Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
  • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
  • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
  • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
  • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
  • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
• • 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
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/221—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having an amino group, e.g. acetylcholine, acetylcarnitine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • 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
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
  • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

• the present invention relates to cationic lipids that make it possible to introduce nucleic acids as active ingredients into various types of cells, tissues or organs.
• the invention further relates to lipid particles containing the cationic lipids and compositions containing the lipid particles and nucleic acids.
• nucleic acid medicines containing nucleic acids as active ingredients have been actively carried out.
• nucleic acid drugs that have target mRNA degradation or function-suppressing effects, including nucleic acids such as siRNA, miRNA, miRNA mimics, and antisense nucleic acids.
• Research is also being conducted on nucleic acid medicines for expressing target proteins in cells, including mRNAs encoding target proteins.
• techniques for introducing nucleic acids into cells, tissues or organs with high efficiency have been developed as drug delivery system (DDS) techniques.
• DDS drug delivery system
• Cationic lipids hydrophilic polymer lipids, helper lipids, and the like are conventionally known as lipids used for the above complex formation.
• cationic lipid for example, compounds described in the following prior art documents are known.
• Patent Document 1 describes a compound represented by the following formula or a salt thereof.
• each R 1 is independently selected from the group consisting of optionally substituted C 8 -C 24 alkyl and optionally substituted C 8 -C 24 alkenyl
• Y 1 and Y 2 are each independently selected from the group consisting of hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted arylalkyl, etc.; optionally substituted C 1 -C 6 alkyl, optionally substituted arylalkyl, etc.
• Y 3 if present, are each independently hydrogen, optionally substituted C 1 - C8 alkyl, optionally substituted arylalkyl, etc.
• m is an integer of 1 to 4
• n is an integer of 0 to 3; 0 or 1, the sum of m, n and p is 4
• k is an integer from 1 to 5
• q is 0 or 1, and so on.
• Patent Document 2 describes a compound represented by the following formula or a salt thereof.
• W represents the formula -NR 1 R 2 or the formula -N + R 3 R 4 R 5 (Z - ), and R 1 and R 2 each independently represent a C 1-4 alkyl group or a hydrogen atom.
• R 3 , R 4 and R 5 each independently represents a C 1-4 alkyl group
• Z — represents an anion
• X represents an optionally substituted C 1-6 alkylene group
• Y A , YB and YC each independently represent an optionally substituted methine group
• LA , LB and LC each independently represent an optionally substituted methylene group or bond
• R A1 , R A2 , R B1 , R B2 , R C1 and R C2 each independently represent an optionally substituted C 4-10 alkyl group.
• Patent Document 3 describes a compound represented by the following formula or a salt thereof.
• n represents an integer of 2 to 5
• R represents a linear C 1-5 alkyl group, a linear C 7-11 alkenyl group or a linear C 11 alkadienyl group
• the wavy line is Each independently indicates cis or trans binding.
• Patent Document 4 describes a compound represented by the following formula or a salt thereof.
• n1 represents an integer of 2 to 6
• n2 represents an integer of 0 to 2
• n3 represents an integer of 0 to 2
• L is -C (O) O- or -NHC (O) O -
• Ra represents a linear C 5-13 alkyl group, a linear C 13-17 alkenyl group or a linear C 17 alkadienyl group
• Rb represents a linear C 2-9 alkyl group.
• Rc represents a hydrogen atom or a linear C 2-9 alkyl group
• Rd represents a hydrogen atom or a linear C 2-9 alkyl group
• Re represents a linear C 2-9 alkyl group
• Rf represents a linear C 2-9 alkyl group.
• Cationic lipids which enable nucleic acids to be introduced into cells with high efficiency, contribute to the creation of nucleic acid medicines with excellent therapeutic effects, which are excellent in terms of drug efficacy and safety (low toxicity).
• cationic lipids that enable the introduction of nucleic acids into various cells are expected to enable the creation of nucleic acid medicines for various diseases occurring in various tissues.
• the inventions of cationic lipids described in Patent Documents 3 and 4 can produce certain effects in terms of highly efficient introduction of nucleic acids into cells and introduction of nucleic acids into various cells.
• improving in vivo degradability that is, improving safety as a drug
• An object of the present invention is to enable introduction of nucleic acids into cells with excellent efficiency and to improve biodegradability (medicine safety) and cationic lipids used therefor. etc. is to be provided.
