WO2018225873A1 - Nanoparticules contenant un acide nucléique - Google Patents

Nanoparticules contenant un acide nucléique Download PDF

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Publication number
WO2018225873A1
WO2018225873A1 PCT/JP2018/022286 JP2018022286W WO2018225873A1 WO 2018225873 A1 WO2018225873 A1 WO 2018225873A1 JP 2018022286 W JP2018022286 W JP 2018022286W WO 2018225873 A1 WO2018225873 A1 WO 2018225873A1
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lipid
nucleic acid
group
siglec
alkyl
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PCT/JP2018/022286
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English (en)
Japanese (ja)
Inventor
上原　啓嗣
剣太朗 畑中
宏徒 岩井
智幸 直井
ジュゼッペ デスティト
レイチェル ソロフ ニュージェント
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協和発酵キリン株式会社
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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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a nucleic acid-containing nanoparticle, a method for introducing a nucleic acid into a cell, a medicine using the nucleic acid-containing nanoparticle, and a method for treating or preventing a disease.
  • Nucleic acid drugs such as plasmid DNA (pDNA), antisense, decoy nucleic acid, ribozyme, siRNA, miRNA, antimiRNA, and mRNA can control genes by promoting or suppressing the expression of all genes in cells. Due to its high versatility, clinical application to various diseases that have been considered difficult to treat is expected. Nucleic acid drugs are expected as next-generation drugs after antibodies and low-molecular-weight drugs because of their high target selectivity and activity in cells. However, it is a problem that nucleic acid drugs are difficult to deliver to target tissues.
  • Targeting compounds include ligands that can bind to receptors expressed extracellularly.
  • Non-Patent Document 1 and Patent Document 1 a lipid comprising an N-acetyl-D-galactosamine (GalNAc) ligand capable of binding to an asialoglycoprotein receptor (ASGPR) highly expressed in hepatocytes and a double-chain lipid / Targeting the liver with nucleic acid nanoparticles has been reported.
  • GalNAc N-acetyl-D-galactosamine
  • ASGPR asialoglycoprotein receptor
  • Non-Patent Document 2 and Patent Document 2 cancer by a lipid / nucleic acid nanoparticle comprising a peptide ligand capable of binding to a prostate membrane specific antigen (PSMA) that is specifically highly expressed in prostate cancer patients and a double-stranded lipid. Targeting to has been reported.
  • PSMA prostate membrane specific antigen
  • Non-Patent Document 3 reports targeting of cancer cells with polymer / nucleic acid nanoparticles having transferrin.
  • Non-Patent Documents 1 to 3 and Patent Documents 1 and 2 application of a targeting compound to a nucleic acid-containing nanoparticle can be expected to improve migration to a target tissue and pharmacological effect.
  • the majority of these target organs and cells are the liver or cancer, and their application range is limited. Accordingly, there is a strong demand for the development of nucleic acid drugs that are effective for target organs and cells other than the liver.
  • Siglec sialic acid-binding-immunoglobulin-likelectin is a receptor that recognizes sialic acid-containing sugar chains belonging to the immunoglobulin superfamily and is expressed on the surface of immune system cells (Non-patent Document 4).
  • Non-Patent Documents 5 and 6 report the use of a complex using an antibody capable of binding to Siglec-2, which is one of Siglec receptors.
  • Patent Document 3 discloses a sugar chain ligand capable of binding to Siglec. Furthermore, Patent Document 4 reports a technique for delivering a low-molecular-weight drug or an antigen by nanoparticles using a sugar chain ligand capable of binding to Siglec.
  • An object of the present invention is to provide nucleic acid-containing nanoparticles and the like that can be delivered to immune system cells.
  • the present invention relates to the following.
  • a lipid containing lipid or a water-soluble unit (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), and a nucleic acid or
  • [2] Contains a lipid or lipid-containing unit with a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) (lipid I), nucleic acid, and cationic lipid (lipid II), or Polymers (Polymer I) containing water-soluble units and cationic units, having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), nucleic acids, and high molecules containing water-soluble units and cationic units Including molecules (polymer II), Nanoparticles.
  • siglec sialic acid-binding immunoglobulin-like lectin
  • lipid II nucleic acid
  • Polymers Polymer I containing water-soluble units and cationic units, having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), nucleic acids, and high molecules containing water-soluble units and cationic units Including
  • Lipid I is a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit are linked via a linker: (N1 is an integer of 1 or more.)
  • Represented by Polymer I has a structure in which a ligand capable of binding to Siglec and a water-soluble unit, or a ligand capable of binding to Siglec, a water-soluble unit, and a cationic unit are linked via a linker: (N2 is an integer of 1 or more.)
  • the nanoparticle according to [1] or [2], represented by: [4] The nanoparticle according to any one of [1] to [3], wherein the ligand is a sugar chain ligand.
  • [5] The nanoparticle according to [4], wherein the sugar chain ligand is a group represented by the following formula (1).
  • Ac represents an acetyl group
  • R 1 represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1 substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. Represents a -C6 alkyl group.
  • R 1 in formula (1) is a group represented by the following structure.
  • Water-soluble unit is polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, chitosan, polyvinylpyrrolidone, polyaspartic acid amide
  • the cationic unit is an amino acid polymer unit containing at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit, according to any one of [1] to [7] Nanoparticles.
  • the nucleic acid is siRNA or mRNA.
  • the nanoparticle according to [10], wherein the siRNA is covalently bonded to cholesterol, tocopherol or a fatty acid via a linker.
  • [12] The nanoparticle according to any one of [1] to [11], which is used for delivery to Siglec-1 (CD169) positive cells.
  • the nanoparticle according to [12], wherein the Siglec-1 (CD169) positive cell is a macrophage, a dendritic cell or a monocyte.
  • [14] [1] A method for introducing a nucleic acid into a cell, using the nanoparticles according to any one of [11].
  • [15] The method according to [14], wherein the cell is a Siglec-1 (CD169) positive cell.
  • the Siglec-1 (CD169) positive cells are macrophages, dendritic cells or monocytes.
  • [17] [1] A pharmaceutical comprising the nanoparticle according to any one of [13].
  • the medicament according to [17] which is for intravenous administration or subcutaneous administration.
  • [19] [17] A method for treating or preventing a disease, comprising administering the medicine according to [18] to a patient in need thereof.
  • the nanoparticle according to [1], comprising a lipid (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) and containing a lipid or a water-soluble unit (lipid I), and a nucleic acid.
  • the lipid according to [2], comprising a lipid (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), including a lipid or a water-soluble unit (lipid I), a nucleic acid, and a cationic lipid (lipid II).
  • lipid I lipid having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), including a lipid or a water-soluble unit (lipid I), a nucleic acid, and a cationic lipid (lipid II).
  • the present invention can provide nucleic acid-containing nanoparticles that are useful as pharmaceuticals and can be delivered to immune system cells.
  • the nanoparticle of the present invention includes a lipid having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), a lipid or a water-soluble unit (lipid I), and a nucleic acid, or A polymer (polymer I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) and containing a water-soluble unit and a cationic unit (polymer I), and a nucleic acid.
  • the nanoparticles of the present invention are nucleic acid-containing nanoparticles.
  • the average particle size of the nanoparticles of the present invention is preferably 1.00 to 2000 nm, more preferably 10.0 to 500 nm, still more preferably 20.0 to 300 nm, and most preferably 20.0 to 200 nm.
  • Siglec is a receptor belonging to the immunoglobulin superfamily and is present on the surface of immune system cells.
  • the nanoparticles of the present invention can be targeted to monocytes, macrophages, or dendritic cells.
  • the nanoparticle of the present invention has, as a model, a structure in which a lipid-containing outer layer is encapsulated in a central part including a nucleic acid (that is, a structure in which a nucleic acid exists inside a particle and a lipid constitutes the outer part of the particle (outer layer)).
  • lipid I and polymer I are present in the outer layer of the nanoparticle, and the ligand capable of binding to Siglec is present in the outward direction of the particle, and the surface of the particle is modified by the ligand. it is conceivable that. This allows the ligand to interact with Siglec.
  • the modification rate (%) by the ligand on the surface of the particle is defined as the molar concentration of lipid I or polymer I relative to the total molar concentration of lipid or polymer constituting the particle (that is, the number of moles of lipid I / particle).
  • the total number of moles of lipid constituting or the number of moles of polymer I / the total number of moles of polymer constituting particles) can be used as an index.
  • the modification rate is preferably 0.2% or more, more preferably 0.4% or more in order for the nanoparticle to reach the target cell and transfer the nucleic acid into the cell.
  • the maximum value of the modification rate is not particularly limited, but is preferably 50% or less, more preferably 30% or less, still more preferably 10% or less, and even more preferably 5% or less.
  • the modification rate is preferably 0.2% or more, more preferably 0.4% in order for the nanoparticles to reach the target cell and transfer the nucleic acid into the cell. Or more, more preferably 1.0% or more, and still more preferably 1.4% or more.
  • the maximum value of the modification rate may be 100% or less.
  • the modification rate in the nucleic acid-containing nanoparticles containing the polymer I is preferably 30 to 100%, more preferably 50 to 100% or less, and even more preferably 70 to 100%.
  • the nanoparticle of the present invention may be a nanoparticle comprising a lipid (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), a lipid containing lipid or a water-soluble unit (lipid I), and a nucleic acid.
  • lipid I lipid having a ligand capable of binding to siglec
  • lipid II cationic lipid
  • Lipid I is a lipid having a ligand capable of binding to Siglec, or a lipid comprising a water-soluble unit having a ligand capable of binding to Siglec.
  • the lipid containing a lipid and a water-soluble unit in the present invention may be a natural type lipid or a non-natural type lipid in which a natural type structure is partially modified, that is, a lipid analog.
  • the polymer I is a polymer including a water-soluble unit and a cationic unit having a ligand capable of binding to Siglec.
  • the lipid having a ligand capable of binding to Siglec can also be referred to as a complex in which a ligand capable of binding to Siglec and a lipid unit are bound.
  • the lipid containing a water-soluble unit having a ligand capable of binding to Siglec can also be referred to as a complex in which a ligand capable of binding to Siglec, a water-soluble unit, and a lipid unit are bound.
  • the polymer containing a water-soluble unit and a cationic unit having a ligand capable of binding to Siglec can also be referred to as a complex in which a ligand capable of binding to Siglec, a water-soluble unit, and a cationic unit are bound.
  • Lipid I preferably has a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit are linked via a linker.
  • the polymer I preferably has a structure in which a ligand capable of binding to Siglec and a water-soluble unit are linked via a linker.
  • a ligand capable of binding to Siglec and a lipid or cationic unit can be linked by a linker.
  • the structure having a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit is linked via a linker can be specifically represented by the following structural formula. Note that one or more ligands that can bind to Siglec in the structural formula may be bound to a linker.
  • N1 and N2 in the structural formula are integers of 1 or more.
  • Lipid I may be a lipid containing a water-soluble unit having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), that is, a ligand capable of binding to siglec and a water-soluble unit It can also be represented by the following structural formula linked via a linker.
  • N1 in the structural formula is an integer of 1 or more.
  • the polymer I a water-soluble unit and a cationic unit may be bonded via a linker, that is, the polymer I can be represented by the following structural formula.
  • N2 in the structural formula is an integer of 1 or more.
  • Lipid I and polymer I may have one or more ligands capable of binding to Siglec as described above, may have two, may have three, or have four It may be.
  • Lipid I and polymer I having two or more ligands capable of binding to Siglec can be obtained by using a linker containing a branched structure, and can be represented by the following structural formula.
  • Lipid I in the following structure may or may not contain a water-soluble unit between the lipid and the linker.
  • a water-soluble unit and a cationic unit may be bonded via a linker.
  • the branched structure contained in the linker is not particularly limited, and examples thereof include the structures (A) to (D) shown below.
  • n1 to m3 each independently represents an integer of 0 to 3)
  • the branched structures (A) to (D) can be provided by the following compounds. Specifically, the branched structure (A) can be provided by utilizing, for example, the branched structure possessed by the compounds (A-1) to (A-5). The branched structure (B) can be provided, for example, by utilizing the branched structure possessed by the compounds (B-1) and (B-2). The branched structure (C) can be provided by using, for example, a branched structure possessed by the compounds (C-1) to (C-3). The branched structure (D) can be provided by utilizing a branched structure possessed by the compound (D-1), for example.
  • Water-soluble units include, for example, polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, chitosan, polyvinylpyrrolidone, polyasparagine It is composed of acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol or the like or derivatives thereof.
  • polyethylene glycol is preferable.
  • the number average molecular weight of the water-soluble unit is not particularly limited, but is preferably 100 to 40000, more preferably 500 to 20000, and further preferably 500 to 5000.
  • the lipid in the lipid I is not particularly limited as long as it is a lipid capable of forming lipid nanoparticles, and a neutral lipid is preferable.
  • Suitable examples of neutral lipids include phospholipids, glycerol lipids, sterols, glyceroglycolipids, glycosphingolipids and sphingoids. These neutral lipids may be used alone or in combination of two or more.
  • Examples of the phospholipid in the neutral lipid constituting the lipid I include phosphatidylcholine (PC) (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3 -Phosphocholine (DSPC), dipalmitoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC) ), Phosphatidylethanolamine (specifically distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE
  • DMPE DMPE
  • POPE palmitoyl oleoyl-phosphatidylethanolamine
  • SOPE 1-stearoyl-2-oleoyl-phosphatidylethanolamine
  • glycerophospholipids specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylglycerol (POPG), lysophosphatidylcholine, etc.
  • sphingophospholipids specifically sphingomyelin, ceramide
  • Phosphoethanolamine ceramide phosphoglycerol
  • ceramide phosphoglycerophosphate etc.
  • glycerophosphonolipid sphingophosphonolipid
  • sphingophosphonolipid natural lecithin (specifically egg yolk lecithin, soybean lec
  • Examples of the glycerol lipid in the neutral lipid constituting the lipid I include, but are not limited to, diacylglycerol.
  • Examples of the glyceroglycolipid in the neutral lipid constituting the lipid I include, but are not limited to, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
  • glycosphingolipid in the neutral lipid constituting the lipid I examples include, but are not limited to, galactosyl cerebroside, lactosyl cerebroside, ganglioside, and the like.
  • Examples of the sphingoid in the neutral lipid constituting the lipid I include, but are not limited to, sphingan, icosasphingan, sphingosine or derivatives thereof.
  • Derivatives include, for example, —NH 2 such as sphingan, icosasphingan, and sphingosine —NHCO (CH 2 ) xCH 3 (wherein x is an integer of 0 to 18, among which 6, 12 or 18 is preferred. However, it is not limited to these.
  • Examples of the sterol in the neutral lipid constituting lipid I include, for example, cholesterol (Chol), dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucosterol, or 3 ⁇ - [N -(N ', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like are exemplified, but not limited thereto.
  • cholesterol cholesterol
  • dihydrocholesterol lanosterol
  • ⁇ -sitosterol campesterol
  • stigmasterol stigmasterol
  • brassicasterol ergocasterol
  • fucosterol or 3 ⁇ - [N -(N ', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like are exemplified, but not limited thereto.
  • Lipid I preferably includes a structure composed of a lipid and a water-soluble unit (hereinafter also referred to as a structure derived from a lipid derivative of a water-soluble polymer).
  • the structure composed of the lipid and the water-soluble unit is preferably derived from a lipid derivative of a water-soluble polymer.
  • the lipid derivative of the water-soluble polymer in the present invention may be the above-described neutral lipid polyethylene glycol lipid derivative.
  • the polyethylene glycol lipid derivative of the neutral lipid includes, for example, a structure in which a polyethylene glycol group is bonded to a hydroxy group contained in the neutral lipid, with or without an amide group or an ester group, or the neutral lipid.
  • a hydroxy group contained in is substituted with an amide group or a carboxyl group, and a polyethylene glycol group is bonded via the substituted amide group or carboxyl group.
  • Specific examples of structures composed of lipids and water-soluble units include structures represented by the following formulas (Z1) to (Z3).
  • Ra and Rb are each independently linear or branched alkyl, alkenyl or alkynyl having 7 to 23 carbon atoms, preferably heptadecanyl, pentadecanyl, tridecanyl, (Z) -heptadecane- 8-enyl, (Z) -tridec-8-enyl, (Z) -pentadeca-8-enyl, (Z) -heptadeca-5-enyl, (Z) -heptadeca-8-enyl, (E) -heptadeca- 8-enyl, (Z) -heptadeca-10-enyl, (8Z, 11Z) -heptadeca-8,11-dienyl, (8Z, 11Z, 14Z) -heptadeca-8,11,14-trienyl, (Z)- Nonadeca-10-enyl, (10Z, 13Z) -nonade
  • Ra ′ and Rb ′ are independently straight-chain or branched alkyl, alkenyl or alkynyl having 7 to 23 carbon atoms, preferably heptadecanyl, pentadecanyl, tridecanyl, (Z) — Heptadeca-8-enyl, (Z) -Tridec-8-enyl, (Z) -Pentadeca-8-enyl, (Z) -Heptadeca-5-enyl, (Z) -Heptadeca-8-enyl, (E)- Heptadeca-8-enyl, (Z) -Heptadeca-10-enyl, (8Z, 11Z) -Heptadeca-8,11-dienyl, (8Z, 11Z, 14Z) -Heptadeca-8,11,14-trienyl, (Z ) -Nonadeca-10-enyl, (10Z, 13Z) -
  • Ra ′′ and Rb ′′ are each independently a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, preferably octadecanyl, hexadecanyl, tetradecanyl, (Z ) -Octadeca-9-enyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E ) -Octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11
  • the lipid derivative of the water-soluble polymer is preferably a lipid derivative of polyethylene glycol, more preferably polyethylene glycol-phosphatidylethanolamine (more specifically 1,2-distearoyl-sn-glycero-3-phospho Ethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG -DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3 -[Methoxy (polyethylene glycol) -2000]), and polyethylene glycol-diacylglycerol (more specifically, 1,2-distearoyl-sn-glycerol
  • the cationic unit in the polymer I is preferably an amino acid polymer unit containing at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit.
  • the amino acid polymer unit containing one or more selected from the group consisting of lysine, arginine and histidine can be obtained by polymerization of an amino acid containing one or more selected from the group consisting of lysine, arginine and histidine.
  • the amino acid polymer unit may be a random copolymer or a block copolymer.
  • the amino acid polymer unit may contain amino acids other than lysine, arginine, and histidine.
  • the amino acid other than lysine, arginine, and histidine is not particularly limited, but is preferably glycine.
  • amino acids constituting the amino acid polymer unit one or more amino acid units selected from the group consisting of lysine, arginine and histidine are preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably it is.
  • amino acid polymer unit include the following structures.
  • n1 is an integer of 2 or more.
  • the cationic unit in the polymer I is oriented so as to wrap the nucleic acid to form particles.
  • the amount of charge of the nucleic acid contained in the nanoparticle varies depending on the size (molecular weight) of the nucleic acid, and the size of the cationic unit necessary for particle formation varies accordingly. Therefore, the number of repeating amino acid monomers represented by n1 may be adjusted based on the molecular weight of the nucleic acid and is not particularly limited, but may be in the range of 1 to 3000, preferably 10 to 2000, and more preferably. Is from 10 to 1000, more preferably from 10 to 100, even more preferably from 10 to 50, and particularly preferably from 10 to 40.
  • the polyethyleneimine unit is a unit made of a polymer obtained by polymerizing ethyleneimine.
  • the polyethyleneimine unit can be represented, for example, as shown in the main chain structure below, but the polyethyleneimine main chain may be cross-linked with other polyethyleneimine structures.
  • n2 is an integer of 1 or more.
  • the number of repeating amino acid monomers represented by n2 may be adjusted based on the molecular weight of the nucleic acid and is not particularly limited, but may be in the range of 1 to 3000, preferably 10 to 2000, and more preferably 50. ⁇ 1000.
  • the polyaminoacrylate unit is a unit made of a polymer obtained by polymerizing methacrylic acid having an amino group such as 2- (dimethylamino) ethyl methacrylate.
  • the polyaminoacrylate unit for example, the following structures are preferably exemplified.
  • n3 is an integer of 1 or more.
  • the number of repeating amino acid monomers represented by n3 may be adjusted based on the molecular weight of the nucleic acid and is not particularly limited, but may be in the range of 1 to 3000, preferably 10 to 2000, and more preferably 50 ⁇ 1000.
  • the ligand is preferably a sugar chain ligand.
  • the sugar chain ligand is preferably a group represented by the following formula (1).
  • Ac represents an acetyl group
  • R 1 represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1 substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. Represents a -C6 alkyl group.
  • Examples of the C6-C12 aryl group in R 1 include a phenyl group, a biphenyl group, and a naphthyl group.
  • the C4-C12 heteroaryl group in R 1 is a furanyl group, thienyl group, thiopyranyl group, isothiochromenyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, pyridyl group, pyralidinyl group, pyrimidinyl group, pyridazinyl group, thiazolyl group , An isothiazolyl group, and a pyranyl group.
  • the C6-C12 aryl group and the C4-C12 heteroaryl group may be substituted with a substituent.
