA Synthesis Kit (manufactured by Invitrogen). Thereafter, the amount of hPLK-1 mRNA was measured by qPCR using Power SYBR TM Green PCR Master Mix (Thermo).
  • the endogenous control gene was human GAPDH (hGAPDH).
  • the primers used are shown in Tables 35 and 36.
  • the amount of hPLK-1 mRNA in the tumor (%) is calculated based on the amount of hPLK-1 mRNA in the tumor of a mouse treated with pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) as 100%, and the amount of hPLK-1 siRNA-loaded lipid nano The amount (%) of hPLK-1 mRNA in the tumor of mice administered with the particles was calculated.
  • Table 37 shows the average particle diameter, PDI, siRNA encapsulation rate, and amount (%) of hPLK-1 mRNA in the tumor of the prepared lipid nanoparticles loaded with hPLK-1 siRNA.
  • the average particle diameter of the hPLK-1siRNA-loaded lipid nanoparticles was 102.4 to 154.1 nm, and the siRNA encapsulation rate was 95% or higher in all cases. Furthermore, particles with a small PDI and high uniformity were formed.
  • the expression level of hPLK-1 in tumors of mice treated with hPLK-1siRNA-loaded lipid nanoparticles was 49.6 to 49.6 in tumors of mice treated with 20mM HEPES buffer (9 wt% sucrose) at pH 7.45. It was 68.6%.
  • an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer solution (50 mM, pH 3.5) adjusted to an mRNA concentration of 46 ⁇ g/mL were introduced into the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution was pumped, and the lipid nanoparticle solution excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.
  • CleanCap registered trademark
  • FLuc mRNA 5 moU
  • CL15F 7-3, CL15F 7-5, CL15F 8-6, CL15F 9-5, CL15F 9-7, CL15F 10-4, CL15F 13-3, CL15F 16-1, CL15F 10- 8, CL15F 11-5, CL15F 11-7, CL15F 11-9, CL15F6, CL15F 9-3, CL15F 10-5, CL15F 11-6, CL15F 12-4 and CL15F 14-2 were used.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of mRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • OS-RC 2 ⁇ 10 6 cells suspended in 100 ⁇ L of RPMI1640 (Sigma-Aldrich, R5886-500 mL) was subcutaneously placed on the right flank of BALB/c-nu/nu mice (Charles River Japan, 7 weeks old, female).
  • -2 cells human kidney cancer cell line, RIKEN, RCB0735)
  • mice were divided into groups based on tumor volume. Group mean tumor volume was 125-140 mm3 .
  • Each Fluc mRNA-loaded lipid nanoparticle (0.1 mg mRNA/kg) or pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) was administered intravenously to mice divided into groups, and Fluc activity in the tumor was measured 24 hours later. Fluc activity was determined by administering VivoGlo Luciferin, In Vivo Grade (Promega, P1041) dissolved at 15 mg/mL in 1 ⁇ PBS (pH 7.4, Gibco) to 1.5 mg per mouse via the tail vein. , measured by an in vivo imaging system (Perkin Elmer, IVIS200). The unit of Fluc activity is the emission intensity per unit area (Avg Radiance [p/s/cm2/sr]) at a maximum emission wavelength of about 560 nm.
  • Tables 38 and 39 show the average particle diameter, PDI, mRNA encapsulation rate, and tumor Fluc activity of the prepared Fluc mRNA-loaded lipid nanoparticles.
  • the average particle diameter of the FlucmRNA-loaded lipid nanoparticles was 84.1 to 303.0 nm.
  • the mRNA inclusion rate was 75% or more in all cases.
  • particles with small PDI and high uniformity were formed in each particle.
  • the Fluc activity in tumors of mice administered with Fluc mRNA-loaded lipid nanoparticles was higher than that of mice treated with pH 7.45, 20 mM HEPES buffer (9 wt% sucrose).
  • an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer solution (50 mM, pH 3.5) adjusted to an mRNA concentration of 46 ⁇ g/mL were introduced into the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution was pumped, and the lipid nanoparticle solution excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.
  • CleanCap registered trademark
  • FLuc mRNA 5 moU
  • CL15F 11-5, CL15F 8-6, and CL15F6 were used as pH-sensitive cationic lipids.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of mRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • mice (Charles River Japan, 5 weeks old, female) were subcutaneously injected into the right flank of 1.5 g/L Sodium bicarbonate (Sigma-Aldrich, S5761) containing Hybri-Care (ATCC, 46-X). -500G) solution and Matrigel (Corning, 354234) at a volume ratio of 1:1. 5 x 10 6 BT-474 cells (human breast cancer cell line, ATCC, HTB -20) was ported. After subcutaneous tumor formation, mice were divided into groups based on tumor volume. Group mean tumor volume was 131-134 mm3 .