• the object of the present invention is to provide a technique for enabling introduction of nucleic acids into various cells and improving in vivo degradability (safety as a drug) and
• the object is to provide a compound or the like used for
• a compound represented by the following formula (I) or a salt thereof [In formula (I), W represents —NR 1 R 2 or —N + R 11 R 12 R 13 (Z ⁇ ); R 1 and R 2 each independently represent H or an optionally substituted C 1-5 alkyl group; R 11 , R 12 and R 13 each independently represent an optionally substituted C 1-5 alkyl group, Z- represents an anion, X represents an optionally substituted C 2-6 alkylene group, R A and R B are each independently an optionally substituted C 1-17 alkyl group, an optionally substituted C 3-17 alkenyl group, an optionally substituted C 15-17 alkadienyl group , or —R 3 —C(O)O—R 4 or —R 3 —OC(O)—R 4 , R C represents -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4 , R 3
• W represents —NR 1 R 2 or —N + R 11 R 12 R 13 (Z ⁇ );
• R 1 and R 2 each independently represent H or an optionally substituted C 1-5 alkyl group;
• R 11 , R 12 and R 13 each independently represent an optionally substituted C 1-5 alkyl group,
• Z- represents an anion
• X represents an optionally substituted C 2-6 alkylene group
• R A1 and R B1 are each independently an optionally substituted C 1-17 alkyl group, an optionally substituted C 3-17 alkenyl group, or an optionally substituted C 15-17 alkadienyl group represents the group
• R C represents -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4
• R 3 represents an optionally substituted C 1-16 alkylene group, an optionally substituted C 4-16 alkenylene group or an optionally substituted C 7-16
• W represents —NR 1 R 2 or —N + R 11 R 12 R 13 (Z ⁇ );
• R 1 and R 2 each independently represent H or an optionally substituted C 1-5 alkyl group;
• R 11 , R 12 and R 13 each independently represent an optionally substituted C 1-5 alkyl group,
• Z- represents an anion,
• X represents an optionally substituted C 2-6 alkylene group,
• R A1 represents an optionally substituted C 1-17 alkyl group, an optionally substituted C 3-17 alkenyl group, or an optionally substituted C 15-17 alkadienyl group,
• R B2 and R C each independently represent -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4 ;
• R 3 represents an optionally substituted C 1-16 alkylene group, an optionally substituted C 4-16 alkenylene group or an optionally substituted C 7-16 alkadieny
• W represents —NR 1 R 2 or —N + R 11 R 12 R 13 (Z ⁇ );
• R 1 and R 2 each independently represent H or an optionally substituted C 1-5 alkyl group;
• R 11 , R 12 and R 13 each independently represent an optionally substituted C 1-5 alkyl group,
• Z- represents an anion,
• X represents an optionally substituted C 2-6 alkylene group,
• R A2 , R B2 and R C each independently represent -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4 ;
• R 3 represents an optionally substituted C 1-16 alkylene group, an optionally substituted C 4-16 alkenylene group or an optionally substituted C 7-16 alkadienyl group,
• R 4 represents H, an optionally substituted C 1-18 alkyl group, an optionally substituted C 3-18 alkenyl group or an optionally substituted C 15-18
• [11] [1] A lipid particle containing the compound of the description or a salt thereof.
• the composition of [12], wherein the nucleic acid is DNA or RNA.
• the composition of [13], wherein the DNA is a nanoplasmid.
• the composition of [13], wherein the DNA is controlled by a CAG promoter or a CMV promoter.
• the compounds represented by formulas (I) to (IV) are sometimes referred to as “compound (I)” to “compound (IV)".
• the “compound represented by formula (I) or a salt thereof” may be referred to as the “compound of the present invention”.
• a lipid particle containing a compound represented by formula (I) or a salt thereof (a compound of the present invention)” is sometimes referred to as a “lipid particle of the present invention.”
• a “composition for nucleic acid transfer containing a nucleic acid and the lipid particles of the present invention” is sometimes referred to as a “composition of the present invention.”
• the present invention makes it possible to introduce nucleic acids into cells, tissues, or organs with excellent efficiency, and to improve in vivo degradability (safety as a drug). Moreover, according to the present invention, nucleic acids can be introduced into various types of cells, tissues, or organs (for example, cancer cells), and their in vivo degradability (safety as a drug) can be improved. INDUSTRIAL APPLICABILITY The present invention makes it possible to obtain medicines or research reagents that introduce nucleic acids into various types of cells, tissues or organs. Furthermore, according to the present invention, when a nucleic acid is introduced into a cell, tissue or organ, the activity (eg, efficacy) of the nucleic acid is expressed with high efficiency.