  • substituents include an alkyl group such as methyl, ethyl, propyl, and isopropyl; an alkoxy group such as methoxy, ethoxy, and propoxy A halogen atom such as chloro and bromo; a hydroxy group; an amino group; a C6-C12 aryl group such as a phenyl group, a biphenyl group and a naphthyl group; a C4-C12 heteroaryl group such as a furanyl group and a thienyl group; a piperidine and a chroman A heterocyclic group; and the like.
  • C6-C12 aryl groups such as phenyl group, biphenyl group and naphthyl group
  • C4-C12 heteroaryl groups such as furanyl group and thienyl group
  • heterocyclic groups such as piperidine and chroman
  • the C6-C12 aryl group and the C4-C12 heteroaryl group may be condensed to form a condensed ring composed of at least two rings.
  • R 1 the "C6-C12 aryl group” and “C4-C12 heteroaryl group” in the C1-C6 alkyl group substituted by C6-C12 aryl or C4-C12 heteroaryl group, the R 1 above , C6-C12 aryl group and C4-C12 heteroaryl group can be exemplified.
  • the “C1-C6 alkyl group” in the C1-C6 alkyl group substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group in R 1 is methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl.
  • R 1 in Formula (1) is preferably a group represented by the following structure.
  • the linker links the ligand and lipid that can bind to Siglec, or the ligand that can bind to Siglec and the water-soluble unit, or the water-soluble unit and the cationic unit. If it does not reduce, it will not restrict
  • the linker structure includes amide group (-NHCO-), amino group (-NH-), ether group (-O-), thioether group (-S-), carbonyl group (-CO-) ester group (-CO)
  • An alkylene group that may contain at least one functional group selected from the group consisting of -O-), a heterocycle, a heteroaromatic ring, and the like is preferable.
  • the number of carbon atoms of the alkylene group is not particularly limited, but is preferably 1 to 10.
  • linker structure I One example of the linker structure is represented by the following linker structure I.
  • Q1 and Q2 are each independently an amide group (-NHCO- or -CONH-), an amino group (-NH-), an ether group (-O-), a thioether group (-S- ), A carbonyl group (—CO—), an ester group (—CO—O— or —O—CO—), a heterocycle, and a heteroaromatic ring, and a and b are each independently 0 Is an integer of 1 to 5, preferably 1 to 5, more preferably 2 to 3, c is 0 or 1, and Z is 1 or more, preferably 1 to 5, more preferably 1 to 2. (It is an integer.)
  • the heterocycle of Q1 and Q2 is not particularly limited, and examples thereof include a 4- to 7-membered heterocycle containing one or more heteroatoms, succinimide and the like.
  • the heteroaromatic ring of Q1 and Q2 is not particularly limited, and examples thereof include triazole, imidazole, tetrazole, pyridine, pyrimidine and the like.
  • Z is 2 or more, a plurality of Q1 and b may be the same or different.
  • the linker existing between the ligand capable of binding to Siglec and the water-soluble unit has a branch point. You may have. Further, in the polymer I, the linker existing between the water-soluble unit and the cationic unit may have a branch point. Therefore, a combination of the above-mentioned branched structures (A) to (D) and linker structure I is also a suitable linker structure. Examples of the linker containing a branched structure include the following linker structures IA to ID.
  • Q1 and Q2 are each independently an amide group (—NHCO— or —CONH—), an amino group (—NH—), an ether group (—O—), a thioether group (—S—), Any one of a carbonyl group (—CO—), an ester group (—CO—O— or —O—CO—), a heterocycle, and a heteroaromatic ring, wherein a1 to a4 and b1 to b4 are each independently And any one of 0 to 5, c1 to c4 are each independently 0 or 1, and Z1 to Z4 are each independently an integer of 1 or more.)
  • the heterocycle of Q1 and Q2 is not particularly limited, and examples thereof include a 4- to 7-membered heterocycle containing one or more heteroatoms, succinimide and the like.
  • the heteroaromatic ring of Q1 and Q2 is not particularly limited, and examples thereof include triazole, imidazole, tetrazole, pyridine, pyrimidine and the like.
  • Z1 to Z4 are 2 or more, a plurality of Q1 and b1 to b4 may be the same or different.
  • linker structure in lipid I linker structure I, linker structure IA, and linker structure IB described above are preferable.
  • Q1 and Q2 in the linker structure I are each independently any of an amide group (—NHCO— or —CONH—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring.
  • a and b are each independently an integer of 0 to 5, c is preferably 0 or 1, and Z is preferably 1 or 2.
  • Q1 and Q2 in the linker structure IA are each independently any one of an amide group (-NHCO- or -CONH-), an amino group (-NH-), a heterocycle, and a heteroaromatic ring, and a1 To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.
  • Q1 and Q2 in the linker structure IB are each independently any one of an amide group (—NHCO— or —CONH—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring;
  • To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.
  • linker structure in lipid I include the following structures.
  • the ligand capable of binding to Siglec is, for example, a group represented by the formula (1), the sugar chain, A linker may be bonded.
  • examples of the linker structure include the following structures.
  • L1 and L2 may be the same or different and represent ligands capable of binding to Siglec.
  • linker structure I As the structure of the linker that links the ligand capable of binding to Siglec and the water-soluble unit in the polymer I, the above-described linker structure I, linker structure IA, and linker structure IB are preferable.
  • Q1 and Q2 in the linker structure I are each independently any one of an amide group (—NHCO—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring, and a and Each b is independently an integer of 0 to 5, c is preferably 0 or 1, and Z is preferably 1 or 2.
  • Q1 and Q2 in the linker structure IA are each independently any one of an amide group (-NHCO- or -CONH-), an amino group (-NH-), a heterocycle, and a heteroaromatic ring, and a1 To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.
  • Q1 and Q2 in the linker structure IB are each independently any one of an amide group (—NHCO— or —CONH—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring;
  • To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.
  • the ligand capable of binding to Siglec is, for example, a group represented by the formula (1), the sugar chain, A linker may be bonded.
  • examples of the linker structure include the following structures.
  • L1 and L2 may be the same or different and represent ligands capable of binding to Siglec.
  • the linker present in the polymer I for linking the water-soluble unit and the cationic unit is preferably the linker structure I described above, and more preferably represented by the following structure.
  • a and b are each independently an integer of 0 to 5, preferably 1 to 5, more preferably 2 to 3.
  • the linker structure I existing for linking the water-soluble unit and the cationic unit is more preferably the following structure.
  • a branch point may be provided in a linker that is present for linking a water-soluble unit and a cationic unit.
  • examples of the structure of the linker include the following structures.
  • lipid I include the following structures.
  • R 1 represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1-C6 alkyl group substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group.
  • Linker represents a linker
  • PEG is a unit containing polyethylene glycol
  • Lipid is a lipid
  • N1 is an integer of 1 or more.
  • Ac represents acetyl.
  • the structure of -PEG-Lipid in the formula is preferably any of the structures represented by the formulas (Z1) to (Z3) described above, and is preferably a structure represented by the formula (Z2). More preferred.
  • the Linker is not particularly limited, but is preferably any of the linker structure I, linker structure IA, and linker structure IB described above, more preferably the linker structure I, and still more preferably the following structure: It is.
  • lipid I preferably has the following structure.
  • R 1 , Q 1 , Q 2, a, b, c, Z, PEG, Lipid, and Ac are the same as above.
  • the lipid I is more preferably the following structure.
  • lipid I the following structure is more preferable.
  • N is an integer of 1 to 300.
  • polymer I examples include the following structures.
  • R 1 represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1-C6 alkyl group substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group.
  • PEG represents A unit containing polyethylene glycol
  • Poly is an amino acid polymer unit, a polyethyleneimine unit, or a polyaminoacrylate unit containing at least one selected from the group consisting of lysine, arginine, and histidine
  • Linker 1 and Linker 2 are (Linker that independently links Poly and PEG.
  • N2 is an integer greater than or equal to 1.
  • Ac represents acetyl.
  • Linker IV 1 is preferably the above-described linker structure I, linker structure IA, and linker structure IB.
  • Preferred examples of the polymer I include the following structures.
  • R 1 , PEG, Poly, Q 1, Q 2, a, b, c, and Ac are the same as described above.
  • Plural Q 1, Q 2, a, b, c may be the same , May be different.
  • R 1 , PEG, Poly, Q 1, Q 2, a, b, c, a 1 to a 3, b 1 to b 3, c 1 to c 3, and Ac are the same as described above.
  • a plurality of R 1 , Q 1 Q2 may be the same or different.
  • R 1 , PEG, Poly, Q 1, Q 2, b, c, a 1 to a 3, b 1 to b 3, c 1 to c 3, and Ac are the same as described above.
  • Multiple R 1 , Q 1 , Q 2 , A1 to a3, b1 to b3, and c1 to c3 may be the same or different.
  • a ′ and a ′′ are each independently an integer of 0 to 5.
  • More preferable polymer I includes the following structures.
  • More preferable polymer I includes the following structures.
  • N1 is 1 to 3000, preferably 10 to 2000, more preferably 10 to 1000, even more preferably 10 to 100, still more preferably 10 to 50, and particularly preferably 10 to 1000. 40)).
  • N1 is 1 to 3000, preferably 10 to 2000, more preferably 10 to 1000, even more preferably 10 to 100, still more preferably 10 to 50, and particularly preferably 10 to 1000. 40)).
  • lipid I and polymer I may exist as lipid I and polymer I, respectively, but lipid I and polymer I may be any one of isomers, and these are optional. The mixture contained in the ratio may be sufficient. Specific examples of isomers include optical isomers.
  • the formula (1) is preferably an isomer represented by the following formula (1 ′).
  • R 1 has the same meaning as R 1 in formula (1).
  • Ac is an acetyl group.
  • Lipid I can be synthesized using organic synthesis techniques with reference to J. Am. Chem. Soc., 2012, 134, 15696. Specifically, as shown in the scheme below, a reaction introduced by performing general derivatization on a reactive group contained in a lipid containing a lipid or a water-soluble unit, or a lipid containing a lipid or a water-soluble unit.
  • the lipid I is obtained by reacting the reactive group with a reactive group in an organic group containing a reactive group bonded to a ligand capable of binding to Siglec (the organic group is a linker in lipid I).
  • the synthesis method is not particularly limited.
  • the combination of reactive groups include a combination of an amino group and a carboxyl group, a combination of a hydroxyl group and a carboxyl group, etc., preferably a combination of an amino group and a carboxyl group, and more preferably a lipid or water-soluble group.
  • This is a combination in which a reactive group of a lipid containing a sex unit is an amino group and a reactive group bonded to a ligand capable of binding to Siglec is a carboxyl group.
  • the reactive carboxyl group may be activated by acid chloride, N-hydroxysuccinimide, N, N'-dicyclohexylcarbodiimide, etc., and applies the general conditions for amide bond formation and dehydration condensation reactions. can do.
  • ligands that can bind to lipids or water-soluble units containing reactive groups and Siglecs containing organic groups containing reactive groups see J. Am. Chem. Soc., 2012, 134, 15696.
  • a commercially available lipid or ligand may be derivatized using an organic synthesis technique.
  • Polymer I can also be synthesized using organic synthesis techniques. Specifically, as shown in the scheme below, the reactive group contained in the polymer containing a water-soluble unit and a cationic unit, or the reactivity introduced by general derivatization of the water-soluble unit.
  • the organic group is a linker in the polymer I
  • the synthesis method is not particularly limited.
  • the combination of the reactive groups is not particularly limited, and examples thereof include a combination of an amino group and a carboxyl group, and a combination of a hydroxyl group and a carboxyl group.
  • the reactive carboxyl group may be activated by acid chloride, N-hydroxysuccinimide, N, N'-dicyclohexylcarbodiimide, etc., and applies the general conditions for amide bond formation and dehydration condensation reactions. can do.
  • Ligand capable of binding to a water-soluble unit containing a reactive group and a polymer containing a cationic unit and a Siglec containing an organic group containing a reactive group are described in J. Am. Chem. Soc., 2012, 134, 15696. And commercially available lipids and ligands may be derivatized using organic synthesis techniques.
  • J. ⁇ Am. ⁇ ⁇ Chem. Soc., 2008, 130, 6680-6681, International Publication No. 2007/056525 and the like are synthesized as ligands that can bind to Siglec containing an organic group containing a reactive group. You can also.
  • a ligand that can bind to Siglec By introducing an organic group containing a reactive group into a ligand that can bind to Siglec, a ligand that can bind to Siglec can be converted into a lipid containing a lipid or a water-soluble unit, or a high water containing a water-soluble unit and a cationic unit. Linkage with molecules becomes possible.
  • the method for introducing an organic group containing a reactive group into a ligand that can bind to Siglec is not particularly limited, and the ligand capable of binding to Siglec and a compound that becomes an organic group containing a reactive group are etherified.
  • the ligand capable of binding to Siglec is a sugar chain ligand
  • a compound that becomes an organic group containing a reactive group at the anomeric position at the end of the sugar chain eg, acetalization
  • An organic group containing a reactive group can be introduced into a ligand capable of binding.
  • a cationic lipid (Lipid II) When the nanoparticles of the present invention are lipid nanoparticles containing lipid, in addition to lipid I, a cationic lipid (lipid II) may be contained.
  • the cationic lipid includes a lipophilic region containing one or more optionally substituted hydrocarbon groups, and at least one primary amino group, secondary amino group, tertiary amino group and / or quaternary ammonium group. It is not particularly limited as long as it is an amphiphilic molecule having a cationic hydrophilic region containing, but may be substituted with a hydrophilic part having one amino group or one quaternary ammonium group which may be substituted, and may be substituted. Mention may be made of lipids having a hydrophobic part having two independent hydrocarbon groups.
  • Examples of the cationic lipid used in the present invention include International Publication No. 2013/089151, International Publication No. 2011/136368, International Publication No. 2014/007398, International Publication No. 2010/042877 or International Publication No. 2010 / And cationic lipids described in No. 054401.
  • cationic lipid used in the present invention include the following formulas (CL-I) to (CL-XVI).
  • R 101 and R 102 are the same or different and are linear or branched C10-C24 alkyl, C10-C24 alkenyl or C10-C24 alkynyl, L 101 and L 102 are hydrogen atoms or together form a single bond or C2-C8 alkylene; L 103 is a single bond, -CO- or -CO-O-, When L 103 is a single bond, X 101 is a hydrogen atom, C1-C6 alkyl, C3-C6 alkenyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino, monoalkylamino, dialkyl C1-C6 alkyl or C3-C6 alkenyl substituted with amino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl,
  • R 103 and R 104 are the same or different and are linear or branched C12-C24 alkyl, C12-C24 alkenyl or C12-C24 alkynyl, p 101 and p 102 are the same or different and are integers of 0 to 3, L 106 and L 107 are hydrogen atoms or together form a single bond or C2-C8 alkylene; L 104 and L 105 are the same or different and are -O-, -CO-O- or -O-CO-, L 108 is a single bond, -CO- or -CO-O- When L 108 is a single bond, X 102 is a hydrogen atom, C1-C6 alkyl, C3-C6 alkenyl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino acids C1-C6 alkyl or C3-C6 al
  • R 105 is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl
  • R 106 is linear or branched C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyl Oxypropyl, C8-C24 alkynyloxyethyl or C8-C24 alkynyloxypropyl
  • X 103 and X 104 are the same or different and are C1-C3 alkyl or taken together to form C2-C8 alkylene, or X 103 is taken together with L 111 to form C2-C8 alkylene.
  • L 111 is a hydrogen atom, C1-C6 alkyl, C3-C6 alkenyl, amino, monoalkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, or the same or different 1 to 3 amino, monoalkylamino, C1-C6 alkyl or C3-C6 alkenyl substituted with hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl, or together with X 103 to form C2-C8 alkylene;
  • L 109 is C1-C6 alkylene
  • L 110 is a single bond or C1-C6 alkylene, provided that the sum of the carbon numbers of L 109 and L 110 is 7 or less, and when L 111 is a hydrogen atom, L 110 is a single bond.
  • R 107 is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl; R 108 is linear or branched C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyl Oxypropyl, C8-C24 alkynyloxyethyl, C8-C24 alkynyloxypropyl, C8-C24 alkyloxyethoxyethyl, C8-C24 alkenyloxyethoxyethyl or C8-C24 alkynyloxyethyl, X 105 is a hydrogen atom or an optionally substituted C1-C4 alkyl)
  • R 109 is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl
  • R 110 is linear or branched C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyl Oxypropyl, C8-C24 alkynyloxyethyl or C8-C24 alkynyloxypropyl
  • L 112 is C1-C3 alkylene
  • X 105 ′ is a hydrogen atom or C1-C3 alkyl
  • R 111 and R 112 are the same or different and may be linear or branched, optionally substituted C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, X 106 and X 107 are the same or different and are C1-C3 alkyl or taken together to form C2-C8 alkylene; p 103 , p 104 and p 105 are the same or different and are 0 or 1, provided that p 103 , p 104 and p 105 are not 0 at the same time, L 113 and L 114 are the same or different and are O, S or NH)
  • R 113 and R 114 are the same or different, linear or branched optionally substituted C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, R 115 is a hydrogen atom, hydroxy, optionally substituted C1-C4 alkyl, C1-C4 alkoxy or C1-C4 acyloxy, X 109 and X 110 are the same or different and are C1-C3 alkyl or taken together to form C2-C8 alkylene; L 115 is -CO-O-, -O-CO-, -NHCO- or -CONH- p 106 is an integer from 0 to 3, p 107 is an integer from 1 to 4)
  • R 116 and R 117 are the same or different and may be linear or branched substituted C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C7-C20 alkyloxy C1-C3 alkyl, C7- C20 alkenyloxy C1-C3 alkyl or C7-C20 alkynyloxy C1-C3 alkyl
  • B 100 is a hydrogen atom, C1-C3 alkyl, hydroxy C2-C4 alkyl, C1-C3 dialkylamino C2-C4 alkyl
  • formula (A) In the formula, X 111 and X 112 are the same or different and are a hydrogen atom or C1-C3 alkyl, or together with the nitrogen atom to which X 111 and X 112 are bonded, a C2-C6 nitrogen-containing heterocycle is formed.
  • P 110 is an integer from 2 to 6), or formula (B) (Wherein X 113 and X 114 are the same or different and are a hydrogen atom or C1-C3 alkyl, or together with the nitrogen atom to which X 113 and X 114 are bonded form a C2-C6 nitrogen-containing heterocycle
  • p 111 is an integer from 1 to 6
  • P 108 is an integer from 0 to 4
  • P 109 is an integer from 1 to 4 (except when P 108 is 0 and P 109 is 1)
  • L 116 is the same or different for each carbon to be bonded and is a hydrogen atom or C1-C3 alkyl
  • L 117 is the same or different for each carbon to be bonded and is a hydrogen atom or C1-C3 alkyl
  • X 115 and X 116 are the same or different and are a hydrogen atom or C1-C3 alkyl
  • L 118 and L 119 are the same or different and may be linear or branched C8-C24 alkylene or C8-C24 alkenylene
  • M 101 and M 102 are the same or different
  • -C C-, -OC (O)-, -C (O) O-, -SC (O)-, -C (O) S-, -OC (S )-, -C (S) O-, -SS-
  • -C (R '' ) N-
  • -N C (R '' )-
  • -C (R '' ) NO-
  • -ON C (R '' )-, -N (R '' ) C (O)-, -C (O) N (R '' )-, -N (R '' ) C (S)-, -
  • X 117 and X 118 are the same or different hydrogen atoms, optionally substituted C1-C6 alkyl, heterocyclyl or polyamine, or X 117 and X 118 together with the nitrogen to which they are attached. In addition to the nitrogen, it may form a 4-7 membered monocyclic heterocycle which may contain 1 or 2 additional heteroatoms selected from N, O and S; R 120 and R 121 are the same or different and may be linear or branched, optionally substituted C4-C24 alkyl or C4-C24 alkenyl)
  • X 119 and X 120 are the same or different and each represents a hydrogen atom, a linear or branched C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl or C6-C20 acyl, R 122 and R 123 are the same or different, linear or branched optionally substituted C1-C30 alkyl, C2-C30 alkenyl or C2-C30 alkynyl, p 112 , p 113 and p 114 are the same or different and are 0 or any positive integer)
  • X 121 and X 122 are the same or different and are a hydrogen atom, cycloalkyl, cycloalkenyl, or X 121 and X 122 together with the nitrogen atom to which they are bonded form a C2-C6 nitrogen-containing heterocycle.