  • Fluc activity was determined by administering VivoGlo Luciferin, In Vivo Grade (Promega, P1041) dissolved at 15 mg/mL in 1 ⁇ PBS (pH 7.4, Gibco) to 1.5 mg per mouse via the tail vein. Measured using an in vivo imaging system (Perkin Elmer, IVIS Spectrum CT).
  • the unit of Fluc activity is the emission intensity per unit area (Avg Radiance [p/s/cm2/sr]) at a maximum emission wavelength of about 560 nm.
  • Table 40 shows the average particle diameter, PDI, mRNA encapsulation rate, and tumor Fluc activity of the prepared Fluc mRNA-loaded lipid nanoparticles.
  • the average particle diameter of the Flucm RNA-loaded lipid nanoparticles was 105.2 to 124.9 nm, and the mRNA encapsulation rate was 95% or more in all cases. Furthermore, particles with a small PDI and high uniformity were formed.
  • the Fluc activity in tumors of mice administered with Fluc mRNA-loaded lipid nanoparticles was higher than that of mice treated with pH 7.45, 20 mM HEPES buffer (9 wt% sucrose).
  • an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer solution (50 mM, pH 3.5) adjusted to an mRNA concentration of 46 ⁇ g/mL were introduced into the microchannel at 3 mL/min and 9 mL/min, respectively.
  • the lipid nanoparticle solution was pumped, and the lipid nanoparticle solution excreted from the channel was collected.
  • the lipid nanoparticle solution was diluted 10 times with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.
  • CleanCap OVA mRNA 5 moU) (TriLink, L-7210-5) was used to prepare OVA mRNA-loaded lipid nanoparticles.
  • CL15F 7-5, CL15F 9-3, CL15F 9-5, CL15F 9-7, CL15F 14-2, CL15F 11-7, CL15F 11-9, CL15F 12-4, CL15F6, CL15F 8-6, CL15F 10-4, CL15F 10-5, CL15F 10-8, CL15F 11-5, CL15F 11-6, and CL15F 13-3, and CL4F 7-3, CL4F 7-5, CL4F 8- 6, CL4F 9-4, CL4F 10-2, CL4F 10-8, CL4F 12-4, CL4F 12-6, CL4F6, CL4F 6-4, CL4F 7-4, CL4F 8-4, CL4F 8-5, CL4F 9-3, CL4F 9-5, CL4F 9-6, CL4F 9-7, CL4F 10-4 and CL4F 11-5 were used.
  • ⁇ Measurement of average particle diameter and PDI of lipid nanoparticles The average particle diameter ( ⁇ -Average) and PDI of lipid nanoparticles in PBS (-) were measured using an analyzer "Zetasizer Nano ZSP" (manufactured by Malvern) that uses a dynamic light scattering method.
  • ⁇ Nucleic acid encapsulation rate of lipid nanoparticles The encapsulation rate of mRNA in lipid nanoparticles was measured using Ribogreen reagent.
  • a solution diluted with a TE buffer so that the concentration of lipid nanoparticles was 8 ⁇ g/mL as a nucleic acid concentration was prepared as a solution for measuring the concentration of nucleic acids on the surface of nanoparticles.
  • a solution for measuring total nucleic acid concentration was prepared by adding 1.2 ⁇ g/mL X-triton100 to the solution so that the concentration of lipid nanoparticles was 1% (w/w).
  • the nucleic acid concentration was calculated using a calibration curve prepared by measuring the fluorescence intensity of a nucleic acid solution containing 1% X-triton100 (nucleic acid concentration: 0 to 2.5 ⁇ g/mL) in the same manner as described above.
  • the nucleic acid encapsulation rate of each lipid nanoparticle was calculated using the following formula.
  • Encapsulation rate % (Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) - Nucleic acid concentration of the solution for measuring the nucleic acid concentration on the surface of nanoparticles ( ⁇ g/mL)) ⁇ Nucleic acid concentration of the solution for measuring the total nucleic acid concentration ( ⁇ g/mL) mL) ⁇ 100
  • mice Each of the prepared OVA mRNA-loaded lipid nanoparticles was administered to mice, and OVA-specific cellular immune responses were examined using Mouse IFN- ⁇ ELISPOT Kit (R&D systems, XEL485). Specifically, C57BL6 mice (Charles River Japan, 9 weeks old, female) were injected with 1 ⁇ g of each prepared OVA mRNA-loaded lipid nanoparticle or pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) at 3-week intervals. Two doses were administered intramuscularly in the left thigh.