• the compound of the present invention is a compound represented by the following formula (I) (compound (I)) or a salt thereof.
• W represents —NR 1 R 2 or —N + R 11 R 12 R 13 (Z ⁇ );
• R 1 and R 2 each independently represent H or an optionally substituted C 1-5 alkyl group;
• R 11 , R 12 and R 13 each independently represent an optionally substituted C 1-5 alkyl group,
• Z- represents an anion
• X represents an optionally substituted C 2-6 alkylene group
• R A and R B are each independently an optionally substituted C 1-17 alkyl group, an optionally substituted C 3-17 alkenyl group, an optionally substituted C 15-17 alkadienyl group , or —R 3 —C(O)O—R 4 or —R 3 —OC(O)—R 4
• R C represents -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4
• R 3 represents an optionally substituted C 1-16 alkylene group, an optionally substituted C 4-16 alkenylene group or an optionally substitute
• C 1-5 alkyl group (R 1 , R 2 , R 11 , R 12 and R 13 ) refers to an alkyl group having 1 to 5 carbon atoms, which may be linear or branched, In the case of the shape, the number of bonds in the branched chain (how many carbon atoms it is bonded to) and the bond position (which carbon atom it is bonded to) are arbitrary.
• C 1-5 alkyl groups include: [C 1 ] methyl; [ C2 ] ethyl; [C 3 ] propyl, 1-methylethyl (aka isopropyl); [C 4 ]butyl, 1-methylpropyl (aka sec-butyl), 2-methylpropyl (aka isobutyl), 1,1-dimethylethyl (aka tert-butyl); [C 5 ] pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl (also known as isopentyl), 1-ethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl (also known as neopentyl ).
• the “optionally substituted C 1-5 alkyl group” of R 1 and R 2 is preferably an “optionally substituted C 1-4 alkyl group”, more preferably an “optionally substituted good C 1-3 alkyl group”.
• the “optionally substituted C 1-5 alkyl group” of R 11 , R 12 and R 13 is preferably an “optionally substituted C 1-3 alkyl group”.
• C 2-6 alkylene group refers to a divalent group derived from an alkyl group having 2 to 6 carbon atoms, which may be linear or branched, and branched if branched The number and position of bonds in the chain are arbitrary.
• C 2-6 alkylene groups include: [C 2 ] ethylene, methylmethylene; [C 3 ] propylene, 1-methylethylene, 2-methylethylene, ethylmethylene; [C 4 ] butylene, 1-methylpropylene, 2-methylpropylene, 3-methylpropylene, 1-ethylethylene, 2-ethylethylene, 1,2-dimethylethylene, propylmethylene; [C 5 ] pentylene, 1-methylbutylene, 2-methylbutylene, 3-methylbutylene, 4-methylbutylene, 1-ethylpropylene, 2-ethylpropylene, 3-ethylpropylene, 1,1-dimethylpropylene, 1, 2-dimethylpropylene, 1,3-dimethylpropylene, 2,2-dimethylpropylene, 2,3-dimethylpropylene, 1-propylethylene, 2-propylethylene, butylmethylene; [C 6 ]hexylene, 1-methylpentylene, 2-methylpentylene, 2-methyl
• the “optionally substituted C 2-6 alkylene group” of X is preferably an “optionally substituted C 2-5 alkylene group”, more preferably an “optionally substituted C 2-6 alkylene group”. 4 alkylene group”.
• C 1-17 alkyl group (R A and R B ) refers to an alkyl group having from 1 to 17 carbon atoms, which may be linear or branched, and if branched, the number of bonds in the branched chain and the binding position is arbitrary.
• C 1-17 alkyl group examples include the following in addition to the above-mentioned specific examples of the C 1-5 alkyl group, but the C 1-17 alkyl group that can be used in the present invention is limited to these Not: [C 6 ]hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl (also known as isohexyl), 1-ethylbutyl, 2-ethylbutyl, 1,1-dimethylbutyl, 1,2-dimethyl butyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl; [C 7 ]heptyl, 1-methylhexyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 1-ethylpentyl, 2-ethylpentyl, 3-ethylpent
• C 3-17 alkenyl group (R A and R B ) refers to an alkenyl group having 3 to 17 carbon atoms, which may be linear or branched, and if branched, the number of bonds in the branched chain and the bonding position are arbitrary, and the position of the carbon-carbon double bond is also arbitrary.