  • L 120 and L 121 may be the same or different and are —O—, —OC (O) — or — (O) CO—, R 124 and R 125 are the same or different and are linear or branched optionally substituted C8-C24 alkyl or C8-C24 alkenyl)),
  • R 126 and R 127 are the same or different and may be linear or branched substituted C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 heteroalkyl, C8-C24 heteroalkenyl or C8-C24 heteroalkynyl
  • X 123 is a hydrogen atom or an optionally substituted C1-C6 alkyl
  • X 124 is C1-C6 alkyl, substituted C1-C6 alkyl substituted with -NR 4a R 4b or optionally substituted C3-C7 heterocyclyl
  • X 123 and X 124 together with the nitrogen atom to which they are attached may form
  • X 125 and X 126 are the same or different hydrogen atoms, optionally substituted C1-C6 alkyl, heterocyclyl or polyamine, or X 125 and X 126 together with the nitrogen to which they are attached, In addition to nitrogen, it may form a 4-7 membered monocyclic heterocycle which may contain 1 or 2 additional heteroatoms selected from N, O and S;
  • R 130 is a hydrogen atom or C1-C6 alkyl;
  • R 128 and R 129 are the same or different and may be linear or branched, optionally substituted C4-C24 alkyl or C4-C24 alkenyl
  • X 127 and X 128 are each independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, X 127 and X 128 together with the nitrogen atom to which they are attached form a heterocyclic ring having 1 to 2 nitrogen atoms;
  • L 122 is -C (O) O-, -OC (O)-, -C (O) N (X 130 )-, -N (X 130 ) C (O)-, -OC (O) O-, -OC (O) N (X 130 )-, -N (X 130 ) C (O) N (X 130 )-, or -N (X 130 ) C (O) O-
  • Each occurrence of X 130 is independently a hydrogen atom or C1-C3 alkyl; a is 1, 2, 3, 4, 5, or 6; b is 0, 1, 2, or 3;
  • X 129 is absent or is hydrogen or C1-C3 alkyl;
  • R 131 and R 132 are chains having at least 4 carbon atoms between the biodegradable group and a tertiary carbon atom marked with an asterisk (*))
  • R 133 and R 134 are the same or different and are each linear or branched C1-C9 alkyl, C2-C11 alkenyl or C2-C11 alkynyl, L 123 and L 124 are the same or different and are each linear C5-C18 alkylene or linear C5-C18 alkenylene, or form a heterocyclic ring with N, L 125 is a single bond or -CO-O-, thereby forming -L 124 -CO-OR 134 ; L 127 is S or O, L 126 is a single bond, or a linear or branched C1-C6 alkylene, or forms a heterocyclic ring with N.
  • L 128 is linear or branched C1-C6 alkylene, and X 131 and X 132 are the same or different and are each hydrogen or linear or branched C1-C6 alkyl)
  • examples of the linear or branched C10-C24 alkyl include decyl, undecyl, dodecyl, tridecyl, 6,10-dimethylundec-2-yl, tetradecyl, Pentadecyl, hexadecyl, heptadecyl, octadecyl, 6,10,14-trimethylpentadecan-2-yl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl and the like, preferably decyl, undecyl, dodecyl, tridecyl, tetradecyl, Examples include pentadecyl, hexadecyl, heptadecyl, and octadecyl, and more preferable examples include tridecyl, tetradecyl,
  • the linear or branched C10-C24 alkenyl may be a linear or branched C10-C24 alkenyl containing 1 to 3 double bonds, such as (Z) -dodec-7-enyl. , (Z) -tetradec-7-enyl, (Z) -tetradec-9-enyl, (Z) -hexadec-4-enyl, (Z) -hexadeca-7-enyl, (E) -hexadeca-7-enyl , (Z) -hexadec-9-enyl, (7Z, 10Z) -hexadec-7,10-dienyl, (7Z, 10Z, 13Z) -hexadec-7,10,13-trienyl, (Z) -octadeca-6 -Enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-eny
  • the linear or branched C10-C24 alkynyl may be a linear or branched C10-C24 alkynyl containing 1 to 3 triple bonds, and examples thereof include deca-9-ynyl and dodeca-4.
  • hexadeca-7-ynyl or octadec-9-ynyl Preferably hexadeca-7-ynyl or octadec-9-ynyl, and more preferably octadec-9-ynyl.
  • R 101 and R 102 are preferably the same linear or branched C10-C24 alkyl, C10-C24 alkenyl or C10-C24 alkynyl, and the same linear It is more preferably a straight or branched C10-C24 alkyl or C10-C24 alkenyl, and even more preferably the same linear C10-C24 alkenyl.
  • C1-C3 alkylene examples include methylene, ethylene, or propylene, preferably methylene or ethylene, and more preferably methylene.
  • Examples of the C1-C6 alkyl include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, cyclopropylmethyl, pentyl, isopentyl, sec-pentyl, neopentyl, tert- Examples include pentyl, cyclopentyl, hexyl, cyclohexyl, etc., preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, hexyl, etc. More preferably, methyl, ethyl, propyl, etc. are mentioned.
  • C3-C6 alkenyl examples include allyl, 1-propenyl, butenyl, pentenyl, hexenyl and the like, preferably allyl and the like.
  • Monoalkylamino and dialkylamino are each one or two identical or different C1-C6 alkyls (as defined above) or amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl Any amino substituted with C1-C6 alkyl (as defined above) substituted with, for example, methylamino, ethylamino, propylamino, butylamino, pentylamino, hexylamino, dimethylamino, diethylamino, ethylmethylamino Methylpropylamino, butylmethylamino, methylpentylamino, hexylmethylamino, aminoethylamino, aminopropylamino, (aminoethyl) methylamino, bis (aminoethyl) amino, etc.,
  • Trialkylammonio includes the same or different C1-C6 alkyl (as defined above), or C1-C6 substituted with amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl.
  • ammonio substituted with alkyl may be used, for example, trimethylammonio, ethyldimethylammonio, diethylmethylammonio, triethylammonio, tripropylammonio, tributylammonio, tripentylammonio, tripentylammonio, Hexylammonio, tris (aminoethyl) ammonio, (aminoethyl) dimethylammonio, bis (aminoethyl) methylammonio and the like can be mentioned, preferably trimethylammonio, triethylammonio, tris (aminoethyl) ammonio (Aminoethyl) dimethyl ammonio or bis (aminoethyl) methyl ammonio, and the like, or more preferably trimethylammonio like.
  • the trialkylammonio may form a salt with a pharmaceutically acceptable anion (as defined above).
  • Alkoxy is substituted with C1-C6 alkyl (as defined above) or C1-C6 alkyl (as defined above) substituted with amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl. It may be hydroxy, for example, methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy, aminoethoxy or methylaminoethoxy, preferably methoxy, ethoxy, aminoethoxy or methylaminoethoxy, More preferably, methoxy etc. are mentioned.
  • Monoalkylcarbamoyl and dialkylcarbamoyl are each one or two identical or different C1-C6 alkyls (as defined above) or amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl Any carbamoyl substituted with C1-C6 alkyl substituted with (as defined above), for example, methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl, pentylcarbamoyl, hexylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethyl Methylcarbamoyl, methylpropylcarbamoyl, butylmethylcarbamoyl, methylpentylcarbamoy
  • L 101 and L 102 are more preferably hydrogen atoms.
  • R 101 and R 102 are the same or different and dodecyl, tetradecyl, (Z) -dodec-7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl.
  • X 101 is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different from 1 to Must be C1-C6 alkyl or C3-C6 alkenyl substituted with 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl Is more preferably a hydrogen atom, methyl, or C1-C6 alkyl or C3-C6 alkenyl substituted with 1 to 3 amino, hydroxy or carbamoyl, which are the same or different, and more preferably a hydrogen atom, methyl, etc. More preferably it is.
  • R 101 and R 102 are the same or different and are tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadec-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, or (11Z, 14Z) -icosa -11,14-dienyl, more preferably (Z) -octadec-9
  • X 101 is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl Or C1-C6 alkyl substituted with 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, the same or different Or a C3-C6 alkenyl, more preferably a hydrogen atom, methyl, or a C1-C6 alkyl or C3-C6 alkenyl substituted with 1 to 3 amino, hydroxy or carbamoyl, which are the same or different.
  • L 101 and L 102 together form a single bond L 103 is —CO— or —CO—O—, preferably —CO—.
  • X 101 is aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-aminopropyl, 1,4-diaminobutyric, 1,5 Jiaminopenchiru, 3-aminopropyl, 4 -Aminobutyl or 5-aminopentyl is preferable, and 1,2-diaminoethyl, 1,3-diaminopropyl, 1,4-diaminobutyl or 1,5-diaminopentyl is more preferable.
  • R 101 and R 102 are the same or different and are tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadec-6-enyl, (Z)- Octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca- Preferably it is 9,12,15-trienyl or (Z) -icosa-11-enyl or (11Z, 14Z) -icosa-11,14-dienyl, (Z) -octadeca-9-enyl or (9Z, More preferably, they are 12Z) -octadeca
  • L 103 is more preferably a single bond.
  • X 101 is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino, monoalkylamino , Dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl substituted C1-C6 alkyl or C3-C6 alkenyl, etc., more preferably a hydrogen atom , Methyl, hydroxymethyl, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxy-3-methoxypropyl, aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 2- (N)
  • X 101 is pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different one to three amino, mono Alkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl substituted C1-C6 alkyl or C3-C6 alkenyl, at least of the substituents More preferably, one is amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl, morpholinyl or the like, and R 3 is aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1 , 3-Diaminopropyl, 3-aminopropyl, 1,
  • L 103 is a single bond and X 101 is a hydrogen atom.
  • R 101 and R 102 are the same or different and dodecyl, tetradecyl, (Z) -dodec-7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl.
  • L 103 is a single bond and X 101 is methyl is one of the more preferable embodiments of the present invention.
  • R 101 and R 102 are the same or different and dodecyl, tetradecyl, (Z) -dodec-7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl.
  • linear or branched C12-C24 alkyl includes, for example, dodecyl, tridecyl, tetradecyl, 2,6,10-trimethylundecyl, pentadecyl, 3,7, 11-trimethyldodecyl, hexadecyl, heptadecyl, octadecyl, 6,10,14-trimethylpentadecan-2-yl, nonadecyl, 2,6,10,14-tetramethylpentadecyl, icosyl, 3,7,11,15-tetra Examples include methylhexadecyl, henicosyl, docosyl, tricosyl, tetracosyl, etc., preferably dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl
  • the linear or branched C12-C24 alkenyl may be a linear or branched C12-C24 alkenyl containing 1 to 3 double bonds, such as (Z) -tridec-8-enyl. , (Z) -tetradec-9-enyl, (Z) -pentadeca-8-enyl, (Z) -hexadeca-9-enyl, (Z) -heptadeca-5-enyl, (Z) -octadeca-6-enyl , (Z) -heptadeca-8-enyl, (Z) -octadeca-9-enyl, (E) -heptadeca-8-enyl, (E) -octadeca-9-enyl, (Z) -heptadeca-10-enyl , (Z) -octadeca-11-enyl, (8Z, 11Z) -heptadeca-8
  • the linear or branched C12-C24 alkynyl may be a linear or branched C12-C24 alkynyl containing 1 to 3 triple bonds, such as dodeca-11-ynyl, tridec-12- Inyl, pentadec-6-ynyl, hexadec-7-ynyl, pentadec-4,6-diynyl, hexadec-5,7-diynyl, heptadec-8-ynyl, octadec-9-ynyl and the like, preferably pentadec-9 6-Inyl, hexadec-7-ynyl, pentadec-4,6-diynyl, hexadec-5,7-diynyl, heptadec-8-ynyl, octadec-9-ynyl, etc., more preferably heptadeca-8-iny
  • C1-C3 alkylene, C1-C6 alkyl and C3-C6 alkenyl in the definition of each group of formula (CL-II) are respectively synonymous with those in formula (CL-I).
  • Monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl have the same meanings as those in formula (CL-I), respectively.
  • R 103 and R 104 are preferably the same linear or branched C12-C24 alkyl, C12-C24 alkenyl or C12-C24 alkynyl, the same linear or branched C12-C24 alkyl, Or C12-C24 alkenyl.
  • L 104 and L 105 are the same -O -, - more preferably CO-O- or -O-CO-.
  • R 103 and R 104 are the same or different and are dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11- Enyl, (11Z
  • R 103 and R 104 are each tridecyl, pentadecyl, heptadecyl, nonadecyl, henicosyl, tricosyl, (Z) -tridec-8-enyl, (Z) -pentadeca-8-enyl, (Z) -heptadeca-5-enyl, (Z) -heptadeca-8-enyl, (E) -heptadeca-8-enyl, (Z) -heptadeca-10-enyl, ( 8Z, 11Z) -heptadeca-8,11-dienyl, (8Z, 11Z, 14Z) -octadeca-8,11,14-trienyl, (Z) -nonadec-10-enyl, (10Z, 13Z) -nonadec-10 , 13-dienyl, (
  • p 101 and p 102 are 0 or 1 at the same time.
  • L 106 and L 107 together form a single bond or C1-C3 alkylene.
  • X 102 is a hydrogen atom, methyl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl , Piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or the same or different 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, More preferably it is C1-C6 alkyl or C3-C6 alkenyl substituted with alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, hydrogen atom, methyl, or the same or different from 1 to 3 C1-C
  • propyl 1,4-diaminobutyl, 1,5-diaminopentyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl or 2-carbamoylethyl.
  • the alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl.
  • Two or three alkyls in dialkylamino, trialkylammonio and dialkylcarbamoyl may be the same or different.
  • L 108 is preferably —CO— or —CO—O—, preferably —CO—.
  • p 101 and p 102 are preferably the same or different and are 1 to 3.
  • X 102 is a hydrogen atom, methyl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidine-4 -Yl, morpholin-2-yl, morpholin-3-yl or the same or different 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, C1-C6 alkyl or C3-C6 alkenyl substituted with pyrrolidinyl, piperidyl or morpholinyl, preferably hydrogen atom, methyl or the same or different from 1 to 3 amino, trialkylammonio, hydroxy or carbamoyl More preferred is a substituted C1-C6 alkyl or C3-C6 alkenyl.
  • Hydrogen atom methyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl, 1,4-diaminobutyl, 1, More preferred are 5-diaminopentyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 2-carbamoylethyl and the like.
  • alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl.
  • Two or three alkyls in dialkylamino, trialkylammonio, and dialkylcarbamoyl may be the same or different.
  • L 108 is preferably a single bond.
  • L 104 and L 105 are preferably —O—.
  • X 102 is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino, monoalkylamino, C1-C6 alkyl or C3-C6 alkenyl substituted with dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl is preferred.
  • 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 2-carbamoylethyl and the like are more preferable.
  • the alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl.
  • Two or three alkyls in dialkylamino, trialkylammonio, and dialkylcarbamoyl may be the same or different.
  • L 104 and L 105 are —O—.
  • L 108 is a single bond and X 102 is a hydrogen atom
  • L 104 and L 105 are preferably the same —CO—O— or —O—CO—, and —CO—O— More preferably.
  • L 104 and L 105 are preferably the same —CO—O— or —O—CO—, and are —CO—O—. Is more preferable.
  • X 102 is pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl or the same or different 1 to 3 amino, monoalkyl
  • it is C1-C6 alkyl or C3-C6 alkenyl substituted with amino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl.
  • X 102 is aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1 , 3-Diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl, 4-a Nobutyl, 1,5-diaminopentyl, 5-aminopentyl, (N, N-dimethylamino) methyl, 2- (N, N-dimethylamino) ethyl, 3- (N, N-dimethylamino) propyl or 1- More preferred are amino-2-hydroxyethyl and the like, aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl,
  • alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C6 alkyl.
  • Two or three alkyls in dialkylamino, trialkylammonio and dialkylcarbamoyl may be the same or different.
  • L 104 and L 105 are preferably the same —CO—O— or —O—CO—, and more preferably —CO—O—.
  • the alkyl moiety in C8-C24 alkyloxyethyl and C8-C24 alkyloxypropyl may be, for example, the linear or Examples thereof include those exemplified for branched C8-C24 alkyl.
  • alkynyl moiety in alkynyloxyethyl and alkynyloxypropyl examples include those exemplified for the linear or branched C8-C24 alkynyl.
  • R 105 and R 106 are preferably the same or different linear or branched C8-C24 alkyl or C8-C24 alkenyl, and are the same or different linear or branched C8-C24 alkenyl. More preferably, it is more preferably the same or different linear C8-C24 alkenyl. R 105 and R 106 are more preferably the same, and in that case, linear or branched C12-C24 alkyl, C12-C24 alkenyl, or C12-C24 alkynyl is preferable. More preferably, the chain is C12-C24 alkenyl. Linear or branched C12-C24 alkyl, C12-C24 alkenyl, and C12-C24 alkynyl have the same meanings as those in formula (CL-II), respectively.
  • R 105 and R 106 are preferably the same or different linear or branched C8-C24 alkyl or C8-C24 alkenyl, and are the same or different linear or branched C8-C24 alkenyl. More preferably, it is more preferably the same or different linear C8-C24 alkenyl. R 105 and R 106 are more preferably the same, and in that case, linear or branched C15-C20 alkyl, C15-C20 alkenyl, or C15-C20 alkynyl is preferable. More preferably, the chain is C15-C20 alkenyl. Linear or branched C15-C20 alkyl, C15-C20 alkenyl, and C15-C20 alkynyl have the same meanings as those in formulas (I) to (IV), respectively, and the same groups are preferable.
  • R 105 and R 106 are different, R 105 is a linear or branched C15-C20 alkyl, C15-C20 alkenyl or C15-C20 alkynyl, and R 106 is a linear or branched C8.
  • -C12 alkyl is preferred.
  • examples of the linear or branched C8-C12 alkyl include octyl, nonyl, decyl, undecyl, and dodecyl, and preferably octyl, decyl, and dodecyl.
  • R 105 is linear C15-C20 alkenyl
  • R 106 is linear C8-C12 alkyl
  • R 105 is (Z) -octadec-9-enyl or (9Z, 12Z) -octadeca More preferably, it is -9,12-dienyl and R 106 is octyl, decyl or dodecyl.
  • R 105 and R 106 are different, R 105 is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, R 106 is C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyloxypropyl, C8-C24 alkynyloxyethyl or C8-C24 alkynyloxypropyl is also preferred.
  • R 105 is a C8-C24 linear alkenyl
  • R 106 is more preferably a C8-C24 alkenyloxy ethyl
  • R 105 is, (Z) - octadec-9-enyl, (9Z , 12Z) -octadeca-9,12-dienyl or (11Z, 14Z) -icosa-11,14-dienyl
  • R 106 is (Z) -octadec-9-enyloxyethyl, (9Z, 12Z)- More preferably, it is octadeca-9,12-dienyloxyethyl or (11Z, 14Z) -icosa-11,14-dienyloxyethyl
  • R 105 is (9Z, 12Z) -octadeca-9,12- Most preferably, it is dienyl and R 106 is (9Z, 12Z) -octadeca-9,12
  • R 105 and / or R 106 are the same or different linear or branched C8-C24 alkyl or C8-C24 alkenyl, the same or different tetradecyl, hexadecyl, (Z) -tetradec-9 -Enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11 -Enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -Icosa-11,14-dienyl or (Z) -doc
  • R 107 and R 108 have the same meanings as R 105 and R 106 , respectively, and the same groups as R 105 and R 106 are preferred.
  • R 107 is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, R 107 and R 108 are identically (9Z, 12Z) -octadeca-9, 12-dienyl is preferred.
  • R 109 and R 110 are synonymous with R 105 and R 106 , respectively, and the same groups as R 105 and R 106 are preferred. However, R 109 and R 110 are preferably the same linear or branched C15-C20 alkyl, C15-C20 alkenyl or C15-C20 alkynyl, and identically (9Z, 12Z) -octadeca-9, More preferred is 12-dienyl.
  • the C1-C3 alkyl in X 103 and X 104 includes, for example, methyl, ethyl, propyl, isopropyl or cyclopropyl, preferably methyl or ethyl, and more preferably methyl.
  • Examples of the C2-C8 alkylene formed by combining X 103 and X 104 include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and the like, preferably butylene, pentylene, hexylene, and the like. More preferred is hexylene.
  • Examples of the C2-C8 alkylene formed by X 103 together with L 111 include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and the like, and preferably propylene, butylene, pentylene, and the like. More preferably, propylene or butylene is used, and propylene is more preferably used.
  • X 103 and X 104 are the same or different and are methyl or ethyl, together form butylene, pentylene or hexylene, or X 103 together with L 111 form ethylene, propylene or butylene It is preferable to do.
  • X 103 and X 104 are the same or different and are preferably methyl or ethyl, or together, form butylene, pentylene or hexylene, and X 103 together with L 111 is ethylene, propylene or It is also preferred that it forms butylene and X 104 is methyl or ethyl.
  • X 103 and X 104 are identically methyl or together form hexylene, X 103 together with L 111 forms propylene or butylene, and X 104 is methyl. It is further more preferable.
  • C1-C6 alkyl, C3-C6 alkenyl, monoalkylamino, alkoxy, mono- alkylcarbamoyl and dialkylcarbamoyl have the same meanings as those in each of the formulas (CL-I).
  • L 111 is a hydrogen atom, C1-C6 alkyl, amino, monoalkylamino, hydroxy, alkoxy or C1-C6 alkyl substituted with 1 to 3 amino, monoalkylamino, or hydroxy or alkoxy, the same or different.
  • X 103 to form C2-C6 alkylene substituted with 1 to 3 amino or hydroxy atoms, hydrogen atom, methyl, amino, methylamino, hydroxy, methoxy, or the same or different More preferably, together with X 103 to form ethylene, propylene or butylene, which is a hydrogen atom, C1-C3 alkyl, or hydroxy, or together with X 103 More preferably it forms propylene or butylene, together with a hydrogen atom or X 103 Most preferably, propylene is formed.