  • the spleen was collected from the mouse euthanized by cervical dislocation and placed in a C tube (Miltenyi Biotec, 130-093-237) containing 3 mL of RPMI1640 (Sigma-Aldrich, R5886-500 mL). and separated using the gentleMACS TM Octo Dissociator with Heaters (Miltenyi Biotec). The separated spleen cells were passed through a 40 ⁇ m cell strainer, washed with RPMI1640, and centrifuged at 300 ⁇ g for 7 minutes at 4° C. After removing the supernatant, red blood cells were lysed using ACK Lysing buffer (Gibco, A10492-01) for 2 minutes at room temperature.
  • ACK Lysing buffer Gibco, A10492-01
  • the reaction was stopped by adding 10 mL of RPMI164011, centrifuged at 300 xg for 7 minutes at 4°C, and the supernatant was completely removed. After removing the supernatant, add RPMI1640 (RPMI1640+FBS+LG/PS) containing 10% FBS (Gibco, 10270-160) and 1% L-Glutamine-Penicillin-Streptomycin solution (Sigma-Aldrich, G1146-100mL). The spleen cells were suspended and the number of cells was counted.
  • RPMI1640+FBS+LG/PS was added to the ELISpot plate, and the filter was expanded by allowing it to stand for 20 minutes or more.
  • 50 ⁇ L of RPMI1640+FBS+LG/PS or RPMI1640+FBS+LG/PS containing 20 ⁇ M of OVA class I peptide (Sigma, sequence: SIINFEKL) was added to each well of the ELISPOT plate.
  • the well to which RPMI1640+FBS+LG/PS was added is used for background detection, and the well to which RPMI1640+FBS+LG/PS containing OVA class I peptide was added is used for OVA-specific IFN- ⁇ detection.
  • the spleen cells whose number was counted were adjusted to 7.5 ⁇ 10 5 cells/mL, and 50 ⁇ L was seeded in each well of an ELISpot plate. Plates seeded with spleen cells were incubated for 20 hours at 37°C, 5% CO2 . After incubation, IFN- ⁇ was detected according to the protocol of Mouse IFN- ⁇ ELISpot Kit. After drying the plate, the number of spots was counted using an ELISpot reader (CTL, ImmunoSpot S6 Analyzer). The number of OVA-specific IFN- ⁇ producing cell spots for each mouse was determined by subtracting the number of OVA-specific IFN- ⁇ detection wells from the background detection well.
  • CTL ImmunoSpot S6 Analyzer
  • the average particle diameter, PDI, siRNA encapsulation rate, and spot number of OVA-specific IFN- ⁇ producing cells of the OVA mRNA-loaded lipid nanoparticles containing the CL15F6 derivative are shown in Tables 41 and 42.
  • Tables 43 and 44 show the average particle diameter, PDI, mRNA encapsulation rate, and spot number of OVA-specific IFN- ⁇ producing cells.
  • the average particle diameter of the OVA mRNA-loaded lipid nanoparticles containing the CL15F6 derivative was 99.5 to 197.3 nm, and the mRNA encapsulation rate was 90% or more in all cases. Furthermore, particles with a small PDI and high uniformity were formed.
  • the average particle diameter of the OVA mRNA-loaded lipid nanoparticles containing the CL4F6 derivative was 80.9 to 175.4 nm, and the mRNA encapsulation rate was 75% or more in all cases. Furthermore, particles with a small PDI and high uniformity were formed.
  • the number of spots of OVA-specific IFN- ⁇ producing cells in mice administered with OVA mRNA-loaded lipid nanoparticles containing CL15F6 derivatives or CL4F6 derivatives was determined by treatment with 20mM HEPES buffer (9 wt% sucrose), pH 7.45. It was detected more frequently than in mice.
  • C57BL6 mice (Charles River Japan, 9 weeks old, female) were injected with 1 ⁇ g of each OVA mRNA-loaded lipid nanoparticle prepared or pH 7.45, 20 mM HEPES buffer (9 wt% sucrose) at 3-week intervals. It was administered intramuscularly into the left thigh once.
  • blood was collected from the heart under isoflurane anesthesia. The collected blood was placed in MiniCollect (registered trademark) CAT Serum (greiner, 450533), mixed by inversion 8 to 10 times, and then allowed to stand for 30 to 90 minutes. After standing, it was centrifuged at 3000 ⁇ g for 10 minutes at room temperature to collect serum.
  • the plate was washed three times with 0.05% Tween20-PBS, and then 50 ⁇ L of serum serially diluted in 1/2 with 4 g/L Block Ace was added to each well and incubated at room temperature for 90 minutes. After that, wash again three times with 0.05% Tween20-PBS, and add 100 ⁇ L each of Anti-IgG (Fc), Mouse, Goat-Poly, HRP (BETHYL, A90-131P) adjusted with 4 g/L Block Ace. It was added to the well and incubated at room temperature for 1 hour.