• C 3-17 alkenyl group examples include the following, but the C 3-17 alkenyl group that can be used in the invention is not limited to these examples: [C 3 ] 1-propenyl, 2-propenyl; [C 4 ] 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl; [C 5 ] 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2- butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1-ethyl-1-propenyl, 2-ethyl-1-propenyl, 1-ethyl
• C 15-17 alkadienyl group (R A and R B ) refers to an alkadienyl group having 15 to 17 carbon atoms, which may be linear or branched, and if branched, the number of bonds in the branched chain and the bonding positions are arbitrary, and the positions of the two carbon-carbon double bonds are also arbitrary.
• C 15-17 alkadienyl group examples include the following, but the C 15-17 alkadienyl group that can be used in the present invention is not limited to these examples: [C 15 ] 1,3-pentadecadienyl, 1,4-pentadecadienyl, 1,5-pentadecadienyl, 1,6-pentadecadienyl, 1,7-pentadecadienyl, 1 ,8-pentadecadienyl, 1,9-pentadecadienyl, 1,10-pentadecadienyl, 1,11-pentadecadienyl, 1,12-pentadecadienyl, 1,13-pentadecadienyl dienyl, 1,14-pentadecadienyl, 2,4-pentadecadienyl, 2,5-pentadecadienyl, 2,6-pentadecadienyl, 2,7-pent
• the “C 1-16 alkylene group” (R 3 ) refers to a divalent group derived from an alkyl group having 1 to 16 carbon atoms, which may be linear or branched. The binding position of the branched chain is arbitrary. Specific examples of the C 1-16 alkylene group include the following in addition to the above-mentioned specific examples of the C 2-6 alkylene group .
• the “optionally substituted C 1-16 alkylene group” of R 3 is preferably an “optionally substituted C 1-10 alkylene group”.
• C 4-16 alkenylene group (R 3 ) refers to a divalent group derived from an alkenyl group having 4 to 16 carbon atoms, which may be linear or branched; The number and position of bonds in the branched chain are arbitrary, and the position of the carbon-carbon double bond is also arbitrary.
• C 4-16 alkenylene group examples include the following, but the C 4-16 alkenylene group that can be used in the present invention is not limited to these examples: [C 4 ] 1-butenylene, 2-butenylene, 3-butenylene, 1-methyl-1-propenylene, 2-methyl-1-propenylene, 1-methyl-2-propenylene, 2-methyl-2-propenylene; [C 5 ] 1-pentenylene, 2-pentenylene, 3-pentenylene, 4-pentenylene, 1-methyl-1-butenylene, 2-methyl-1-butenylene, 3-methyl-1-butenylene, 1-methyl-2- butenylene, 2-methyl-2-butenylene, 3-methyl-2-butenylene, 1-methyl-3-butenylene, 2-methyl-3-butenylene, 3-methyl-3-butenylene, 1-ethyl-1-propenylene, 2-ethyl-1-propenylene, 1-ethyl-2-propenylene, 2-ethyl-3-propeneny
• C 7-16 alkadienyl group (R 3 ) refers to a divalent group derived from an alkadienyl group having 7 to 16 carbon atoms, which may be linear or branched, and in the case of branched The number and position of bonds in the branched chain are arbitrary, and the position of the carbon-carbon double bond is also arbitrary.
• C 7-16 alkadienyl group examples include the following, but the C 7-16 alkadienyl group that can be used in the present invention is not limited to these examples: [C 7 ] 1,3-heptadienylene, 1,4-heptadienylene, 1,5-heptadienylene, 1,6-heptadienylene, 2,4-heptadienylene, 2,5-heptadienylene, 2,6-heptadienylene, 3,5- heptadienylene, 3,6-heptadienylene; [C 8 ] 1,3-octadienylene, 1,4-octadienylene, 1,5-octadienylene, 1,6-octadienylene, 1,7-octadienylene, 2,4-octadienylene, 2,5-octadienylene, 2,6- Octadienylene, 2,7-octadienylene
• C 1-18 alkyl group refers to an alkyl group having 1 to 18 carbon atoms, which may be linear or branched; is optional. Specific examples of the C 1-18 alkyl group include the following in addition to the specific examples of the above-mentioned "C 1-17 alkyl group” (R A and R B ).