  • L 109 and L 110 as the C1-C6 alkylene, such as methylene, ethylene, propylene, butylene, etc. pentylene or hexylene and the like, preferably methylene or ethylene and the like.
  • L 109 is preferably methylene, ethylene, propylene, or the like, more preferably methylene, ethylene, or the like.
  • L 110 is preferably a single bond, methylene, ethylene, or the like, and is a single bond, methylene, or the like. More preferably.
  • the sum of the carbon numbers of L 109 and L 110 is preferably 1 to 3, and more preferably 2.
  • X 103 and X 104 are the same or different, such as methyl or ethyl
  • L 111 is a hydrogen atom, methyl, amino, methylamino, hydroxy, methoxy, or the same or different 1 ⁇ 3 amino or hydroxy substituted methyls or the like, or X 103 and X 104 together form pentylene, hexylene or heptylene, etc.
  • L 111 is a hydrogen atom, methyl, amino, methylamino, X 103 and L 111 together form propylene, butylene, pentylene or the like, such as hydroxy, methoxy or methyl substituted with 1 to 3 amino or hydroxy identically or differently
  • X 104 preferably is methyl or ethyl and the like
  • X 103 and X 104 is methyl
  • L 111 is a hydrogen atom, X 103 and X 104 are together It forms a pentylene or hexylene or
  • the C1-C4 alkyl in X 105 ′ includes, for example, methyl, ethyl, propyl, isopropyl, cyclopropyl and the like, preferably methyl, ethyl, isopropyl and the like More preferably, methyl or ethyl is exemplified.
  • X 105 ′ is more preferably a hydrogen atom or methyl, and most preferably a hydrogen atom.
  • L 112 as the C1-C3 alkylene, e.g., methylene, ethylene or propylene and the like, preferably methylene or ethylene and the like.
  • Examples of the C1-C4 alkyl in the optionally substituted C1-C4 alkyl in R 115 of the formula (CL-VII) include, for example, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert -Butyl, cyclobutyl, cyclopropylmethyl, etc. are mentioned, Preferably methyl, ethyl etc. are mentioned, More preferably, methyl is mentioned.
  • the alkyl part of C1-C4 alkoxy which may be substituted has the same meaning as the C1-C4 alkyl.
  • substituent in the optionally substituted C1-C4 alkyl include amino, monoalkylamino, dialkylamino, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, and piperidine 4-yl, morpholin-2-yl, morpholin-3-yl, hydroxy, alkoxy, alkoxycarbonyl, hydroxycarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, nitro, cyano, fluoro, chloro, bromo and the like.
  • alkyl moiety in monoalkylamino, dialkylamino, alkoxy, alkoxycarbonyl, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl.
  • Two alkyls in dialkylamino and dialkylcarbamoyl may be the same or different.
  • acyl in the optionally substituted C1-C4 acyloxy examples include formyl, acetyl, propanoyl, 2-methylpropanoyl, cyclopropanoyl, butanoyl, and preferably acetyl and the like.
  • C1-C4 acyloxy examples include amino, monoalkylamino, dialkylamino, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl and piperidine 4-yl, morpholin-2-yl, morpholin-3-yl, hydroxy, alkoxy, alkoxycarbonyl, hydroxycarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, nitro, cyano, fluoro, chloro, bromo and the like.
  • alkyl moiety in monoalkylamino, dialkylamino, alkoxy, alkoxycarbonyl, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl.
  • Two alkyls in dialkylamino and dialkylcarbamoyl may be the same or different.
  • R 111 and R 112 are preferably the same linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, and the same linear or branched More preferably, it is C8-C24 alkyl or C8-C24 alkenyl.
  • R 111 and R 112 are the same or different and are octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, (Z) -tetradec-9-enyl, (Z) -hexadec-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12 -Dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,
  • X 106 and X 107 are preferably the same or different and are preferably methyl or ethyl, more preferably methyl.
  • Examples of the C2-C8 alkylene formed by combining X 106 and X 107 include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and the like, preferably butylene, pentylene, hexylene, and the like. More preferred is butylene or pentylene.
  • X 106 and X 107 are preferably the same methyl or taken together to form butylene, pentylene or hexylene.
  • p 103 and 104 are preferably 0 at the same time, and p 105 is preferably 1.
  • L 113 and L 114 are preferably O at the same time.
  • R 113 and R 114 are preferably the same linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, the same linear or branched C8-C24 alkyl or More preferably, it is C8-C24 alkenyl.
  • C1-C3 alkyl and C2-C8 alkylene in X 109 and X 110 are respectively synonymous with those in the formula (CL-VI).
  • R 115 is preferably a hydrogen atom, hydroxy, methyl, methoxy or the like, more preferably a hydrogen atom or hydroxy, and even more preferably a hydrogen atom.
  • L 115 is preferably —O—CO—.
  • p 106 is 0 or 1
  • p 107 is an integer of 2 ⁇ 4
  • p 106 is 0 or 1
  • more preferably p 107 is 3.
  • L 115 is the case of -CO-O-, p 106 is 0, it is preferable that p 107 is an integer of 2 ⁇ 4, p 106 is 0, more that p 107 is 3 preferable.
  • each group of formula (CL-VIII) to formula (CL-XVI) may be synonymous with that in formula (CL-I) to formula (CL-VII).
  • each group in the formula (CL-VIII) is in WO 2016/002753
  • each group in the formula (CL-X) is in WO 2009/129385
  • each group in the formula (CL-XI) is In International Publication No.2013 / 149140
  • each group in formula (CL-XII) is in International Publication No.2009 / 129395
  • each group in formula (CL-XIII) is in International Publication No.2013 / 059496
  • -XIV) in International Publication No. 2011/149733
  • each group in Formula (CL-XV) in International Publication No.2011 / 153493
  • each group in Formula (CL-XVI) in International Publication No.
  • a preferred embodiment of each group described correspondingly may be used.
  • L 118 and L 119 in formula (CL-IX) are the same or different and are preferably linear or branched C8-C24 alkylene or C8-C24 alkenylene, more preferably linear or branched C8. -C20 alkylene or C8-C20 alkenylene.
  • C1-C6 alkyl (CL-X) in X 117 and X 118, heterocyclyl or polyamines, halogen atom, R ', OR', SR 1 is selected ', CN, CO 2 R' or from CONR '2 It may be substituted with up to 3 substituents.
  • the monocyclic heterocycle is a halogen atom, R ′, OR ′, SR ′, CN, CO 2 R ′ or CONR ′. It may be substituted with 1 to 3 substituents selected from 2 .
  • R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.
  • R 120 and R 121 in formula (CL-X) are the same or different and are preferably linear or branched C4-C24 alkyl or C4-C24 alkenyl, more preferably linear or branched C4- C20 alkyl or C4-C20 alkenyl.
  • C4-C24 alkyl or C4-C24 alkenyl may be substituted with one or more substituents selected from a halogen atom, R ′, OR ′, SR ′, CN, CO 2 R ′ or CONR ′ 2.
  • R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.
  • R 124 and R 125 in formula (CL-XII) are the same or different and are preferably linear or branched C8-C24 alkyl or C8-C24 alkenyl, more preferably linear or branched C14. -C20 alkyl or C14-C20 alkenyl.
  • X 125 and X 126 C1-C6 alkyl, heterocyclyl or polyamine in the formula (CL-XIV) are selected from halogen atoms, R ′, OR ′, SR ′, CN, CO 2 R ′ or CONR ′ 2 It may be substituted with up to 3 substituents.
  • X 125 and X 126 in formula (CL-XIV) contain, in addition to the nitrogen to which they are attached, one or two additional heteroatoms selected from N, O and S.
  • the monocyclic heterocycle is a halogen atom, R ′, OR ′, SR ′, CN, CO 2 R ′ or CONR ′.
  • R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.
  • R 128 and R 129 in formula (CL-XIV) are the same or different and are preferably linear or branched C4-C24 alkyl or C4-C24 alkenyl, more preferably linear or branched C4. -C20 alkyl or C4-C20 alkenyl. C4-C24 alkyl or C4-C24 alkenyl may be substituted with one or more substituents selected from a halogen atom, R ′, OR ′, SR ′, CN, CO 2 R ′ or CONR ′ 2. .
  • R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.
  • R 130 in formula (CL-XIV) is a hydrogen atom or C1-C6 alkyl.
  • the cationic lipid in the present invention is preferably the formula (CL-I), (CL-II), (CL-III), (CL-IV), (CL-V), (CL-VIII), (CL- IX) is a cationic lipid.
  • cationic lipid used in the present invention are shown in Tables 1 to 7 below, but the cationic lipid of the present invention is not limited thereto.
  • the lipid represented by the formula (CL-I) can be obtained by the method described in International Publication No. 2013/089151, or a method analogous thereto.
  • the lipid represented by the formula (CL-II) can be obtained by the method described in International Publication No. 2011/136368 or a method analogous thereto.
  • the lipids represented by formula (CL-III), formula (CL-IV) and formula (CL-V) can be obtained by the method described in International Publication No. 2014/007398 or a method analogous thereto.
  • the lipid represented by the formula (CL-VI) can be obtained by the method described in International Publication No. 2010/042877 or a method analogous thereto.
  • the lipid represented by the formula (CL-VII) can be obtained by the method described in International Publication No. 2010/054401, or a method analogous thereto.
  • the lipid represented by the formula (CL-VIII) can be obtained by the method described in International Publication No. 2016/002753 or a method analogous thereto.
  • the lipid represented by the formula (CL-IX) can be obtained by the method described below or a method analogous thereto.
  • R 118 , R 119 , M 101 , M 102 , L 118 and L 119 have the same meanings as defined above, and X in IX-IIIa and IX-IIIb is the same or different, Represents a leaving group such as iodine atom, trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyloxy, p-toluenesulfonyloxy, R 135 is a hydrogen atom, methyl or ethyl, and R 136 is a hydrogen atom or methyl Or R 135 and R 136 together with adjacent carbons form a cyclopropyl ring (provided that when R 135 is a hydrogen atom or ethyl, R 136 is not methyl))
  • Step 26 and Step 27 Compound (IX-IIa) is obtained by reacting 2-amino-2-methyl-1,3-propanediol and compound (IX-IIIa) at room temperature in the presence of 1 to 10 equivalents of a base without solvent or in a solvent. It can be produced by reacting at a temperature between 200 ° C. for 5 minutes to 100 hours. Further, compound (CL-IXa) is obtained by reacting compound (IX-IIa) and compound (IX-IIIb) in the presence of 1 to 10 equivalents of base without solvent or in a solvent at a temperature between room temperature and 200 ° C. Thus, it can be produced by reacting for 5 minutes to 100 hours.
  • solvent examples include dichloromethane, 1,2-dichloroethane, toluene, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, pyridine and the like, and these may be used alone or in combination. it can.
  • Examples of the base include sodium methoxide, potassium tert-butoxide, sodium hydride, lithium diisopropylamide, hexamethyldisilazane lithium, hexamethyldisilazane sodium, n-butyllithium and the like.
  • Compound (IX-IIIa) and Compound (IX-IIIb) are commercially available products or known methods (for example, “5th edition Experimental Chemistry Course 13 Synthesis of Organic Compounds I”, 5th edition, p.374, Maruzen ( 2005)) or a method based thereon.
  • 2-Amino-2-methyl-1,3-propanediol can be obtained as a commercial product.
  • Step 28 Compound (CL-IXb) is obtained by mixing compound (CL-IXa) with 2 to 20 equivalents of compound (IX-IV), preferably 1 equivalent to a large excess of reducing agent and preferably 1 to 10 equivalents in a solvent. It can be produced by reacting at a temperature between ⁇ 20 ° C. and 150 ° C. for 5 minutes to 72 hours in the presence of an equivalent amount of acid.
  • solvent examples include methanol, ethanol, tert-butyl alcohol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N , N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, water and the like, and these may be used alone or in combination.
  • Examples of the reducing agent include sodium triacetoxyborohydride and sodium cyanoborohydride.
  • Examples of the acid include hydrochloric acid and acetic acid.
  • Compound (IX-IV) can be obtained as a commercial product.
  • R 118 , R 119 , M 101 , M 102 , L 118 , L 119 , R 135 , R 136 and X are as defined above, R 137 is as defined in X 115 , and PG is protected) Represents a group.
  • Step 29 Compound (IX-IIb) is compound (CL-IXa) that is commonly used in synthetic organic chemistry (e.g., Protective Groups in Organic Synthesis, third edition, Green (TWGreene ) By John Wiley & Sons Inc. (1999), etc.].
  • Process 30 Compound (IX-IIc) is obtained by reacting Compound (IX-IIb) and Compound (IX-IIIc) in the presence of 1 to 10 equivalents of a base without solvent or in a solvent at a temperature between ⁇ 20 ° C. and 150 ° C. Thus, it can be produced by reacting for 5 minutes to 72 hours.
  • solvent examples include dichloromethane, 1,2-dichloroethane, toluene, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, pyridine, N, N-dimethylformamide, N, N-dimethylacetamide and the like. These may be used alone or in admixture.
  • Examples of the base include sodium methoxide, potassium tert-butoxide, sodium hydride, lithium diisopropylamide, hexamethyldisilazane lithium, hexamethyldisilazane sodium, n-butyllithium, potassium carbonate, cesium carbonate, triethylamine and the like.
  • Compound (IX-IIIc) can be obtained as a commercial product.
  • Step 31 Compound (CL-IXc) can be obtained by removing protecting group PG of compound (IX-IIc) by an appropriate method.
  • Methods for removing protecting groups include those commonly used in organic synthetic chemistry (for example, Protective Groups in Organic Synthesis, third edition, TW Greene, John The removal method described in Wiley & Sons Inc. (1999), etc.] can be used, whereby the target compound can be produced.
  • Compound (CL-IXd) is obtained by combining compound (CL-IXc) with 1 to 10 equivalents of compound (IX-IV), preferably 1 equivalent to a large excess of a reducing agent and preferably 1 to 10 equivalents in a solvent. It can be produced by reacting at a temperature between ⁇ 20 ° C. and 150 ° C. for 5 minutes to 72 hours in the presence of an equivalent amount of acid.
  • Examples of the solvent, reducing agent, and acid include those exemplified in Step 28.
  • R 118 , R 119 , M 101 , M 102 , L 118 , L 119 , R 135 , R 136 , R 137 and PG are as defined above, and B and B ′ are linear or branched.
  • Step 33 Compound (IX-IId) can be produced by reacting compound (IX-IIc ′) and an oxidizing agent in a solvent at a temperature between ⁇ 20 ° C. and 150 ° C. for 5 minutes to 72 hours.
  • oxidizing agent examples include ozone, osmium tetroxide / sodium periodate, osmium tetroxide / lead tetraacetate, and the like.
  • Examples of the solvent include those exemplified in Step 28.
  • Compound (IX-IIc ′) can be produced by the method described in Production Method 2.
  • Step 34 Compound (IX-IIe) can be produced by reacting compound (IX-IId) and an oxidizing agent in a solvent at a temperature between ⁇ 20 ° C. and 150 ° C. for 5 minutes to 72 hours.
  • oxidizing agent examples include Jones reagent, pyridinium dichromate, ruthenium tetroxide, sodium chlorite and the like.
  • Solvents include tert-butyl alcohol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetone, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N- Examples thereof include dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, water and the like, and these can be used alone or in combination.
  • Step 35 and Step 36 Compound (IX-IIf) is obtained by reacting Compound (IX-IIe) and Compound (IX-Va) at room temperature and in the presence of 1 to 10 equivalents of a condensing agent and 1 to 10 equivalents of a base without solvent or in a solvent. It can be produced by reacting at a temperature between 0 ° C. and 5 minutes to 100 hours. Further, Compound (IX-IIc ′′) is obtained by combining Compound (IX-IIf) and Compound (IX-Vb) in the absence of solvent or in a solvent in the presence of 1 to 10 equivalents of condensing agent and 1 to 10 equivalents of base. The reaction can be carried out at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.
  • solvent examples include dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, pyridine and the like can be mentioned, and these can be used alone or in combination.
  • condensing agent examples include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N, N′-dicyclohexylcarbodiimide, 4- (4,6-dimethoxy-1,3,5-triazine-2- ⁇ ⁇ yl). ) -4-Methylmorpholinium chloride n hydrate, 1H-benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, O- (7-azabenzotriazol-1-yl) -N , N, N ′, N ′,-tetramethyluronium hexafluorophosphate and the like.
  • Examples of the base include potassium carbonate, cesium carbonate, triethylamine, N, N-diisopropylethylamine, N-methylmorpholine, pyridine and the like.
  • Compound (IX-Va) and compound (IX-Vb) can be obtained as commercial products.
  • the compound (IX-IIc ′′) in the case where R 118 and R 119 are the same can be obtained by using 2 equivalents or more of the compound (IX-Va) in Step 35.
  • Step 37 Compound (CL-IXc ′) is obtained by removing the protecting group PG of compound (IX-IIc ′′) by an appropriate method.
  • Methods for removing protecting groups include those commonly used in organic synthetic chemistry (for example, Protective Groups in Organic Synthesis, third edition, TW Greene, John The removal method described in Wiley & Sons Inc. (1999), etc.] can be used, whereby the target compound can be produced.
  • Step 38 Compound (CL-IXd ′) is obtained by combining compound (CL-IXc ′) with 1 to 10 equivalents of compound (IX-IV), preferably 1 equivalent to a large excess of reducing agent in a solvent and preferably 1 It can be produced by reacting at a temperature between ⁇ 20 ° C. and 150 ° C. for 5 minutes to 72 hours in the presence of ⁇ 10 equivalents of acid.
  • Examples of the solvent and the acid include those exemplified in Step 28.
  • compounds other than the compounds (CL-IXa) to (CL-IXd) can be obtained by adopting materials and reagents suitable for the structure of the target compound. It can be produced according to the production method or by applying a general production method commonly used in organic synthetic chemistry.
  • the intermediates and target compounds in each of the above production methods should be isolated and purified by subjecting them to separation and purification methods commonly used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, and various recrystallization chromatography. Can do.
  • the intermediate can be subjected to the next reaction without any particular purification.
  • X 115 and X 116 are the same or different and each represents a hydrogen atom or C1-C3 alkyl.
  • X 115 and X 116 are the same or different and are preferably a hydrogen atom, methyl, ethyl, or propyl, more preferably a hydrogen atom or methyl.
  • the combination of (X 115 , X 116 ) is preferably (hydrogen atom, hydrogen atom), (hydrogen atom, methyl), (methyl, methyl), more preferably (hydrogen atom, methyl), (methyl, Methyl).
  • L 118 and L 119 are the same or different and are linear or branched C8-C24 alkylene or C8-C24 alkenylene.
  • L 118 and L 119 are the same or different and are alkylene, they are preferably linear C8-C24 alkylene, more preferably linear C8-C20 alkylene, and even more preferably linear C8-C12 alkylene.
  • L 118 and L 119 are the same or different and are preferably octylene, nonylene, undecylene, tridecylene, pentadecylene, and more preferably octylene, nonylene, undecylene.
  • L 118 and L 119 are the same or different and are alkenylene, they are preferably linear C8-C24 alkenylene, more preferably linear C10-C20 alkenylene, and even more preferably linear C10-C12 alkenylene.
  • L 118 and L 119 are the same or different, preferably (Z) -undec-9-enylene, (Z) -tridec-11-enylene, (Z) -tetradec-9-enylene, (Z) -hexadeca-9 -Enylene, (Z) -octadeca-9-enylene, (Z) -octadeca-11-enylene, (9Z, 12Z) -octadeca-9,12-dienylene.
  • L 118 and L 119 are preferably the same.
  • the bond of each structure of M 101 and M 102 will be described by taking —OC (O) — as an example, which means that the structure is R 118 —OC (O) —L 118 .
  • M 101 and M 102 are preferably the same.
  • R 'in M 101 and M 102' and R '''the same or different is a hydrogen atom or a C1-C3 alkyl.
  • R ′′ and R ′ ′′ are preferably a hydrogen atom, methyl, ethyl or propyl, more preferably a hydrogen atom or methyl, and even more preferably a hydrogen atom.
  • R 118 and R 119 are the same or different and are linear or branched C1-C16 alkyl or C2-C16 alkenyl.
  • R 118 and R 119 are the same or different and are alkyl, they are preferably linear C1-C16 alkyl, more preferably linear C2-C9 alkyl.
  • R 118 and R 119 are the same or different and are preferably pentyl, octyl, nonyl, decyl, dodecyl.
  • R 118 and R 119 are the same or different and are alkenyl, they are preferably linear C 2 -C 16 alkenyl, more preferably linear C 3 -C 9 alkenyl.
  • R 118 and R 119 are the same or different, preferably (Z) -hept-2-ene, (Z) -oct-2-ene, (Z) -non-2-ene, (Z) -nona-3 -Ene, nona-8-ene, (Z) -dodec-2-ene, (Z) -tridec-2-ene.
  • R 118 and R 119 are preferably the same.
  • R 118 -M 101 -L 118 and R 119 -M 102 -L 119 are the same or different, and R 118 and R 119 , M 101 and M 102 , and L 118 and L 119 are from the structure described for each group. It may be a combination of R 118 -M 101 -L 118 and R 119 -M 102 -L 119 are preferably the same.