  • Anti-IgG Fc
  • Mouse Mouse
  • Goat-Poly HRP
  • the plate was washed three times with 0.05% Tween20-PBS, 100 ⁇ L of TMB solution (05298-80, Nacalai Teix) was added, and the plate was incubated at room temperature for 30 minutes. After incubation, 100 ⁇ L of 1M sulfuric acid was added to each well, and the absorbance at 450 nm was measured using EnSight (Perkin Elmer). The minimum absorbance of OVA-specific IgG was set to 0.3, and the average value for each group was calculated. When the Log2 titer exceeded 28, 28 was taken as the measured value.

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  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dispersion Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Inorganic Chemistry (AREA)
  • Nanotechnology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention aborde le problème de la fourniture de nanoparticules lipidiques qui sont utiles en tant que composition pharmaceutique destinée à être administrée pour un cancer ou en tant que composition immunostimulante. Le problème est résolu par une composition pharmaceutique destinée à être administrée pour un cancer, une composition immunostimulante, ou similaire qui contient : un lipide cationique sensible au pH représenté par la formule (I) ; un stéréoisomère de celui-ci ; ou un mélange de stéréoisomères. [Dans la formule (I), a représente un nombre entier de 3 à 5 ; b représente 0 ou 1 ; R1 et R2 représentent chacun indépendamment un groupe représenté par la formule générale (A) (dans la formule (A), R11 et R12 représentent chacun indépendamment un groupe alkyle en C1-15 à chaîne linéaire ou à chaîne ramifiée ; c représente 0 ou 1 ; v représente un nombre entier de 4 à 12) ; X représente un groupe représenté par la formule générale (B) (dans la formule (B), d représente un nombre entier de 0 à 3 ; et R3 et R4 représentent chacun indépendamment un groupe alkyle en C1-4 ou un groupe alcényle en C2-4 (ledit groupe alkyle en C1-4 ou ledit groupe alcényle en C2-4 pouvant avoir un ou deux atomes d'hydrogène substitués par des groupes phényle), mais R3 et R4 peuvent être liés l'un à l'autre pour former un hétérocycle non aromatique de 5 à 7 chaînons (ledit cycle peut avoir un ou deux atomes d'hydrogène substitués par un groupe alkyle en C1-4 ou un groupe alcényle en C2-4) ou un groupe hétérocyclique non aromatique de 5 à 7 chaînons (ledit groupe étant lié à (O-CO)b- par l'intermédiaire d'un atome de carbone, et un ou deux atomes d'hydrogène dudit cycle pouvant être substitués par un groupe alkyle en C1-4 ou un groupe alcényle en C2-4)].
PCT/JP2023/013553 2022-03-31 2023-03-31 Composition pharmaceutique destinée à être administrée pour un cancer, ou composition immunostimulante WO2023191050A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2018230710A1 (fr) * 2017-06-15 2018-12-20 国立大学法人北海道大学 Structure de membrane lipidique pour administration intracellulaire d'arnsi
WO2022071582A1 (fr) * 2020-10-02 2022-04-07 国立大学法人北海道大学 Nanoparticule lipidique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018230710A1 (fr) * 2017-06-15 2018-12-20 国立大学法人北海道大学 Structure de membrane lipidique pour administration intracellulaire d'arnsi
WO2022071582A1 (fr) * 2020-10-02 2022-04-07 国立大学法人北海道大学 Nanoparticule lipidique

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HAJJ KHALID A., BALL REBECCA L., DELUTY SARAH B., SINGH SHRIDHAR R., STRELKOVA DARIA, KNAPP CHRISTOPHER M., WHITEHEAD KATHRYN A.: "Branched‐Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH", SMALL, WILEY, HOBOKEN, USA, vol. 15, no. 6, 1 February 2019 (2019-02-01), Hoboken, USA, pages 1805097, XP055812239, ISSN: 1613-6810, DOI: 10.1002/smll.201805097 *
HASHIBA KAZUKI, SATO YUSUKE, TAGUCHI MASAMITSU, SAKAMOTO SACHIKO, OTSU AYAKA, MAEDA YOSHIKI, SHISHIDO TAKUYA, MURAKAWA MASAO, OKAZ: "Branching Ionizable Lipids Can Enhance the Stability, Fusogenicity, and Functional Delivery of mRNA", SMALL SCIENCE, vol. 3, no. 1, 1 January 2023 (2023-01-01), pages 2200071, XP093096223, ISSN: 2688-4046, DOI: 10.1002/smsc.202200071 *
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