• the 1-18 alkyl groups are not limited to: [C 18 ] octadecyl, 1-methylheptadecyl, 2-methylheptadecyl, 3-methylheptadecyl, 4-methylheptadecyl, 5-methylheptadecyl, 6-methylheptadecyl, 7-methylheptadecyl, 8- methylheptadecyl, 9-methylheptadecyl, 10-methylheptadecyl, 11-methylheptadecyl, 12-methylheptadecyl, 13-methylheptadecyl, 14-methylheptadecyl, 15-methylheptadecyl, 16-methylhepta Decyl, 1-ethylhexadecyl, 2-ethylhexadecyl, 3-ethylhexadecyl, 4-ethylhexadecyl, 5-ethylhex
• the “optionally substituted C 1-18 alkyl group” of R 4 is preferably an “optionally substituted C 1-16 alkyl group”, more preferably an “optionally substituted C 1 ⁇ 14 alkyl group”.
• C 3-18 alkenyl group refers to an alkenyl group having 3 to 18 carbon atoms, which may be linear or branched, and in the case of a branched chain, the number and position of bonds in the branched chain is arbitrary, and the position of the carbon-carbon double bond is also arbitrary.
• Specific examples of the C 3-18 alkenyl group include the following in addition to the specific examples of the above-mentioned "C 3-17 alkenyl group” (R A and R B ).
• 3-18 alkenyl groups are not limited to: [C 18 ] 1-octadecenyl, 2-octadecenyl, 3-octadecenyl, 4-octadecenyl, 5-octadecenyl, 6-octadecenyl, 7-octadecenyl, 8-octadecenyl, 9-octadecenyl, 10-octadecenyl, 11-octadecenyl, 12- Octadecenyl, 13-octadecenyl, 14-octadecenyl, 15-octadecenyl, 16-octadecenyl, 17-octadecenyl. Since the C 3-18 alkenyl group contains one carbon-carbon double bond, it can have either cis-type or trans-type structure.
• the “optionally substituted C 3-18 alkenyl group” of R 4 is preferably an “optionally substituted C 5-18 alkenyl group”, more preferably an “optionally substituted C 7 ⁇ 18 alkenyl group”.
• C 15-18 alkadienyl group refers to an alkadienyl group having 15 to 18 carbon atoms, which may be linear or branched; is arbitrary, and the position of the carbon-carbon double bond is also arbitrary.
• Specific examples of the C 15-18 alkadienyl group include the following in addition to the specific examples included in the above-mentioned "C 15-17 alkadienyl group” (R A and R B ).
• the C 15-18 alkadienyl groups that can be formed are not limited to these examples: [C 18 ] 1,3-octadecadienyl, 1,4-octadecadienyl, 1,5-octadecadienyl, 1,6-octadecadienyl, 1,7-octadecadienyl, 1 ,8-octadecadienyl, 1,9-octadecadienyl, 1,10-octadecadienyl, 1,11-octadecadienyl, 1,12-octadecadienyl, 1,13-octadecadienyl dienyl, 1,14-octadecadienyl, 1,15-octadecadienyl, 1,16-octadecadienyl, 1,17-octadecadienyl, 2,4-octadecadieny
• each alkadienyl group (R 4 ) may have includes, for example, a halogen atom, a cyano group, a nitro group, an acyl group (e.g., formyl group, acetyl group, propionyl group), further substituted an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group
• substitutional substituent examples include a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, and a sulfanyl group.
• Z ⁇ is not particularly limited as long as it is an anion that can be electrically bonded when W is ⁇ N + R 11 R 12 R 13 , but pharmacologically acceptable anions are preferred.
• Suitable examples of Z- include halide ions (fluoride, chloride, bromide and iodide); anions of inorganic acids such as nitrate, sulfate, phosphate; formate, acetate ions, trifluoroacetate, phthalate, fumarate, oxalate, tartrate, maleate, citrate, succinate, malate, methanesulfonate, benzenesulfonate, p- Anions of organic acids such as toluenesulfonate; or aspartate.
• Compound (I) is a compound (I) according to embodiments of R A and R B , specifically R A and R B are each an optionally substituted C 1-17 alkyl group that does not contain an ester bond, a substituted an optionally substituted C 3-17 alkenyl group or an optionally substituted C 15-17 alkadienyl group (hereinafter referred to as “R A/B Group 1”), or —R 3 containing an ester bond -C(O)O-R 4 or -R 3 -OC(O)-R 4 (hereinafter referred to as "R A/B Group 2”) can be classified into the following three types. can.