  • R 118 -M 101 -L 118 and R 119 -M 102 -L 119 are the same or different, preferably (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca -9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9 , 12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-dienyl and (Z) -docosa-13-enyl, more preferably The group consisting of (Z) -hexadec-9-enyl, (Z) -octadeca-9
  • n is an integer of 1-4
  • the lipid represented by the formula (CL-X) can be obtained by the method described in International Publication No. 2009/129385 or a method analogous thereto.
  • the lipid represented by the formula (CL-XI) can be obtained by the method described in International Publication No. 2013/1491401, or a method analogous thereto.
  • the lipid represented by the formula (CL-XII) can be obtained by the method described in International Publication No. 2009/129395 or a method analogous thereto.
  • the lipid represented by the formula (CL-XIII) can be obtained by the method described in International Publication No. 2013/059496 or a method analogous thereto.
  • the lipid represented by the formula (CL-XIV) can be obtained by the method described in International Publication No. 2011/149733 or a method analogous thereto.
  • the lipid represented by the formula (CL-XV) can be obtained by the method described in International Publication No. 2011/153493 or a method analogous thereto.
  • the lipid represented by the formula (CL-XVI) can be obtained by the method described in International Publication No. 2015/074085 or a method analogous thereto.
  • neutral lipids include phospholipids, glycerol lipids, sterols, glyceroglycolipids, glycosphingolipids, lipids containing water-soluble units, and sphingoids. These neutral lipids may be used alone or in combination of two or more.
  • the total number of molecules of neutral lipids is not particularly limited, but is preferably 0.05 times the molar amount or more relative to the total number of moles of lipids. Preferably it is 0.10 times mole amount or more, More preferably, it is 0.20 times mole amount or more, More preferably, it is 0.30 times mole amount or more.
  • the total number of neutral lipid molecules is not particularly limited, but is preferably 0.75 times or less, more preferably 0.70 times or less, more preferably 0.65 times or less, and even more preferably 0.60 times or less. It is.
  • PC phosphatidylcholine
  • EPC egg yolk phosphatidylcholine
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • glyceroglycolipid in the neutral lipid examples include, but are not limited to, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
  • glycosphingolipid in the neutral lipid examples include, but are not limited to, galactosyl cerebroside, lactosyl cerebroside, ganglioside, and the like.
  • Examples of the sphingoid in the neutral lipid include, but are not limited to, sphingan, icosasphingan, sphingosine, and derivatives thereof.
  • the derivative for example, —NH 2 such as sphingan, icosasphingan or sphingosine —NHCO (CH 2 ) xCH 3 (wherein x is an integer of 0 to 18, among which 6, 12 or 18 is preferable. However, it is not limited to these.
  • sterols in neutral lipids include cholesterol (Chol), dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucostosterol, or 3 ⁇ - [N- (N ′, N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like, but are not limited thereto.
  • the lipid containing a water-soluble unit is a lipid derivative or a fatty acid derivative of a water-soluble polymer.
  • the water-soluble polymer lipid derivative or fatty acid derivative include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, and polyglycerin. , Chitosan, polyvinyl pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol etc.
  • Examples thereof include salts formed by binding with fatty acids such as acid, myristic acid or lauric acid, salts thereof, and the like. More preferably, lipid derivatives or fatty acid derivatives such as polyethylene glycol or polyglycerin and the like. Cited et salts, more preferable example is a lipid derivative or fatty acid derivatives and salts thereof polyethylene glycol.
  • lipid derivatives or fatty acid derivatives of polyethylene glycol include polyethylene glycolated lipids [specifically, polyethylene glycol-phosphatidylethanolamine and polyethylene glycol-diacylglycerol (more specifically, 1,2-distearoyl-sn-glycero -3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)- 2000] (PEG-DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn -Glycerol, methoxypolyethylene glycol-2000 (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene
  • lipid derivative or fatty acid derivative of polyglycerin examples include polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine) or polyglycerin fatty acid esters, and more preferably, polyglycerinized lipids. Can be mentioned.
  • the total number of water-soluble polymer lipid derivatives and fatty acid derivative molecules in the nucleic acid-containing nanoparticles is not particularly limited, but is 0.005 times the molar amount of the total lipid or more.
  • the molar amount is preferably 0.01 to 0.30 times the molar amount, more preferably 0.02 to 0.25 times the molar amount, still more preferably 0.03 to 0.20 times the molar amount, and more preferably 0.04 to 0.15 times the molar amount. It is even more preferred that it is 0.04 to 0.12 times the molar amount.
  • the nanoparticles of the present invention may contain a polymer, for example, protein, albumin, dextran, polyfect, chitosan, dextran sulfate, such as poly-L-lysine, polyethyleneimine, polyaspartic acid.
  • a micelle comprising at least one polymer such as styrene maleic acid copolymer, isopropylacrylamide-acryl pyrrolidone copolymer, polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or a salt thereof.
  • a polymer for example, protein, albumin, dextran, polyfect, chitosan, dextran sulfate, such as poly-L-lysine, polyethyleneimine, polyaspartic acid.
  • a micelle comprising at least one polymer such as styrene maleic acid copolymer, isopropylacrylamide-acryl pyrroli
  • the polymer salt includes, for example, metal salts, ammonium group salts, acid addition salts, organic amine addition salts, amino acid addition salts, and the like.
  • the metal salt include, but are not limited to, alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt.
  • ammonium group salts include, but are not limited to, salts such as ammonium groups or tetramethylammonium groups.
  • the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate or phosphate, and organic acid salts such as acetate, maleate, fumarate or citrate. It is not limited to.
  • organic amine addition salts include, but are not limited to, addition salts such as morpholine or piperidine.
  • amino acid addition salts include, but are not limited to, addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid, or lysine.
  • nucleic acid-containing lipid nanoparticles of the present invention may contain, for example, a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant.
  • the neutral lipid in the present invention includes the above lipid derivatives and fatty acid derivatives.
  • a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant is a part of the molecule and other components of the composition, such as hydrophobic affinity, static It is a substance with a two-sided property that has the property of binding by electrical interaction, etc., and the other part has the property of binding to the solvent at the time of production of the composition, for example, hydrophilic affinity, electrostatic interaction, etc. Preferably there is.
  • lipid derivatives or fatty acid derivatives of sugars, peptides or nucleic acids include sugars such as sucrose, sorbitol, and lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides, peptides such as glutathione, or, for example, DNA, Nucleic acids such as RNA, plasmid, siRNA, ODN and the like and neutral lipids listed in the definition of the composition or fatty acids such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. Is mentioned.
  • sugar lipid derivative or fatty acid derivative examples include glyceroglycolipid and glycosphingolipid mentioned in the definition of the composition.
  • surfactant examples include polyoxyethylene sorbitan monooleate (specifically polysorbate 80 etc.), polyoxyethylene polyoxypropylene glycol (specifically Pluronic F68 etc.), sorbitan fatty acid ester (specifically Sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid ester or polyethylene glycol alkyl ether, etc.
  • polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester, polyethylene glycol alkyl ether or the like is used.
  • cationic lipid in addition to the above-described cationic lipid (lipid II), another cationic lipid may be used.
  • lipid II and another cationic lipid are combined and simply referred to as a cationic lipid.
  • Examples of cationic lipids other than lipid II include N- [1- (2,3-dioleyloxy) propyl] disclosed in JP-A-61-161246 (US Pat. No. 5,049,386).
  • DOTMA N-trimethylammonium chloride
  • DORIE Dimethyl-N-hydroxyethylammonium bromide
  • DOSPA 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid
  • Etc. disclosed in International Publication No.
  • One embodiment of the present invention is a lipid nanoparticle containing a lipid (lipid I) having a ligand capable of binding to Siglec and containing a nucleic acid, for example, a lipid composed of lipid I, a cationic lipid, and a nucleic acid A nanoparticle; a lipid nanoparticle containing a complex of a lipid I and a cationic lipid and a neutral lipid and a nucleic acid; a lipid nanoparticle containing the complex and a lipid membrane containing lipid I; And the like, and the like. Lipid nanoparticles containing the complex in lipid nanoparticles containing a lipid membrane.
  • the complex examples include a complex of a membrane composed of a nucleic acid and a lipid bilayer, a complex of a nucleic acid and a liposome, a complex of a nucleic acid and a micelle, and preferably a complex of a nucleic acid and a micelle or Examples include a complex of a nucleic acid and a liposome.
  • the lipid membrane may be a lipid single membrane (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane).
  • the lipid membrane may contain a cationic lipid or a neutral lipid.
  • Examples of the form of the complex include a complex of a nucleic acid and a membrane composed of a single lipid (single molecule) layer (reverse micelle), a complex of a nucleic acid and a liposome, a complex of a nucleic acid and a micelle, and the like.
  • a complex of a nucleic acid and a membrane composed of a lipid monolayer or a complex of a nucleic acid and a liposome can be mentioned.
  • Examples of the lipid nanoparticle containing the complex and the lipid membrane that encapsulates the complex include liposomes that encapsulate the complex and the complex with a lipid bilayer.
  • one or more kinds of cationic lipids may be mixed and used.
  • the lipid nanoparticles of the present invention can contain nucleic acids, but can also contain compounds that are chemically similar to nucleic acids (for example, peptide nucleic acids).
  • the total lipid is the total of lipid I and lipids other than lipid I.
  • nucleic acid-containing nanoparticles of the present invention can be optionally subjected to surface modification with a water-soluble polymer, for example [Radasic, edited by F. Martin, “Stealth Liposomes”. (Stealth Liposomes) ”(USA), CRC Press Inc, 1995, p.93-102].
  • a water-soluble polymer for example [Radasic, edited by F. Martin, “Stealth Liposomes”. (Stealth Liposomes) ”(USA), CRC Press Inc, 1995, p.93-102].
  • examples include glycerin, chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol, etc., preferably polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, etc.
  • polyethylene glycol, polyglycerin and the like can be mentioned, but not limited thereto.
  • a lipid derivative or fatty acid derivative (as defined above) of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant or the like can be used.
  • the surface modification is one of methods in which the lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant is contained in the nucleic acid-containing lipid nanoparticles of the present invention.
  • the polymer II is a polymer including a water-soluble unit and a cationic unit.
  • the water-soluble unit and the cationic unit may be bonded via a linker, that is, can be represented by the following structural formula.
  • water-soluble units examples include polyethylene glycol units, polyglycerin units, polyethyleneimine units, polyvinyl alcohol units, polyacrylic acid units, polyacrylamide units, oligosaccharide units, dextrin units, water-soluble cellulose units, dextran units, and chondroitin sulfate.
  • water-soluble units examples include units, polyglycerin units, chitosan units, polyvinylpyrrolidone units, polyaspartic acid amide units, poly-L-lysine units, mannan units, pullulan units, oligoglycerol units and the like.
  • a polyethylene glycol unit is preferable.
  • the number average molecular weight of the water-soluble unit is not particularly limited, but is preferably 100 to 5000, and more preferably 500 to 3000.
  • the cationic unit in polymer II is preferably an amino acid polymer unit containing at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit.
  • the amino acid polymer unit containing one or more selected from the group consisting of lysine, arginine and histidine can be obtained by polymerization of an amino acid containing one or more selected from the group consisting of lysine, arginine and histidine.
  • the amino acid polymer unit is composed of two or more amino acids, it may be a random copolymer or a block copolymer. Examples of the amino acid polymer unit include the following structures.
  • n1 is an integer of 2 or more.
  • N1 is preferably 2 to 100, more preferably 5 to 50.
  • the polyethyleneimine unit is a unit made of a polymer obtained by polymerizing ethyleneimine.
  • the polyethyleneimine unit can be represented, for example, as shown in the main chain structure below, but the polyethyleneimine main chain may be cross-linked with other polyethyleneimine structures.
  • n2 is an integer of 1 or more.
  • N2 is preferably 2 to 100, more preferably 5 to 50.
  • the polyaminoacrylate unit is a unit made of a polymer obtained by polymerizing methacrylic acid having an amino group such as 2- (dimethylamino) ethyl methacrylate.
  • the polyaminoacrylate unit for example, the following structures are preferably exemplified.
  • n3 is an integer of 1 or more.
  • the linker in polymer II is not particularly limited as long as it can link a water-soluble unit and a cationic unit, but a linker suitably used for lipid I and polymer I is also suitable for a linker in polymer II. is there.
  • the nucleic acid used in the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides and / or molecules having functions equivalent to nucleotides, for example, ribonucleic acid that is a polymer of ribonucleotides.
  • RNA deoxyribonucleic acid
  • DNA deoxyribonucleic acid
  • chimeric nucleic acids composed of RNA and DNA and nucleotides in which at least one nucleotide of these nucleic acids is replaced with a molecule having a function equivalent to that nucleotide A polymer etc. are mentioned.
  • the nucleic acid of the present invention also includes a derivative containing at least a part of the structure of a molecule obtained by polymerizing nucleotides and / or molecules having functions equivalent to nucleotides.
  • uracil U and thymine T can be replaced with each other.
  • nucleotide derivatives examples include nucleotide derivatives.
  • the nucleotide derivative may be any molecule as long as it is a modified nucleotide, for example, it improves nuclease resistance or stabilizes from other degradation factors compared to RNA or DNA.
  • a molecule in which ribonucleotides or deoxyribonucleotides are modified is preferably used.
  • nucleotide derivatives include sugar moiety-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and the like.
  • the sugar moiety-modified nucleotide may be any nucleotide as long as it is modified or substituted with an arbitrary substituent on a part or all of the sugar sugar chemical structure, or substituted with an arbitrary atom. 2'-modified nucleotides are preferably used.
  • Examples of the modifying group in the sugar moiety-modified nucleotide include 2′-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-alkenyl, 2′-substituted alkenyl, 2′-halogen and 2′-O-cyano.
  • sugar-modified nucleotides include, for example, a crosslinked structure-type artificial nucleic acid (BNA) having a structure in which a modification group at the 2 ′ position is crosslinked to a carbon atom at the 4 ′ position, more specifically, the 2 ′ position.
  • BNA crosslinked structure-type artificial nucleic acid
  • LNA Locked Nucleic Acid
  • ENA Ethylene bridged nucleic acid
  • PNA Peptide nucleic acid
  • OPNA oxypeptide nucleic acid
  • PRNA peptide ribonucleic acid
  • alkyl 2′-O-alkenyl, 2′-O-substituted alkenyl, 2′-Se-alkyl or 2′-Se-substituted alkyl, 2′-cyano, 2′-fluoro, 2′-chloro, 2'-bromo, 2'-trifluoromethyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-isopropyl, 2'-O-trifluoromethyl, 2'-O- [2- (Methoxy) ethyl], 2'-O- (3-aminopropyl), 2'-O- [2- (N, N-dimethylaminooxy
  • the preferred range of the modifying group in the sugar moiety-modified nucleotide can also be defined from its size, preferably from the size of fluoro to the size of -O-butyl, and from the size of -O-methyl- Those corresponding to the size of O-ethyl are more preferred.
  • alkyl in the modifying group in the sugar-modified nucleotide examples include C1-C6 alkyl, and more specifically, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl. And C1-C6 alkyl such as neopentyl or hexyl.
  • alkenyl in the modifying group in the sugar moiety-modified nucleotide examples include C3-C6 alkenyl, and more specifically, C3-C6 alkenyl such as allyl, 1-propenyl, butenyl, pentenyl, hexenyl and the like.
  • halogen in the modifying group in the sugar moiety-modified nucleotide examples include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
  • amino acids in amino acid residues include aliphatic amino acids (specifically, glycine, alanine, valine, leucine, isoleucine, etc.), hydroxy amino acids (specifically, serine, threonine, etc.), acidic amino acids (specifically, Aspartic acid, glutamic acid, etc.), acidic amino acid amides (specifically, asparagine, glutamine, etc.), basic amino acids (specifically, lysine, hydroxylysine, arginine, ornithine, etc.), sulfur-containing amino acids (specifically, Specifically, cysteine, cystine, methionine and the like) or imino acid (specifically, proline, 4-hydroxyproline and the like) and the like.
  • aliphatic amino acids specifically, glycine, alanine, valine, leucine, isoleucine, etc.
  • hydroxy amino acids specifically, serine, threonine, etc.
  • acidic amino acids specifically, Aspart
  • Examples of the substituted alkyl in the modified group in the sugar-modified nucleotide and the substituent in the substituted alkenyl include halogen (as defined above), hydroxy, sulfanyl, amino, oxo, -O-alkyl (the alkyl portion of the -O-alkyl is The same as C1-C6 alkyl in the modifying group), -S-alkyl (the alkyl part of the -S-alkyl is synonymous with C1-C6 alkyl in the modifying group), -NH-alkyl (the alkyl of -NH-alkyl) Part is synonymous with C1-C6 alkyl in the modifying group), dialkylaminooxy (the two alkyl parts of the dialkylaminooxy are the same or different and are synonymous with C1-C6 alkyl in the modifying group), dialkylamino (the dialkylamino The two alkyl moieties are the same or different and have the same meaning
  • the phosphodiester bond-modified nucleotide is any nucleotide that has been modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the phosphodiester bond of the nucleotide, or with any atom.
  • a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond
  • Examples include nucleotides in which a diester bond is substituted with a phosphoramidate bond.
  • any or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom may be used.
  • oxygen atom is substituted with sulfur atom
  • hydrogen atom is substituted with C1-C6 alkyl group
  • methyl group is substituted with hydrogen atom or C2-C6 alkyl group
  • amino group is C1-C6 Examples include those protected with a protecting group such as an alkyl group and a C1-C6 alkanoyl group.
  • nucleotide derivatives nucleotides or sugar moieties
  • nucleotide derivatives modified with at least one of phosphodiester bonds or bases lipids, phospholipids, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and Examples include dyes and other chemical substances added.
  • 5'-polyamine-added nucleotide derivatives examples include fluorescent dye (Cy3) addition nucleotide derivatives, red fluorescent dye (Cy5) addition nucleotide derivatives, fluorescein (6-FAM) addition nucleotide derivatives, and biotin addition nucleotide derivatives.
  • a nucleotide or a nucleotide derivative is an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, or a structure combining at least two of these with other nucleotides or nucleotide derivatives in the nucleic acid.
  • a cross-linked structure such as
  • the nucleic acid used in the present invention preferably has a molecular weight of 1,000 kDa or less, more preferably 100 kDa or less, and even more preferably 30 kDa or less.
  • the nucleic acid used in the present invention preferably includes a nucleic acid that supplements a target gene expressed in normal cells and a nucleic acid that suppresses the expression of the target gene, and more preferably RNA interference ( Examples include nucleic acids having an inhibitory effect on target gene expression using RNAi).
  • the nucleic acid used in the present invention may be mRNA.
  • mRNA refers to mRNA transcribed from template DNA, and the encoded protein (including peptide) is not particularly limited.
  • the mRNA may encode a glycoprotein or a fusion protein.
  • the number of bases of mRNA is not particularly limited.
  • the mRNA may be a homologous sequence capable of synthesizing the target protein, and may have a plurality of bases deleted, substituted, inserted or added.
  • the nucleic acid in the nanoparticles of the present invention is introduced into the cell.
  • Such cells are preferably mammalian Siglec-1 (CD169) positive cells.
  • Siglec-1 (CD169) positive cells include macrophages, dendritic cells or monocytes.
  • the target gene in the present invention may be appropriately selected according to the type of disease intended for treatment, for example, a gene capable of repairing or correcting a defect causing the disease that is the purpose of treatment, and the cause of the disease. And genes that suppress the expression of factors and receptors.
  • the disease include diseases in which Siglec-1 (CD169) -positive cells are seen, and diseases involving macrophages, dendritic cells or monocytes.
  • nucleic acid for example, a nucleic acid containing a base sequence complementary to a partial base sequence of mRNA of a gene encoding a protein (target gene) and suppressing the expression of the target gene
  • Any nucleic acid may be used, such as double-stranded nucleic acid such as siRNA (short interference RNA) and miRNA (micro RNA), single-stranded nucleic acid such as shRNA (short hairpin RNA), antisense nucleic acid, and ribozyme. Double-stranded nucleic acids are preferred.
  • a nucleic acid containing a base sequence complementary to a part of the base sequence of the target gene mRNA is called an antisense strand nucleic acid
  • a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is a sense strand.
  • a sense strand nucleic acid refers to a nucleic acid capable of forming a double strand forming part by pairing with an antisense strand nucleic acid, such as a nucleic acid itself consisting of a partial base sequence of a target gene.
  • a double-stranded nucleic acid refers to a nucleic acid in which two strands are paired and have a duplex forming part.
  • the double-stranded forming part refers to a part where nucleotides constituting the double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand.
  • the base pair constituting the duplex forming part is usually 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, further preferably 15 to 21 base pairs, and 15 to 19 base pairs. Base pairs are particularly preferred.
  • the antisense strand nucleic acid of the duplex forming part for example, a nucleic acid comprising a partial sequence of the mRNA of the target gene, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in the nucleic acid is substituted.
  • a nucleic acid that is deleted or added and has the activity of suppressing the expression of the target protein is preferably used.