• the first type is a compound (compound (II)) represented by the following formula (II).
• Formula (II) corresponds to formula (I) where R A and R B are R A/B Group 1 (does not contain an ester bond), where R A and R B are each represented by R They are denoted as A1 and R B1 . Definitions of symbols other than R A1 and R B1 in formula (II) are the same as in formula (I).
• Compound (II) is a cationic lipid with only one of the three side chains, ie the side chain containing R 2 C , and two ester bonds.
• the second type is a compound (compound (III)) represented by the following formula (III).
• Formula (III) is represented by formula (I) when RA is RA /B Group 1 (without an ester bond) and RB is RA /B Group 2 (with an ester bond) , and RA and RB in this case are denoted as RA1 and RB2 , respectively. Definitions of symbols other than R A1 and R B2 in formula (III) are the same as in formula (I).
• Compound (III) is a cationic lipid in which two of the three side chains, namely the side chain containing R 1 C and the side chain containing R 2 B2 , have two ester bonds.
• the third type is a compound represented by the following formula (IV) (compound (IV)).
• Formula (IV) is represented by formula (I) wherein R A and R B are -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4 (R A/B Group 2 ), and RA and RB in this case are denoted as RA2 and RB2 , respectively.
• R A2 , R B2 and R C are all defined as -R 3 -C(O)O-R 4 or -R 3 -OC(O)-R 4 . Definitions of symbols other than R A2 and R B2 in formula (IV) are the same as in formula (I).
• Compound (IV) is a cationic lipid in which all three side chains, i.e., the side chain containing RC , the side chain containing RB2 and the side chain containing RA2 , all have two ester bonds. be.
• the salt of compound (I) is preferably a pharmacologically acceptable salt, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a basic or acidic Examples include salts with amino acids.
• salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts and ammonium salts. Preferred are sodium salts, potassium salts, calcium salts and magnesium salts, and more preferred are sodium salts and potassium salts.
• Suitable examples of salts with organic bases include trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, Salts with dicyclohexylamine and N,N-dibenzylethylenediamine can be mentioned.
• salts with inorganic acids include salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid. Preferred are salts with hydrochloric acid and salts with phosphoric acid.
• Suitable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfonic acid. , and salts with p-toluenesulfonic acid.
• salts with basic amino acids include salts with arginine, lysine and ornithine.
• salts with acidic amino acids include salts with aspartic acid and glutamic acid.
• the compounds of the present invention can be used as cationic lipids.
• Cationic lipids can form complexes with multiple molecules in a solvent or dispersion medium.
• the complex may contain other components in addition to the compound of the present invention. Examples of such other components include other lipid components and nucleic acids.
• the other lipid components include structured lipids that can form lipid particles.
• structured lipids include: sterols (e.g., cholesterol, cholesterol esters, cholesterol hemisuccinate, etc.); Phospholipids (e.g., phosphatidylcholines (e.g., dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, lysophosphatidylcholine, dioleoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, dilinolenoylphosphatidylcholine, MC-1010 (NOF CORPORATION), MC-2020 (NOF CORPORATION) , MC-4040 (NOF CORPORATION), MC-6060 (NOF CORPORATION), MC-8080 (NOF CORPORATION), etc.), phosphatidylserine (e.g., dipalmitoylphosphati
• the ratio of the compound of the present invention and the structured lipid in the mixed lipid component forming the lipid particles of the present invention can be appropriately adjusted depending on the purpose and application.
• the ratio of the structured lipid is usually 0.008 to 4 mol, preferably 0.4 to 1.5 mol, per 1 mol of the compound of the present invention.
• the compound of the present invention is usually 1 to 4 mol
• the sterol is usually 0 to 3 mol
• the phospholipid is usually 0 to 2 mol
• the polyethylene glycol lipid is usually The ratio is between 0 and 1 mol.
• a more preferred embodiment when the compound of the present invention is used in combination with other lipid components is 1 to 1.5 mol of the compound of the present invention, 0 to 1.25 mol of sterols, and 0 to 0.5 mol of phospholipid. and polyethylene glycol lipid in a ratio of 0-0.125 moles.
• the compound of the present invention can be used to produce the lipid particles of the present invention.
• the lipid particle of the present invention means a complex that does not contain nucleic acid among the above complexes.
• the shape of the lipid particles of the present invention is not particularly limited. complexes, homogeneously or non-uniformly dispersed complexes in a dispersion medium, and the like.
• the lipid particles of the present invention include, for example, the lipid particles and nucleic acids (especially substances useful for pharmaceutical use or research purpose use) a nucleic acid) and a composition of the invention.
• the composition of the invention can be used as a medicine or reagent. In the composition of the present invention, it is preferable that as much of the nucleic acid as possible is encapsulated in the lipid particles (that is, the encapsulation rate is high).
• nucleic acid may be any molecule as long as it is a polymer of nucleotides and molecules having functions equivalent to the nucleotides. Polymers in which nucleotides and deoxyribonucleotides are mixed, and nucleotide polymers containing nucleotide analogues can be mentioned, and nucleotide polymers containing nucleic acid derivatives can also be used. Also, the nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. A double-stranded nucleic acid also includes a double-stranded nucleic acid in which one strand hybridizes to the other strand under stringent conditions.
• nucleotide analogue compared with RNA or DNA, ribonuclease is used for improving or stabilizing nuclease resistance, for increasing affinity with complementary strand nucleic acid, for increasing cell permeability, or for visualization.
• Any molecule can be used as long as it is a molecule obtained by modifying nucleotides, deoxyribonucleotides, RNA or DNA.
• Nucleotide analogues may be naturally occurring molecules or non-natural molecules, and examples thereof include sugar moiety-modified nucleotide analogues, phosphodiester bond-modified nucleotide analogues, and the like.
• the sugar moiety-modified nucleotide analogue may be any one obtained by adding or substituting an arbitrary chemical structural substance to part or all of the chemical structure of the sugar of a nucleotide.
• the phosphodiester bond-modified nucleotide analogue may be any one obtained by adding or substituting an arbitrary chemical substance to part or all of the chemical structure of the phosphodiester bond of a nucleotide.
• Examples include nucleotide analogs substituted with phosphorothioate bonds, nucleotide analogs substituted with N3'-P5' phosphoramidate bonds, etc. [Cell Engineering, 16, 1463-1473 (1997)] [RNAi method and antisense method, Kodansha (2005)].
• nucleic acid derivative a different chemical is added to the nucleic acid in order to improve the nuclease resistance compared to the nucleic acid, to stabilize the nucleic acid, to increase the affinity with the complementary strand nucleic acid, to increase the cell permeability, or to make the nucleic acid visible.
• Any molecule may be used as long as it is a molecule to which a substance is added, and specific examples thereof include 5′-polyamine addition derivatives, cholesterol addition derivatives, steroid addition derivatives, bile acid addition derivatives, vitamin addition derivatives, Cy5 addition derivatives, and Cy3 addition derivatives. , 6-FAM-added derivatives, and biotin-added derivatives.
• Nucleic acids in the present invention are not particularly limited. It may also be a nucleic acid for the purpose of ⁇ treating a disease, etc.''), or a nucleic acid for regulating the expression of a desired protein that does not contribute to the treatment, etc. of a disease but is useful for research. may
• disease-related genes or polynucleotides
• McKusick-Nathans Institute of Genetic Medicine Johns Hopkins University (Baltimore, Md.) and National Available from the Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), and the like.
• nucleic acids in the present invention include single-stranded DNA, double-stranded DNA, siRNA, miRNA, miRNA mimic, antisense nucleic acid, ribozyme, mRNA, gRNA, decoy nucleic acid, aptamer, and the like. It may be a modified analogue or derivative.
• the nucleic acid is preferably DNA such as single-stranded DNA, double-stranded DNA, siRNA, mRNA, gRNA, RNA, or analogues or derivatives obtained by artificially modifying these.
• siRNA means a double-stranded RNA of 10 to 30 bases, preferably 15 to 25 bases, or an analogue thereof containing a complementary sequence.
• the siRNA preferably has 1-3, more preferably 2 overhangs at the 3' end.
• the complementary sequence portions may be fully complementary or may contain non-complementary bases, but are preferably fully complementary.
• the siRNA in the present invention is not particularly limited, and for example, siRNA for knocking down the gene expression of disease-related genes can be used.
• a disease-associated gene refers to any gene or polynucleotide that produces a transcription or translation product at an abnormal level or in an abnormal morphology in cells derived from diseased tissue compared to non-disease control tissue or cells. .
• siRNA for regulating the expression of a desired protein useful for research can also be used.
• mRNA means RNA containing a base sequence translatable into protein.