  • the single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of a series of 15 to 30 bases (nucleosides), preferably 15 to 29 bases, more preferably 15 to 27 bases, and further preferably 15 to 25 bases. 17 to 23 bases are particularly preferred, and 19 to 21 bases are most preferred.
  • the antisense strand, the sense strand, or both of the nucleic acids constituting the double-stranded nucleic acid have an additional nucleic acid that does not form a duplex on the 3 ′ side or 5 ′ side following the duplex forming part. May be.
  • the part that does not form a double chain is also referred to as a protrusion (overhang).
  • the double-stranded nucleic acid having an overhang for example, one having an overhang of 1 to 4 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. Those having a protruding portion made of a base are preferably used, and those having a protruding portion made of dTdT or UU are more preferably used.
  • the overhang can have only the antisense strand, only the sense strand, and both the antisense strand and the sense strand, but a double-stranded nucleic acid having an overhang on both the antisense strand and the sense strand is preferably used. .
  • Sequence that matches part or all of the base sequence of the target gene mRNA following the duplex formation part, or part or all of the base sequence of the complementary strand of the target gene mRNA following the duplex formation part Sequences that match may be used.
  • a nucleic acid that suppresses the expression of a target gene for example, a nucleic acid molecule that generates the above double-stranded nucleic acid by the action of a ribonuclease such as Dicer (International Publication No. 2005/089287), a 3 ′ end or a 5 ′
  • a double-stranded nucleic acid or the like that does not have a terminal protruding portion can also be used.
  • the antisense strand has a sequence of at least the 1st to 17th bases (nucleosides) from the 5 ′ end to the 3 ′ end, and the mRNA of the target gene More preferably, the antisense strand has a sequence of bases 1 to 19 from the 5 ′ end to the 3 ′ end, The base sequence is complementary to the 19-base sequence of the gene mRNA, or the base sequence 1 to 21 is the base sequence complementary to the 21-base sequence of the target gene mRNA.
  • the sequence of the 1st to 25th bases is a sequence of bases complementary to the sequence of 25 consecutive bases of the mRNA of the target gene.
  • ribose substituted with a modifying group at the 2'-position is preferably included.
  • ribose substituted with a modifying group at the 2′-position means that the hydroxyl at the 2′-position of ribose is substituted with the modifying group, and the configuration is the same as the hydroxy at the 2′-position of ribose. Although it may be present or different, the configuration is preferably the same as that of the 2′-hydroxy of ribose.
  • Examples of the modifying group in ribose substituted with a modifying group at the 2′-position include those exemplified in the definition of the modifying group in the 2′-modified nucleotide in the sugar moiety-modified nucleotide and the hydrogen atom, such as 2′-cyano, 2 ′ -Halogen, 2'-O-cyano, 2'-alkyl, 2'-substituted alkyl, 2'-O-alkyl, 2'-O-substituted alkyl, 2'-O-alkenyl, 2'-O-substituted alkenyl , 2'-Se-alkyl, 2'-Se-substituted alkyl, etc.
  • 2'-cyano, 2'-fluoro, 2'-chloro, 2'-bromo, 2'-trifluoromethyl, 2'-O -Methyl, 2'-O-ethyl, 2'-O-isopropyl, 2'-O-trifluoromethyl, 2'-O- [2- (methoxy) ethyl], 2'-O- (3-aminopropyl ), 2'-O- [2- (N, N-dimethyl) aminooxy] ethyl, 2'-O- [3- (N, N-dimethylamino) propyl], 2'-O- ⁇ 2- [ 2- (N, N-dimethylamino) ethoxy] ethyl ⁇ , 2'-O- [2- (methyl Amino) -2-oxoethyl], 2'-Se-methyl, hydrogen atom and the like are more preferable, 2'-O-methyl, 2'-O-ethyl
  • the nucleic acid used in the present invention includes a derivative in which an oxygen atom or the like contained in a phosphoric acid part, an ester part or the like in the structure of the nucleic acid is substituted with another atom such as a sulfur atom.
  • the sugar that binds to the 5 'terminal base of the antisense strand and the sense strand has a 5'-positioned hydroxy group, either a phosphate group or the above-mentioned modifying group, or an in vivo nucleolytic enzyme, etc. It may be modified by a group that is converted to a modifying group.
  • the sugar that binds to the base at the 3 ′ end of the antisense strand and the sense strand is such that the 3′-position hydroxy is a phosphate group or the above-mentioned modifying group, or an in vivo nucleolytic enzyme, etc. It may be modified by a group that is converted to a modifying group.
  • the single-stranded nucleic acid for example, 15 to 27 bases (nucleoside) of the target gene, preferably 15 to 25 bases, more preferably 15 to 23 bases, still more preferably 15 to 21 bases, particularly preferably 15 A nucleic acid comprising a sequence complementary to a sequence consisting of ⁇ 19 bases, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base is substituted, deleted or added in the nucleic acid, and the target protein expression inhibitory activity Any nucleic acid may be used as long as it has a nucleic acid.
  • the single-stranded nucleic acid is preferably composed of a series of 10 to 30 bases (nucleosides), more preferably 10 to 27 bases, further preferably 10 to 25 bases, particularly preferably 10 to 23 bases. Nucleic acids are preferably used.
  • the single-stranded nucleic acid one obtained by linking the antisense strand and the sense strand constituting the double-stranded nucleic acid via a spacer sequence (spacer oligonucleotide) may be used.
  • the spacer oligonucleotide is preferably a 6- to 12-base single-stranded nucleic acid molecule, and the sequence at the 5 'end is preferably 2 Us.
  • An example of a spacer oligonucleotide is a nucleic acid having the sequence UUCAAGAGA. Either the antisense strand or the sense strand connected by the spacer oligonucleotide may be on the 5 'side.
  • the single-stranded nucleic acid is preferably, for example, a single-stranded nucleic acid such as shRNA having a duplex forming part by a stem-loop structure.
  • Single-stranded nucleic acids such as shRNA are usually 50 to 70 bases in length.
  • a nucleic acid having a length of 70 bases or less, preferably 50 bases or less, more preferably 30 bases or less, designed to produce the above single-stranded nucleic acid or double-stranded nucleic acid by the action of ribonuclease or the like may be used. Good.
  • the nucleic acid used in the present invention can be obtained using a known RNA or DNA synthesis method and RNA or DNA modification method.
  • nucleic acid-containing lipid nanoparticles of the present invention can contain not only nucleic acids but also compounds that are chemically similar to nucleic acids (anionic polymers such as anionic peptides).
  • the lipid nanoparticles of the present invention can be produced by a known production method or a method similar thereto, and may be produced by any production method.
  • a known method for preparing liposomes can be applied to the production of a composition containing liposome, which is one of the compositions.
  • Known liposome preparation methods include, for example, Bangham et al.'S liposome preparation method [“J. Mol. Biol.”, 1965, Vol. 13, p.238- 252], ethanol injection method [“J. Cell Biol.”, 1975, Vol. 66, pp.
  • liposomes for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, or the like can be used.
  • an antioxidant such as citric acid, ascorbic acid, cysteine or ethylenediaminetetraacetic acid (EDTA), for example, an isotonic agent such as glycerin, glucose or sodium chloride can be added.
  • EDTA ethylenediaminetetraacetic acid
  • isotonic agent such as glycerin, glucose or sodium chloride
  • Liposomes can also be produced by dissolving lipids or the like in an organic solvent such as ethanol and distilling off the solvent, and then adding physiological saline or the like and stirring to form liposomes.
  • the lipid nanoparticles of the present invention may be prepared, for example, by dissolving a cationic lipid in chloroform in advance, and then adding an aqueous solution of nucleic acid and methanol and mixing to form a complex of cationic lipid / nucleic acid. A layer is taken out, and lipid I, polyethylene glycolated phospholipid, neutral lipid, and water are added to form a water-in-oil (W / O) emulsion and processed by the reverse phase evaporation method (special method).
  • W / O water-in-oil
  • nucleic acid is dissolved in an acidic aqueous electrolyte solution.
  • an acidic aqueous electrolyte solution for example, a mixture of lipid I and cationic lipid or lipid I, cationic lipid and neutral lipid (in ethanol) is added.
  • concentration of the encapsulated lipid nanoparticles such as the nucleic acid is reduced to 20 v / v%, sizing filtration, and excess ethanol is removed by dialysis.
  • grain A method for removing nucleic acid attached to the surface of a child Japanese Patent Publication No. 2002-501511 and “Biochimica et Biophysica Acta”, 2001, 1510, p.152- 166) and the like.
  • lipid nanoparticles of the present invention a lipid nanoparticle containing a complex of lipid I and a combination of cationic lipid and / or neutral lipid and a nucleic acid or the like or a complex of cationic lipid and nucleic acid is encapsulated
  • Lipid nanoparticles composed of lipid membranes containing lipid I can be produced, for example, according to the production methods described in WO02 / 28367 and WO2006 / 080118.
  • lipid nanoparticles of the present invention for example, a complex of lipid I, a cationic lipid, and a nucleic acid, or a combination of a lipid I, a cationic lipid, a neutral lipid, and a nucleic acid, or a cationic Lipid nanoparticles encapsulating lipid / nucleic acid complexes with lipid membranes containing lipid I and / or cationic lipids and neutral lipids are described in WO 02/28367 and WO 2006/080118. Each composite can be manufactured according to the manufacturing method.
  • a cationic lipid and a nucleic acid are mixed in water or a 0 to 40% ethanol aqueous solution, and the complex containing the cationic lipid and the nucleic acid is dispersed without dissolving (the first lipid solution).
  • One or more kinds of cationic lipids may be used as the cationic lipid in the first lipid solution and the second lipid solution.
  • a complex of lipid I, a cationic lipid and a nucleic acid a complex of lipid I and a cationic lipid in combination with a neutral lipid and a nucleic acid, or a complex of a cationic lipid and a nucleic acid
  • lipid nanoparticles encapsulated with lipid membranes containing lipid I and / or cationic lipids and neutral lipids and electrostatic interaction between the nucleic acid in the complex and the cationic lipid in the lipid membrane
  • the lipid nanoparticles of the present invention also include those in which the structure of the complex and the membrane is mutated due to the action or fusion of the cationic lipid in the complex and the cationic lipid in the lipid membrane.
  • the nucleic acid-containing lipid nanoparticle of the present invention contains lipid II
  • it can be produced, for example, by a production method including the following steps (a) to (c).
  • a step of preparing (c) a step of mixing the first lipid solution and the second lipid solution and further adding water or an aqueous buffer solution
  • the method for producing the nucleic acid-containing lipid nanoparticles of the present invention include the following methods. Production of a complex of a nucleic acid (as defined above), preferably a double-stranded nucleic acid and a liposome containing a cationic lipid, according to the production method described in WO 02/28367 and WO 2006/080118 Then, the complex is dispersed in water or an aqueous solution of 0 to 40% ethanol without dissolving it (first lipid solution), and a lipid containing a water-soluble unit having a ligand capable of binding to Siglec separately (lipid I) Cationic lipid and neutral lipid are dissolved in an aqueous ethanol solution (second lipid solution), and the first lipid solution and the second lipid in an equal amount or volume ratio of 1: 1 to 10: 1 Lipid nanoparticles containing the nucleic acid and lipid I can also be obtained by mixing the solution or adding water as appropriate.
  • a nucleic acid as
  • the lipid nanoparticle is preferably a composition containing a lipid membrane containing lipid I encapsulating a complex of lipid I, a cationic lipid and a nucleic acid and a complex of a cationic lipid and a nucleic acid, or the nucleic acid And a lipid membrane containing lipid I encapsulating the complex (reverse micelle) composed of a lipid monolayer containing the cationic lipid.
  • the lipid membrane in these cases may be a lipid monolayer (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane).
  • the former may be added to the latter, or the latter may be added to the former. Moreover, you may add the former and the latter simultaneously to a container, stirring. Furthermore, the former and the latter can be mixed in-line. In this case, for example, a T-connector or the like can be used as the in-line mixing device.
  • the liposome in the complex of the nucleic acid of the present disclosure and the liposome is preferably a liposome whose size is adjusted in advance to an average particle size of 10 nm to 400 nm, more preferably 20 nm to 110 nm, and further preferably 30 nm to 80 nm.
  • the complex and / or lipid membrane may contain a neutral lipid and / or a polymer.
  • the first lipid solution may have an ethanol concentration of 0 to 70% as long as a complex of liposomes and the nucleic acid can be formed.
  • the complex does not dissolve after mixing the first lipid solution and the second lipid solution
  • the second lipid solution You may mix by the ratio used as the ethanol density
  • the first lipid solution and the second lipid solution are in such a ratio that the complex does not dissolve, the lipid in the second lipid solution does not dissolve, and the ethanol concentration becomes an ethanol aqueous solution of 10 to 60%.
  • the first lipid solution and the second lipid may be mixed in a ratio that results in an ethanol concentration that does not dissolve the complex after mixing the first lipid solution and the second lipid solution.
  • the solution may be mixed and water may be added to obtain an ethanol concentration at which the lipid in the second lipid solution is not dissolved.
  • the complex of the nucleic acid and the liposome in the first lipid solution of the present disclosure is prepared by mixing the first lipid solution and the second lipid solution, and further adding water appropriately, and then adding the cationic lipid.
  • the form may be changed to a complex of a membrane (reverse micelle) composed of a lipid monolayer and a nucleic acid.
  • the composition containing lipid I, the nucleic acid and the cationic lipid obtained by the production method of the present disclosure is preferably a composition containing a complex of a cationic lipid and a nucleic acid and a lipid membrane encapsulating the complex Or a complex of a lipid monolayer containing a cationic lipid (reverse micelle) and a nucleic acid, and a lipid membrane encapsulating the complex, and the lipid membrane contains lipid I and a cationic lipid.
  • the average particle size of the complex in the lipid nanoparticle of the present invention or the lipid membrane encapsulating the complex can be freely selected as desired, but the average particle size described below is preferable.
  • a method for adjusting the average particle size for example, an extrusion method, a method of mechanically crushing large multilamellar liposomes (MLV) or the like (specifically using a manton gourin, a microfluidizer, etc.) [Müller (RH Muller, S. Benita, B. Bohm, “Emulsion and Nanosuspensions for the Formulation of Poorly Soluble Drugs) ”, Scientific Publishers Stuttgart, 1998, p.267-294].
  • the nucleic acid in step (a) of the production method of the present invention may be the nucleic acid itself, or may be dissolved in distilled water in advance to form a nucleic acid aqueous solution.
  • the nucleic acid concentration in the aqueous solution is preferably 0.1 to 1000 mg / mL, more preferably 1 to 500 mg / mL, and further preferably 10 to 100 mg / mL. .
  • the nucleic acid concentration in the first solution may be appropriately adjusted depending on the type and molecular weight of the nucleic acid, but is usually 0.01 to 150 mg / mL, preferably 0.05 to 30 mg / mL, more preferably 0.3 to 10 mg / mL. It is.
  • the concentration of the cationic lipid (lipid II) in the first solution may be appropriately adjusted according to the type of nucleic acid, molecular weight, and amount used, but is usually 0.01 to 400 mmol / L, preferably 0.2 to 50 mmol. / L, more preferably 1 to 20 mmol / L.
  • the concentration of lipid I in the second solution may be appropriately adjusted according to the target modification rate by lipid I in the nanoparticles, but is usually 0.00001 to 6 mmol / L, preferably 0.02 to 4 mmol / L, and more Preferably it is 0.05-3 mmol / L.
  • the concentration of the cationic lipid (lipid II) in the second solution may be appropriately adjusted according to the type of nucleic acid, molecular weight, and amount used, but is usually 0.01 to 100 mmol / L, preferably 0.1 to 80 mmol. / L, more preferably 0.8 to 50 mmol / L.
  • the lipid may be one or more, preferably a neutral lipid, usually 0.01 to 100 mmol / L, preferably 0.1 to 80 mmol / L, more preferably 0.8 to 50 mmol / L.
  • the temperature at which the organic solvent solution containing the nucleic acid and lipid is prepared is not particularly limited as long as the nucleic acid and lipid are dissolved, but is preferably 10 to 60 ° C, more preferably 20 to 50 ° C, and further preferably 20 to 30 ° C. preferable. In addition, when heating at 30 degreeC or more, the solubility of a nucleic acid and a lipid increases and a lipid nanoparticle can be manufactured with a smaller solvent amount.
  • the water-miscible organic solvent is preferably alcohol, dimethyl sulfoxide, tetrahydrofuran, acetone, acetonitrile or a mixture thereof, and more preferably an alcohol.
  • the alcohol is preferably methanol, ethanol, propanol, butanol or a mixture thereof.
  • C1-C6 alcohols such as methanol, ethanol, propanol, butanol and the like containing 0 to 50% (v / v) water or a mixture thereof are preferable.
  • Ethanol or propanol containing 0 to 50% (v / v) water is more preferred, and ethanol containing 0 to 50% (v / v) water is more preferred.
  • % (v / v) indicates the volume percentage of the solute in the total volume of the solution, and so on.
  • an inorganic acid such as hydrochloric acid, acetic acid, phosphoric acid, or a salt of these acids can be added to the solvent in the organic solvent solution containing nucleic acid and lipid.
  • the pH of the solvent is preferably 1 to 7, more preferably 1 to 5, and further preferably 2 to 4.
  • the volume of water or aqueous buffer solution used is not particularly limited, but is 0.5% relative to the volume of the organic solvent solution of nucleic acid and lipid. ⁇ 100 times is preferable, 1.5 to 20 times is more preferable, and 2.0 to 10 times is more preferable.
  • the concentration of the organic solvent after adding water or an aqueous buffer solution is not particularly limited, but is preferably 50% (v / v) or less, more preferably 40% (v / v) or less with respect to the obtained solution. Preferably, it is more preferably 30% (v / v) or less, and most preferably 20% / (v / v) or less.
  • the aqueous buffer solution is not particularly limited as long as it has a buffering action, and examples thereof include a phosphate buffer aqueous solution, a citrate buffer aqueous solution, and an acetate buffer aqueous solution.
  • the temperature at the time of the above addition operation is not particularly limited, but is preferably 10 to 60 ° C, more preferably 20 to 50 ° C, and further preferably 20 to 30 ° C.
  • the organic solvent concentration from 70% 70 (v / v) to 50% (v / v) within 1 minute, more preferably within 0.5 minutes, 0.1% More preferably, it is changed within minutes, and most preferably within 0.05 minutes.
  • the nucleic acid-containing lipid nanoparticle of the present invention does not contain a cationic lipid (lipid II), it can be produced, for example, by a production method including the following steps (a ′) to (c ′).
  • a ' Aqueous solution A is prepared by mixing water and a lipid having a ligand capable of binding to nucleic acid and siglec (Sialic acid-binding immunoglobulin-like lectin) or a lipid containing a water-soluble unit (lipid I).
  • Process (b ′) preparing a lipid solution B of an organic solvent miscible with water of other lipids; and (c ′) contacting the aqueous solution A with the lipid solution B
  • the ratio of the number of moles of total lipid to the number of moles of nucleic acid is preferably 50 or more, and preferably 100 to 1,000. More preferably, it is more preferably from 120 to 800, even more preferably from 140 to 600, and most preferably from 200 to 500.
  • the average particle size of the nucleic acid-containing nanoparticles of the present invention can be further adjusted after the preparation of lipid nanoparticles.
  • a method for adjusting the average particle size for example, an extrusion method, a method of mechanically pulverizing large multilamellar liposomes (MLV) or the like (specifically using a manton gourin, a microfluidizer, etc.) RHMuller, S. Benita, B. Bohm, “Emulsion and ⁇ Nanosuspensions for the Emulsion and Nanosuspensions for the Formulation of Poorly Soluble Drugs) ”, Scientific Publishers Stuttgart, 1998, p.267-294].
  • MLV large multilamellar liposomes
  • the size of the nucleic acid-containing nanoparticles of the present invention can be measured by, for example, a dynamic light scattering method.
  • the nanoparticles of the present invention can be produced by mixing nucleic acid, polymer I and polymer II. That is, one of the present invention is a method for producing nucleic acid-containing polymer nanoparticles.
  • the method for producing nucleic acid-containing polymer nanoparticles includes a step of mixing nucleic acid, polymer I and polymer II. In the above mixing, the nucleic acid, the polymer I, and the polymer II may be mixed in any order, and examples thereof include the following methods.
  • Method 1 Method of adding polymer I and polymer II to nucleic acid solution or adding nucleic acid to solution containing polymer I and polymer II
  • Method 2 Method of simultaneously mixing nucleic acid solution, solution containing polymer I, and solution containing polymer II
  • the polymer I and the polymer II when the polymer I and the polymer II are added to the nucleic acid solution, the polymer I and the polymer II may be a solution of a mixture of the polymer I and the polymer II.
  • the nucleic acid when the nucleic acid is added to the solution containing the polymer I and the polymer II, the nucleic acid is preferably a nucleic acid solution.
  • the solvent of each solution in Method 1 and Method 2 include water or a mixed solvent of water and an organic solvent miscible with water.
  • the concentration of nucleic acid, polymer I and polymer II in each solution in the above method 1 and method 2 depends on the type of nucleic acid, molecular weight, amount used, or target modification rate by polymer I in nanoparticles. What is necessary is just to adjust suitably.
  • the water-miscible organic solvent is preferably alcohol, dimethyl sulfoxide, tetrahydrofuran, acetone, acetonitrile or a mixture thereof, and more preferably an alcohol.
  • the alcohol is preferably methanol, ethanol, propanol, butanol or a mixture thereof.
  • C1-C6 alcohols such as methanol, ethanol, propanol, butanol and the like containing 0 to 50% (v / v) water or a mixture thereof are preferable.
  • Ethanol or propanol containing 0 to 50% (v / v) water is more preferred, and ethanol containing 0 to 50% (v / v) water is more preferred.
  • the solution of the mixture when a solution of a mixture of polymer I and polymer II is added to the nucleic acid solution, or when a nucleic acid solution is added to a solution containing polymer I and polymer II, the solution of the mixture, Alternatively, the speed at which the nucleic acid solution is added is not particularly limited. In addition, in the method 2, even when the nucleic acid solution, the solution containing the polymer I, and the solution containing the polymer II are added simultaneously, the speed of adding each solution is not particularly limited.
  • the nucleic acid-containing nanoparticles of the present invention can be introduced into cells by introducing the nucleic acid-containing nanoparticles of the present invention into mammalian cells.
  • nucleic acid-containing nanoparticles of the present invention into mammalian cells in vivo may be performed according to known transfection procedures that can be performed in vivo.
  • the nucleic acid-containing nanoparticles of the present invention are intravenously administered to mammals including human beings, for example, delivered to organs or sites where tumors or inflammation has occurred, and the cells of the present invention are delivered into cells of the delivery organs or sites.
  • Nucleic acids in nucleic acid-containing nanoparticles can be introduced.
  • the organ or site where cells expressing Siglec-1 are not particularly limited, but for example, liver, lung, lymph, spleen, stomach, large intestine, intestine, liver, lung, spleen, pancreas, kidney, bladder, skin , Blood vessels, eyeballs and the like.
  • the nucleic acid-containing nanoparticles of the present invention can be intravenously administered to mammals including humans, for example, delivered to the liver, stomach, lungs, kidneys, pancreas and / or spleen, and within the cells of the delivery organ or site.
  • the nucleic acid in the nucleic acid-containing nanoparticle of the present invention can be introduced.
  • the liver, lung, lymph, kidney, intestine and / or spleen are preferable.
  • the nucleic acid in the nucleic acid-containing nanoparticle of the present invention is a nucleic acid having a target gene expression suppressing action using RNA interference (RNAi)
  • RNAi RNA interference
  • the nucleic acid that suppresses the expression of the target gene in a mammalian cell in vivo. Etc. can be introduced, and the expression of the target gene can be suppressed.
  • the administration subject is preferably a human.
  • the target gene in the nucleic acid-containing nanoparticle of the present invention is a gene expressed in cells positive for Siglec-1, and is a gene expressed in, for example, the liver, lung, lymph, kidney, intestine and / or spleen If present, the nucleic acid-containing nanoparticles of the present invention are used as a therapeutic or prophylactic agent for diseases related to the liver, lung, lymph, kidney, intestinal tract and / or spleen, preferably as a therapeutic or prophylactic agent for diseases related to the liver. Can be used.
  • the present invention also provides a method for treating diseases related to liver, lung, lymph, kidney, intestinal tract and / or spleen, etc., wherein the nucleic acid-containing nanoparticles of the present invention described above are administered to mammals.
  • the administration subject is preferably a person, more preferably a person suffering from a disease related to the liver, lung, lymph, kidney, intestine and / or spleen.
  • the nucleic acid-containing nanoparticles of the present invention involve cells positive for Siglec-1, and are evaluated in vivo for therapeutic or prophylactic agents for diseases related to liver, lung, lymph, kidney, intestinal tract and / or spleen. It can also be used as a tool to verify the effectiveness of suppressing target genes in a model.
  • the nucleic acid-containing nanoparticles of the present invention can be used, for example, to stabilize a nucleic acid in a biological component such as a blood component (for example, blood, digestive tract, etc.), to reduce side effects, or to a tissue or organ containing a target gene expression site. It can also be used as a preparation for the purpose of increasing drug accumulation.
  • a biological component such as a blood component (for example, blood, digestive tract, etc.)
  • a tissue or organ containing a target gene expression site for example, to reduce side effects, or to a tissue or organ containing a target gene expression site. It can also be used as a preparation for the purpose of increasing drug accumulation.
  • the administration route is the most effective administration in the treatment. It is desirable to use the route, and examples thereof include parenteral or oral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular or intravenous, preferably intravenous, subcutaneous or intramuscular. Intravenous administration can be mentioned, and intravenous administration is more preferable.
  • the dose varies depending on the disease state, age, administration route, etc. of the administration subject, but for example, it may be administered so that the daily dose converted to nucleic acid is about 0.1 ⁇ g to 1000 mg.
  • a preparation suitable for intravenous administration or intramuscular administration for example, an injection can be mentioned, and a dispersion of the composition prepared by the above-described method can be used as it is, for example, in the form of an injection or the like.
  • the dispersion can be used after removing the solvent by, for example, filtration, centrifugation, etc.
  • the dispersion can be used after lyophilization, and / or mannitol, lactose, trehalose, maltose, glycine, etc. It is also possible to use a dispersion obtained by adding the above-mentioned excipient by lyophilization.
  • an injection for example, water, acid, alkali, various buffers, physiological saline or amino acid infusion, etc. are mixed with the dispersion of the composition or the composition after removing or lyophilizing the solvent. It is preferable to prepare an injection. Further, for example, an injection can be prepared by adding an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA or an isotonic agent such as glycerin, glucose or sodium chloride. Further, for example, it can be cryopreserved by adding a cryopreservative such as glycerin.
  • an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA
  • an isotonic agent such as glycerin, glucose or sodium chloride.
  • it can be cryopreserved by adding a cryopreservative such as glycerin.
  • the present invention also relates to a nanoparticle for use in the treatment of a disease; a pharmaceutical composition for use in the treatment of a disease; use of a nanoparticle for the treatment of a disease; a nanoparticle in the manufacture of a medicament for the treatment of a disease A nanoparticle for use in the manufacture of a medicament for the treatment of a disease; a method of treating or preventing a disease, comprising administering an effective amount of the nanoparticle to a subject in need thereof.
  • Reference Examples 1 to 50 show the synthesis of cationic lipids.
  • Reference example 2 N-methyl-N, N-bis (2-((Z) -hexadec-9-enyloxy) ethyl) amine (compound CL-2) To a toluene (2 mL) suspension of sodium hydride (oil, 60%, 222 mg, 5.55 mmol) in a toluene (2 mL) solution of N-methyldiethanolamine (Tokyo Chemical Industry Co., Ltd., 82.6 mg, 0.693 mmol) was added with stirring, and a solution of (Z) -hexadeca-9-enyl methanesulfonate (530 mg, 1.66 mmol) in toluene (2 mL) was added dropwise.
  • sodium hydride oil, 60%, 222 mg, 5.55 mmol
  • N-methyldiethanolamine Tokyo Chemical Industry Co., Ltd., 82.6 mg, 0.693 mmol
  • (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (manufactured by Nu-Chek Prep, Inc., 2.458 g, 7.13 mmol) was added, and the mixture was stirred for 2 hours under heating reflux.
  • a saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered.
  • reaction solution is diluted with ethyl acetate, washed with 2 mol / L aqueous sodium hydroxide solution and then with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give methyl ((9Z, 12Z) -octadeca
  • a crude product of -9,12-dienyl) amine was obtained.
  • To the obtained crude product add (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (0.93 g, 2.70 mmol) and 50% aqueous sodium hydroxide solution (0.960 g, 12.0 mmol). The mixture was heated and stirred at 135 ° C. for 60 minutes.
  • cyclopropylmagnesium bromide manufactured by Sigma-Aldrich, 0.5 mmol / L. 1.06 mL, 0.529 mmol
  • cyclopropylmagnesium bromide manufactured by Sigma-Aldrich, 0.5 mmol / L. 1.06 mL, 0.529 mmol
  • a saturated aqueous ammonium chloride solution was added to the reaction mixture, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and filtered.
  • lithium bromide (Sigma-Aldrich, 0.108 g, 1.24 mmol) and chlorotrimethylsilane (Tokyo Kasei Kogyo, 0.135 g, 1.24 mmol) were additionally added, and the mixture was stirred for 1 hour.
  • a saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the aqueous layer was extracted with hexane. The organic layer was dried over anhydrous magnesium sulfate and filtered.
  • Reference Example 27 4- (Dimethylamino) butyl di ((9Z, 12Z) -oxadec-9,12-dien-1-yl) carbamate (Compound CL-27) CL-27 was synthesized by the method described in International Publication No. 2014/007398.
  • Reference Example 30 (6Z, 9Z, 28Z, 31Z) -N, N-Dimethylheptatriaconta-6,9,28,31-tetraene-19-amine (Compound CL-30) CL-30 was synthesized by the method described in International Publication No. 2010/054405.
  • Reference Example 31 N, N, 2-trimethyl-1,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propan-2-amine (Compound CL-31) 2-Methyl-1,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propan-2-amine (0.240 g, 0.399 mmol) obtained in Step 1 of Reference Example 9 Is dissolved in a mixed solvent of 1,2-dichloroethane (1 mL) and methanol (1 mL), and formaldehyde (Wako Pure Chemical Industries, 37% aqueous solution, 0.144 mL, 1.99 mmol), sodium triacetoxyborohydride (Tokyo) Kasei Kogyo Co., Ltd., 0.211 g, 0.997 mmol) was added, and the mixture was stirred overnight at room temperature.
  • 1,2-dichloroethane 1 m
  • Reference Example 37 3- (Dimethylmino) propyl di ((Z) -octadeca-9-enyl) carbamate (Compound CL-37) Process 1 Add (Z) -octadeca-9-enyl methanesulfonate (1.04 g, 3.00 mmol) to ammonia (Tokyo Chemical Industry Co., Ltd., approx. 2 mol / L methanol solution, 12.0 mL, 24.0 mmol) And stirred at 130 ° C. for 3 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted 5 times with chloroform.
  • ammonia Tokyo Chemical Industry Co., Ltd., approx. 2 mol / L methanol solution, 12.0 mL, 24.0 mmol
  • the obtained residue was dissolved in a small amount of n-hexane / ethyl acetate (1/4), adsorbed on a pad of amino-modified silica gel, eluted with n-hexane / ethyl acetate (1/4), and concentrated under reduced pressure.
  • Reference Example 51 (Synthesis of lipid having ligand capable of binding to Siglec) A lipid having a ligand capable of binding to Siglec represented by the following formula was synthesized according to the method described in J. Am. Chem. Soc., 2012, 134, 15696. Hereinafter, the lipid synthesized in Reference Example 51 is also referred to as Siglec-1L-PEG-DSPE. Siglec-1L-PEG-DSPE obtained by synthesis agreed with the spectrum data described in J. Am. Chem. Soc., 2012, 134, 15696.
  • Reference Examples 52 to 63 show the synthesis of a cationic polymer SPP-1 to 12 having a ligand ⁇ ⁇ (Siglec-1L) capable of binding to Siglec.
  • Each PEG part described below is not a single molecular weight but has a distribution in the molecular weight, but in the MS spectrum, it was calculated by using the average molecular weight value (5000 kg or 2000) of each PEG part.
  • Process 2-1 After adding 10 M acetic acid-N, N-dimethylformamide solution (2.0 ⁇ L) to the compound Int-1 solution obtained in step 1, K 15 GC peptide (Toray, 11 mg, 5.3 ⁇ mol, N, N-dimethylformamide (100 ⁇ L) solution having a retention time of 0.17 minutes (HPLC condition A)) was added and allowed to stand for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was diluted with water, and the solvent was replaced with water by Vivaspin6 5K (manufactured by Sartorius).
  • Reference Example 54 Synthesis process 2-3 of Siglec-1L-PEG5000-R 30 GC (compound SPP-3) 2-3 A compound Int-1 solution was obtained in the same manner as in Step 1 of Reference Example 52 using Siglec-1L (5.2 mg, 5.6 ⁇ mol) and SUNBRIGHT MA-050TS (20 mg, 3.8 ⁇ mol). Using the reaction solution and R 30 GC peptide (Toray Industries, Inc., 18 mg, 3.8 ⁇ mol, retention time; 0.15 min (HPLC condition A)), compound SPP-3 in the same manner as in step 2-1 of Reference Example 52 (28 mg, 2.5 ⁇ mol, 66% for 2 steps, retention time; 1.01 min (HPLC condition A)) was obtained as a white solid.
  • R 30 GC peptide Toray Industries, Inc., 18 mg, 3.8 ⁇ mol, retention time; 0.15 min (HPLC condition A)
  • reaction solution was diluted with water, and the solvent was replaced with water by Amicon Ultra-4 3K (manufactured by Waters). Purified with Sep-Pak (registered trademark) 6cc C18 Cartridge (Waters, eluted with 30% acetonitrile aqueous solution), and freeze-dried compound SPP-4 (7.2 mg, 1.0 ⁇ mol, 77%, retention time; 3.30 minutes ( HPLC condition B)) was obtained as a white solid.
  • Sep-Pak registered trademark
  • 6cc C18 Cartridge Waters, eluted with 30% acetonitrile aqueous solution
  • Reference Example 56 Synthesis step 1-2 of PEG5000-K 30 GC (compound SPP-5) Using SUNBRIGHT ME-050MA (6.0 mg, 1.1 ⁇ mol) and K 30 GC peptide (6.9 mg, 1.7 ⁇ mol), Compound SPP-5 was obtained as a white solid in the same manner as in Step 1-1 of Reference Example 55 ( 6.0 mg, 0.65 ⁇ mol, 59%, retention time; 3.04 minutes (HPLC condition B)).
  • Process 4 Using compound Int-4 obtained in Step 3 (3.3 mg, 1.7 ⁇ mol, retention time; 1.12 minutes (HPLC condition A), 5.95 minutes (HPLC condition B)) and SUNBRIGHT MA-050TS (6.0 mg, 1.1 ⁇ mol) In the same manner as in Step 1 of Reference Example 52, a compound Int-5 solution (retention time; 5.54 minutes (HPLC condition B)) was obtained.
  • Process 5-1 Using the compound Int-5 solution obtained in step 4 and K 15 GC peptide (3.5 mg, 1.7 ⁇ mol), compound SPP-7 (1.2 mg, 0.13 ⁇ mol, 2 steps) in the same manner as in step 2 of Reference Example 52 12%, retention time; 1.12 min (HPLC condition A)) was obtained as a white solid.
  • Reference Example 63 ((Siglec-1L) 2 -PEG2000) 2 -R 30 GC (SPP-12) Synthesis Process 2-3 Compound Int-4 (4.9 mg, 2.5 ⁇ mol) obtained in Step 3 of Reference Example 58 and SUNBRIGHT 2TS-GL2-020MA4 (2.5 mg, 1.0 ⁇ mol) were used in the same manner as in Step 1 of Reference Example 61. -6 solution was obtained. Using the reaction solution and R 30 GC peptide (5.8 mg, 1.2 ⁇ mol), compound SPP-12 (6.3 mg, 0.57 ⁇ mol, 2 steps 57%, retention time; retention time; 0.92 in the same manner as in step 2-1 of Reference Example 61 Minute (HPLC condition A)) was obtained as a white solid.
  • Examples 1 to 6 and Comparative Example 1 are preparation examples in which the content of Siglec-1L-PEG-DSPE in nanoparticles, that is, the modification rate of Siglec-1L-PEG-DSPE in nanoparticles was changed. is there.
  • Example 1 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, Compound CL-8 obtained in Reference Example 8, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing nanoparticles as follows.
  • the nucleic acid used was a siRNA that suppresses the expression of the ⁇ 2-microglobulin gene, which comprises a sense strand (5′-AGCAAGGACUGGUCUUUCUAUCUCU-3 ′) and an antisense strand (5′-AGAGAUAGAAAGACCAGUCCUUGCU-3 ′) base sequence (hereinafter, “ Also referred to as “B2M siRNA”).
  • B2M siRNA PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
  • B2M siRNA was dissolved in distilled water and prepared to 24 mg / mL.
  • Each lipid contains hydrochloric acid and ethanol so that the concentration ratio of CL-8 to PEG-DSPE (CL-8 / PEG-DSPE Na) is 57.26 (mmol / L) /5.521 (mmol / L) It was suspended in an aqueous solution and stirred and heated repeatedly with a vortex mixer to obtain a uniform suspension. This suspension was passed through a 0.2 ⁇ m polycarbonate membrane filter and a 0.05 ⁇ m polycarbonate membrane filter at room temperature to obtain a dispersion of lead particles. The average particle size of the lead particles obtained with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern Instruments) was measured and confirmed to be within the range of 30 nm to 100 nm.
  • a particle size measuring device Zetasizer Nano ZS, manufactured by Malvern Instruments
  • a dispersion of nucleic acid complex particles was prepared.
  • the concentration ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol, and Siglec-1L-PEG-DSPE CL-8 / PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG-
  • Each lipid was weighed so that DSPE) was 14.72 (mmol / L) /1.082 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.007 (mmol / L), and 90 vol%
  • a solution of lipid membrane constituents was prepared by dissolving in ethanol.
  • the lipid membrane constituent solution and the nucleic acid complex particle dispersion are mixed at a volume ratio of 1: 1, and then several times the amount.
  • distilled water was mixed to obtain a crude preparation.
  • the obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), further replacing the solvent with physiological saline, and filtered in a clean bench using a 0.2 ⁇ m filter (manufactured by Toyo Roshi Kaisha, Ltd.). Furthermore, the siRNA concentration of the obtained preparation was measured, and the preparation 1 was obtained by diluting with physiological saline so that the siRNA concentration was 10 ⁇ M.
  • Example 2 A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows. Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /1.067 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.022 (mmol / L), Formulation 2 was obtained in the same manner as Example 1.
  • Example 3 A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows. Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /1.024 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.065 (mmol / L), Formulation 3 was obtained in the same manner as Example 1.
  • Example 4 A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows. Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /0.893 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.196 (mmol / L), Formulation 4 was obtained in the same manner as Example 1.
  • Example 5 A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows. Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /0.500 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.589 (mmol / L), Formulation 5 was obtained in the same manner as Example 1.
  • Example 6 A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows. Concentration ratio of CL-8, DOPE, cholesterol, and Siglec-1L-PEG-DSPE for the lipid membrane component solution of Formulation 1 (CL-8 / DOPE / cholesterol / Siglec-1L-PEG-DSPE) A formulation 6 was obtained in the same manner as in Example 1 except that 14.72 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /1.089 (mmol / L) was used.
  • THP-1 human-derived monocytic cell line
  • Siglec-1 was induced on the cell membrane by culturing in 10% FBS RPMI1640 medium containing 500 U / mL Recombinant Human IFN-alpha 2 protein (R & D, 11105-1) for 24 hours. -1 described as THP-1).
  • Each nucleic acid-containing lipid nanoparticle is diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 30, 10, 3 or 1 nmol / L, and a 96-well suspension cell culture plate After dispensing 20 ⁇ L each into (Corning, 3474), Siglec-1-induced THP-1 suspended in Optimem was added so that the cell number would be 12500/80 ⁇ L / well, 37 ° C., 5%
  • Each nucleic acid-containing lipid nanoparticle was introduced into cells by culturing for 4 hours under CO 2 conditions. Moreover, Siglec-1-induced THP-1 that was not treated as a negative control group was cultured in the same manner.
  • the cultured cells were centrifuged at room temperature at 1800 rpm for 3 minutes, the culture supernatant was carefully removed, 10% FBS RPMI1640 medium was added, and the cells were further cultured at 37 ° C., 5% CO 2 for 20 hours.
  • the cultured cells were washed with ice-cold phosphate buffered saline (Nacalai Tesque, 14249-24), and Super Prep Cellulosis and RT Kit Forkyu PC (catalog number SCQ-201, manufactured by Toyobo Co., Ltd.)
  • total RNA was collected and cDNA was prepared by reverse transcription using the obtained total RNA as a template.
  • B2M beta-2 microglobulin
  • GAPDH glyceraldehyde 3-phosphate dehydrogenase
  • the B2M mRNA expression rate was determined from the B2M mRNA semi-quantitative value, with the B2M mRNA semi-quantitative value in the negative control measured as 1.
  • Table 9 shows the results of expressing the expression rate of the obtained B2M mRNA as a suppression rate with respect to the B2M mRNA expression rate of the negative control.
  • Formulation 1-6 into which the targeting element (Siglec-1L-PEG-DSPE) was introduced, showed improved gene expression suppression activity as compared with Formulation 7 not containing the targeting element. Moreover, the improvement of the gene expression suppression activity depending on the introduction amount of the targeting element was shown.
  • Examples 7 to 8 and Comparative Example 2 are preparation examples in which PEG lipid derivatives are changed.
  • Example 7 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-8 (CL-8) obtained in Reference Example 8, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
  • PEG-DMPE was obtained from NOF.
  • Example 8 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-8 (CL-8), 1,2-distearoyl-sn-glycero-3- [methoxy (polyethylene) obtained in Reference Example 8 Glycol) -2000] (PEG-DSG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
  • PEG-DSG was obtained from NOF.
  • a preparation 9 was obtained in the same manner as in Example 5 except that the PEG-DSPE used in the preparation 5 was changed to PEG-DSG.
  • Test Example 4 The target gene expression inhibitory effect on nucleic acid-containing lipid nanoparticles on human cell lines was evaluated. For each nucleic acid-containing lipid nanoparticle obtained in Examples 7 and 8 and Comparative Example 2, the target gene expression inhibitory action was evaluated in the same manner as in Test Example 2. Table 11 shows the results of expressing the expression rate of the obtained B2M mRNA as a suppression rate with respect to the B2M mRNA expression rate of the negative control.
  • ND represents Not Detected.
  • preparation 8 with the targeting element (Siglec-1L -PEG-DSPE) improved gene expression suppression activity compared to preparation 10 without the targeting element (Siglec-1L -PEG-DSPE) showed that.
  • Preparation 9 in which the PEG lipid derivative was converted showed the same gene expression inhibitory activity as Preparation 8.
  • Example 9 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-10 (CL-10) obtained in Reference Example 10, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows. Preparation 11 was obtained in the same manner as in Example 7, except that CL-8 of preparation 8 was changed to CL-10.
  • Example 10 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and Compound CL-26 (CL-26) obtained in Reference Example 26, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
  • a preparation 12 was obtained in the same manner as in Example 7, except that CL-8 of the preparation 8 was changed to CL-26.
  • Example 11 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-6 (CL-6) obtained in Reference Example 6, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
  • Formulation 13 was obtained in the same manner as in Example 7, except that CL-8 in formulation 8 was changed to CL-6.
  • Example 12 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-7 (CL-7) obtained in Reference Example 7, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
  • Formulation 14 was obtained in the same manner as in Example 7, except that CL-8 of formulation 8 was changed to CL-7.
  • Example 13 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and Compound CL-9 (CL-9), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine obtained in Reference Example 9 N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows. Formulation 15 was obtained in the same manner as in Example 7, except that CL-8 was replaced with CL-9.
  • Test Example 6 The target gene expression inhibitory effect on nucleic acid-containing lipid nanoparticles on human cell lines was evaluated. For each of the nucleic acid-containing lipid nanoparticles obtained in Examples 9 to 13 and Comparative Examples 3 to 7, the target gene expression inhibitory action was evaluated in the same manner as in Test Example 2. Table 13 shows the results of expressing the expression rate of the obtained B2M mRNA as a suppression rate with respect to the B2M mRNA expression rate of the negative control.
  • the preparations 11 to 15 in which the targeting element (Siglec-1L-PEG-DSPE) was introduced were compared with the preparations 16 to 20 that did not contain the corresponding targeting element (Siglec-1L -PEG-DSPE).
  • the gene expression suppression activity was improved.
  • Example 7 The target gene expression inhibitory action of nucleic acid-containing lipid nanoparticles on human primary monocyte cells was evaluated. About each nucleic acid-containing lipid nanoparticle obtained in Example 10 and Comparative Example 4, the target gene for healthy human-derived CD14-positive monocyte cells (Untouched Frozen NPB-CD14 + Monocytes, manufactured by Allcells, PB011F) by the following method, respectively. The expression inhibitory action was measured.
  • Healthy human-derived CD14 positive monocyte cells are 10% fetal bovine serum, 1% MEM Non-Essential Amino Acids Solution (Gibco, 11140-050), 1 mM sodium pyruvate (Gibco, 11360-070), 50 ⁇ M Monocyte cells using RPMI1640 medium (hereinafter referred to as human monocyte basal medium) and DNase I solution (DNase I Solution, StemCell Technology, 07900) containing 2-mercaptoethanol (Gibco, 21985-023) Thawed according to the protocol attached to After that, inoculate a 96-well suspension cell culture plate at a density of 100,000 cells / 100 ⁇ L / well in human monocyte basal medium containing 1000 U / mL Recombinant Human IFN-alpha 2 protein, 37 ° C, 5% CO By culturing for 24 hours under two conditions, Siglec-1 was induced on the cell membrane (hereinafter referred to as Siglec-1-induced human primary monocyte
  • each nucleic acid-containing lipid nanoparticle is diluted with Optimem to a final concentration of 30, 10, 3 or 1 nmol / L, and 20 ⁇ L is added to each well under conditions of 37 ° C and 5% CO 2
  • Each of the nucleic acid-containing lipid nanoparticles was introduced into the cells by culturing for 4 hours. Further, Siglec-1-induced primary human monocytes that were not treated as a negative control group were cultured in the same manner.
  • FITC anti-human ⁇ 2-microglobulin Antibody Biolegend, 316304
  • flow cytometry buffer diluted 4-fold with flow cytometry buffer, and dispensed into a 96-well U-bottom plate (Falcon, 353077) at 20 ⁇ L / well.
  • Siglec-1-derived human primary monocytes after standing for 15 minutes were added at 80 ⁇ L / well, and the mixture was allowed to stand at 4 ° C. for 60 minutes to react the B2M protein on the cell membrane with the antibody.
  • the Siglec-1-derived primary human monocytes after the antibody reaction were washed three times with a flow cytometry buffer, and then the fluorescence intensity at 530 nm with respect to the excitation light at 488 nm of each cell was measured using the BD FACS Canto II Flow Cytometer ( The expression level of B2M protein on the cell membrane was measured by measuring using BD). Similarly, the expression level of B2M protein was determined with the B2M protein expression level in the negative control measured in the same manner as 1. Table 14 shows the results of expressing the expression rate of the obtained B2M protein as a suppression rate with respect to the B2M mRNA expression rate of the negative control.
  • the preparation 12 containing Siglec-1L-PEG-DSPE is the corresponding preparation 17 containing no Siglec-1L -PEG-DSPE.
  • the gene expression inhibitory activity was remarkably strong.
  • Example 14 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, Compound CL-8 obtained in Reference Example 8, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing nanoparticles as follows. As the nucleic acid, CleanCap TM EGFP mRNA (5moU) expressing green fluorescent protein (GFP) obtained from TriLink BioTechnologies, LLC was used (hereinafter also referred to as “GFP mRNA”).
  • GFP mRNA Green fluorescent protein
  • PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
  • GFP mRNA was dissolved in distilled water and adjusted to 1 mg / mL for use.
  • Each lipid contains hydrochloric acid and ethanol so that the concentration ratio of CL-8 to PEG-DSPE (CL-8 / PEG-DSPE Na) is 57.26 (mmol / L) /5.521 (mmol / L) It was suspended in an aqueous solution and stirred and heated repeatedly with a vortex mixer to obtain a uniform suspension. This suspension was passed through a 0.2 ⁇ m polycarbonate membrane filter and a 0.05 ⁇ m polycarbonate membrane filter at room temperature to obtain a dispersion of lead particles.
  • the average particle size of the lead particles obtained with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern Instruments) was measured and confirmed to be within the range of 30 nm to 100 nm.
  • a dispersion of nucleic acid complex particles was prepared.
  • the concentration ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol, and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG-
  • CL-8 / PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG- Each lipid is weighed so that DSPE) is 14.72 (mmol / L) /0.500 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.589 (mmol / L), and 90 vol%
  • a solution of lipid membrane constituents was prepared by dissolving in ethanol.
  • the lipid membrane constituent solution and the nucleic acid complex particle dispersion are mixed at a volume ratio of 1: 1, and then several times the amount.
  • distilled water was mixed to obtain a crude preparation.
  • the obtained crude preparation was concentrated using Vivaspin (GE Healthcare), further substituted with physiological saline, and filtered in a clean bench using a 0.2 ⁇ m filter (Toyo Roshi Kaisha, Ltd.). Furthermore, the GFP mRNA concentration of the obtained preparation was measured, and the preparation 21 was obtained by diluting with physiological saline so that the GFP mRNA concentration was 60 ⁇ g / mL.
  • Example 15 Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, Compound CL-8 obtained in Reference Example 8, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing nanoparticles as follows. As the nucleic acid, CleanCap TM FLuc mRNA (5moU) expressing firefly luciferase obtained from TriLink BioTechnologies, LLC was used (hereinafter also referred to as “FLuc mRNA”).
  • PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
  • FLuc mRNA was dissolved in distilled water and adjusted to 1 mg / mL.
  • a preparation 22 was obtained in the same manner as in Example 14 except that the GFP mRNA in the preparation 21 was changed to FLuc mRNA.
  • THP-1 human-derived monocyte cell line
  • Siglec-1 was induced on the cell membrane by culturing in 10% FBS RPMI1640 medium containing 500 U / mL Recombinant Human IFN-alpha 2 protein (R & D, 11105-1) for 48 hours (hereinafter, Siglec -1 described as THP-1).
  • Each nucleic acid-containing lipid nanoparticle is diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 1, 0.3, 0.1, or 0.03 ⁇ g / mL, and a 96-well suspension cell culture plate After dispensing 20 ⁇ L each into (Corning, 3474), Siglec-1-induced THP-1 suspended in Optimem was added so that the cell number would be 12500/80 ⁇ L / well, 37 ° C., 5% Each nucleic acid-containing lipid nanoparticle was introduced into cells by culturing for 24 hours under CO 2 conditions. Moreover, Siglec-1-induced THP-1 that was not treated as a negative control group was cultured in the same manner.
  • the cultured cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and then the cells were treated with 0.05% sodium azide (Nacalai Tesque, 13160-94), 0.02% EDTA (Ambion, AM9269G). And 1% bovine serum albumin-containing phosphate buffered saline (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer).
  • flow cytometry buffer 1% bovine serum albumin-containing phosphate buffered saline (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer).
  • the preparation 21 containing the targeting element (Siglec-1L -PEG-DSPE) was compared to the human cell line THP-1 that induced the receptor Siglec-1 It showed strong gene expression enhancing activity.
  • Example 10 The target gene expression inhibitory action of nucleic acid-containing lipid nanoparticles on human primary monocyte cells was evaluated. About each nucleic acid-containing lipid nanoparticle obtained in Example 14 and Comparative Example 8, the target gene for CD14-positive monocyte cells derived from healthy subjects (Untouched Frozen NPB-CD14 + Monocytes, manufactured by Allcells, PB011F) by the following methods, respectively. The expression enhancing action was measured.
  • Healthy human-derived CD14 positive monocyte cells are 10% fetal bovine serum, 1% MEM Non-Essential Amino Acids Solution (Gibco, 11140-050), 1 mM sodium pyruvate (Gibco, 11360-070), 50 ⁇ M Using RPMI1640 medium (hereinafter referred to as human monocyte basal medium) containing 2-mercaptoethanol (Gibco, 21985-023) and DNase I solution (DNase I Solution, StemCell Technology, 07900), monocytes Thawed according to the protocol attached to the cells.
  • human monocyte basal medium containing 2-mercaptoethanol (Gibco, 21985-023) and DNase I solution (DNase I Solution, StemCell Technology, 07900)
  • Siglec-1-induced human primary monocyte cells After 48 hours of culture, the cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and Optimem was added at 80 ⁇ L / well.
  • each nucleic acid-containing lipid nanoparticle is diluted with Optimem to a final concentration of 1, 0.3 or 0.1 ⁇ g / mL, and 20 ⁇ L is added to each well, and the mixture is added under conditions of 37 ° C. and 5% CO 2.
  • each nucleic acid-containing lipid nanoparticle was introduced into a cell.
  • Siglec-1-induced primary human monocytes that were not treated as a negative control group were cultured in the same manner.
  • the cultured cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and then the cells were treated with 0.05% sodium azide (Nacalai Tesque, 13160-94), 0.02% EDTA (Ambion, AM9269G). And 1% bovine serum albumin-containing phosphate buffered saline (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer).
  • flow cytometry buffer 1% bovine serum albumin-containing phosphate buffered saline (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer).
  • the preparation 21 using Siglec-1L-PEG-DSPE is the corresponding preparation 23 containing no Siglec-1L -PEG-DSPE. Compared with, it showed strong gene expression enhancing activity.
  • THP-1 human-derived monocyte cell line
  • Siglec-1 was induced on the cell membrane by culturing in 10% FBS RPMI1640 medium containing 2000 U / mL Recombinant Human IFN-alpha 2 protein (R & D, 11105-1) for 48 hours (hereinafter Siglec -1 described as THP-1).
  • Each nucleic acid-containing lipid nanoparticle is diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 0.3 or 0.1 ⁇ g / mL, and a 96-well suspension cell culture plate (coning) 3474), and then add Siglec-1-induced THP-1 suspended in Optimem to a cell number of 12500/80 ⁇ L / well, and at 37 ° C and 5% CO 2
  • Each of the nucleic acid-containing lipid nanoparticles was introduced into the cells by culturing for 24 hours. Moreover, Siglec-1-induced THP-1 that was not treated as a negative control group was cultured in the same manner.
  • the preparation 22 that introduced the targeting element (Siglec-1L -PEG-DSPE) against the human cell line THP-1 that induced the receptor Siglec-1 showed strong gene expression enhancing activity.
  • Formulation 25 containing Siglec-1L-PEG-lipid containing no cationic lipid was prepared as follows.
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • the nucleic acid has a base sequence of a sense strand (5'-GCCAGACUUUGUUGGAUUUGA-3 ') and an antisense strand (5'-AAmAUmCCmAAmCAmAAmGUmCUmGGmCmUmU-3') (m represents 2'-OMe-RNA), and the antisense strand 3 '
  • PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
  • HPRT-1 siRNA-cho was dissolved in distilled water and stored at 24 mg / mL. Each lipid was adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) was 0.4730 (mmol / L) /9.119 (mmol / L) /2.977 (mmol / L). Dissolved in ethanol. On the other hand, the concentration ratio of Siglec-1L-PEG-lipid and HPRT-1 siRNA-cho (Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho) is 0.06058 (mmol / L) /0.001048 (mmol / L). Diluted with distilled water.
  • Syringe pumps are filled with lipid solutions and aqueous solutions containing Siglec-1L-PEG-lipid and nucleic acid in syringes (manufactured by Hamilton), and the flow rates of each solution are 3.000 mL / min and 9.000 mL / min.
  • a crude preparation was obtained through a microreactor mixer (manufactured by YMC). The obtained crude preparation was concentrated using Vivaspin (GE Healthcare), further substituted with physiological saline, and filtered in a clean bench using a 0.2 ⁇ m filter (Toyo Roshi Kaisha, Ltd.). Further, the HPRT-1 siRNA-cho concentration of the obtained preparation was measured, and diluted with physiological saline so that the HPRT-1 siRNA-cho concentration was 10 mM, whereby a preparation 25 was obtained.
  • Formulation 26 containing Siglec-1L-PEG-lipid containing no cationic lipid was prepared as follows. Lipids and nucleic acids were the same as in Example 16. Each lipid is adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) is 0.2365 (mmol / L) /4.559 (mmol / L) /1.489 (mmol / L). Dissolved in ethanol.
  • the concentration ratio of Siglec-1L-PEG-lipid and HPRT-1 siRNA-cho is 0.02983 (mmol / L) /0.005159 (mmol / L). Diluted with distilled water. Otherwise, the preparation 26 was obtained in the same manner as in Example 16.
  • Formulation 27 containing Siglec-1L-PEG-lipid containing no cationic lipid was prepared as follows. Lipids and nucleic acids were the same as in Example 16. PEG-DSPE / DOPE / Cholesterol / Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho molar ratio (PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho) was dissolved in a mixed solution of t-BuOH / distilled water so as to be 0.5202 ( ⁇ mol) /9.914 ( ⁇ mol) /3.237 ( ⁇ mol) /0.1946 ( ⁇ mol) /0.03365 ( ⁇ mol) and freeze-dried.
  • lipid / nucleic acid powder To the obtained lipid / nucleic acid powder, physiological saline was added so as to have a nucleic acid concentration of 1 mg / mL, followed by stirring with a vortex stirring mixer to obtain a suspension. This suspension was passed through a 0.4 ⁇ m polycarbonate membrane filter, a 0.2 ⁇ m polycarbonate membrane filter, and a 0.1 ⁇ m polycarbonate membrane filter at room temperature to prepare a dispersion of nucleic acid complex particles. The resulting nucleic acid complex particle dispersion was filtered in a clean bench using a 0.2 ⁇ m filter (Toyo Roshi Kaisha, Ltd.). Furthermore, the HPRT1 siRNA-cho concentration of the obtained preparation was measured, and diluted with physiological saline so that the HPRT1 siRNA-cho concentration was 10 mM, whereby a preparation 27 was obtained.
  • Formulation 28 containing no cationic lipid and Siglec-1L-PEG-lipid was prepared as follows. Lipids and nucleic acids were the same as in Example 16. Each lipid is adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) is 0.6676 (mmol / L) /9.260 (mmol / L) /3.023 (mmol / L). Dissolved in ethanol. On the other hand, it was diluted with distilled water so that HPRT-1 siRNA-cho was 0.001048 (mmol / L). Otherwise, the preparation 28 was obtained in the same manner as in Example 16.
  • Formulation 29 containing no cationic lipid and Siglec-1L-PEG-lipid was prepared as follows. Lipids and nucleic acids were the same as in Example 16. Each lipid is adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) is 0.3338 (mmol / L) /4.630 (mmol / L) /1.512 (mmol / L). Dissolved in ethanol. On the other hand, it was diluted with distilled water so that HPRT-1 siRNA-cho was 0.005239 (mmol / L). Otherwise, the preparation 29 was obtained in the same manner as in Example 16.
  • Formulation 30 containing no cationic lipid and Siglec-1L-PEG-lipid was prepared as follows. Lipids and nucleic acids were the same as in Example 18. The molar ratio of PEG-DSPE, DOPE, cholesterol, and HPRT-1 siRNA-cho (PEG-DSPE Na / DOPE / cholesterol / HPRT-1 siRNA-cho) is 0.7148 ( ⁇ mol) /9.914 ( ⁇ mol) /3.237 ( ⁇ mol) It was dissolved in a mixed solution of t-BuOH / distilled water so as to be /0.03365 ( ⁇ mol) and lyophilized. Otherwise, the procedure of Example 18 was repeated to obtain a preparation 30.
  • Example 19 A preparation containing a polymer (polymer I) containing a water-soluble unit and a cationic unit and having a ligand capable of binding to nucleic acid and siglec (Sialic acid-binding immunoglobulin-like lectin) was prepared as follows. Using the Siglec-1L-PEG5000-K 15 GC (compound SPP-1) obtained in Reference Example 52, a nucleic acid complex was produced as follows.
  • Nucleic acid is sense strand (5'-A (F) ⁇ G (M) ⁇ G (F) A (M) C (F) U (M) G (F) G (M) U (F) C (M) U (F) U (M) C (F) U (M) A (F) U (M) C (F) U (M) ⁇ C (F) ⁇ U (M) -3 ') And antisense strand (5'-A (F) ⁇ G (M) ⁇ A (F) G (M) A (F) U (M) A (F) G (M) A (F) A (M) G (F) A (M) C (F) C (M) A (F) G (M) U (F) C (M) U (M) ⁇ U (F) ⁇ G (M) -3 ') (M is 2'-OMe-RNA, (F) is 2'-F-RNA, ⁇ is Phosphorothioated
  • siRNA-2 was dissolved in distilled water, stored at 24 mg / mL, and diluted with 10 mM HEPES as necessary.
  • Siglec-1L-PEG5000-K15GC (compound SPP-1) was dissolved in 10 mM HEPES so as to be 10 mg / mL.
  • Formulation 31 was made by mixing siRNA-2 and adjusting the nucleic acid concentration to a final concentration of 10 ⁇ M.
  • SEQ ID NO: 1 shows the base sequence of the B2M siRNA sense strand.
  • SEQ ID NO: 2 shows the base sequence of B2M siRNA antisense strand.
  • SEQ ID NO: 3 shows the base sequence of HPRT1 siRNA-cho sense strand.
  • SEQ ID NO: 4 shows the base sequence of HPRT1 siRNA-cho antisense strand.
  • SEQ ID NO: 5 shows the base sequence of B2M-2 siRNA sense strand.
  • SEQ ID NO: 6 shows the base sequence of the B2M-2 siRNA antisense strand.

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Abstract

La présente invention concerne des nanoparticules contenant : un lipide (lipide I) qui comprend une unité liposoluble ou hydrosoluble et a un ligand capable de se lier à Siglec (lectine de type immunoglobuline de liaison à l'acide sialique), et un acide nucléique; ou un polymère (polymère I) qui comprend une unité hydrosoluble et une unité cationique et a un ligand capable de se lier à Siglec (lectine de type immunoglobuline de liaison à l'acide sialique), et un acide nucléique. Ces nanoparticules sont des nanoparticules contenant un acide nucléique qui peuvent être administrées à des cellules du système immunitaire.
PCT/JP2018/022286 2017-06-09 2018-06-11 Nanoparticules contenant un acide nucléique WO2018225873A1 (fr)

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US11773061B2 (en) 2021-09-14 2023-10-03 Renagade Therapeutics Management Inc. Cyclic lipids and methods of use thereof

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