• the mRNA in the present invention is not particularly limited as long as it can express a desired protein in cells.
• the above-mentioned mRNA is preferably mRNA that is useful for pharmaceutical use (e.g., disease treatment use) and/or research purpose use. mRNA for.
• gRNA means guide RNA corresponding to the CRISPR system.
• the gRNA in the present invention may be in the form of one RNA in which crRNA and tracrRNA are linked, that is, chimeric RNA (also called single guide RNA, sgRNA, etc.), It may be in the form of single RNAs (a combination of two RNAs or a combination of more RNAs).
• DNA means DNA containing a nucleotide sequence that can be transcribed into mRNA.
• the DNA in the present invention is not particularly limited as long as it is DNA that can be transcribed into desired mRNA in cells.
• the above DNA is preferably DNA that is useful for medical use (e.g., gene therapy use) and/or research purpose use.
• Examples of such DNA include plasmid DNA (pDNA), single-stranded DNA (ssDNA ), nanoplasmid, minicircle DNA, closed-end DNA (ceDNA), doggy bone DNA (dbDNA), ministring DNA (msDNA), linear DNA (linDNA) and the like.
• DNAs include, for example, DNAs for intracellularly expressing marker proteins such as luciferase.
• the DNA in the present invention may contain enhancers or promoters.
• the enhancer or promoter in the present invention is not particularly limited as long as it can control transcription to desired mRNA in cells.
• Examples of the promoter or enhancer include ApoE/hAAT enhancer or promoter, CAG promoter, CMV (Cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter or enhancer, SV40 promoter, DHFR (Dihydrofolate reductase) promoter, EF1 ⁇ promoter, EF and CBA (Chicken ⁇ -Actin) promoter, PGK (Phosphoglycerate Kinase) promoter, hSYN (human synapsin) promoter, MND promoter, RSV (Rous Sarcoma Virus LTR) promoter, Chicken beta actin + intron promoter, TRE (tetracyc line-responsive element) promoter , UBC (Ubiquitin C) promoter,
• the above diseases are not particularly limited, and include, for example, the following diseases (1) to (7).
• "( )" indicates an example of a disease-related gene, unless a specific disease example is described.
• Nucleic acids in the present invention also include nucleic acids that regulate the expression levels of these disease-associated genes (or proteins encoded by them).
• Hematologic disease anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT), lymphoma bulbar syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5), bleeding disorders (TBXA2R, P2RX1, P2X1), factor H and factor H-like 1 deficiency (HF1, CFH, HUS) , Factor V and Factor VIII deficiency (MCFD2), Factor VII deficiency (F7), Factor X deficiency (F10), Factor XI deficiency (F11), Factor XII deficiency (F12, HAF), Factor XIIIA deficiency (F13A1, F13A), factor XIIIB
• Inflammatory/immune diseases AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1), autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A), combined immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4), HIV infection (CCL5, SCYA5, D17S135E, TCP228, IL10, CSIF, CMKBR2, CCR2, DMKBR5, CCCKR5, CCR5), immunodeficiency (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (IL10, IL-1, IL-13, IL -17,
• ALS SOD1, ALS2, STEX, FUS, TARDBP, VEGF
• Alzheimer's disease APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU , URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3), autism (BZRAP1, MDGA2, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2), fragile X syndrome (FMR2, FXR1, FXR2, mGLUR5), Huntington's disease (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP,
• eye diseases age-related macular degeneration (Abcr, Ccl2, cp, Timp3, cathepsin D, Vldlr, Ccr2), cataracts (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BSFP2, CP49, CP47, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1), corneal opacities (APOA1, TGFB1, CSD2, CDGG1, CSD, BIGH3, CDG2, TASTD2, TROP2, M1S1, VSX1,
• Neoplastic disease malignant tumor, neovascular glaucoma, infantile hemangioma, hereditary angioedema, multiple myeloma, chronic sarcoma, metastatic melanoma, Kaposi's sarcoma, vascular proliferation, cachexia, metastasis of breast cancer etc., cancer (e.g., colon cancer (e.g., familial colon cancer, hereditary nonpolyposis colon cancer, gastrointestinal stromal tumor, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.) , mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), breast cancer (e.g., invasive duct
• composition of the present invention as a medicament can be produced by a method known in the pharmaceutical technical field using a pharmaceutically acceptable carrier.
• Formulations of the above-mentioned medicament include, for example, preparations for parenteral administration (e.g., liquids such as injections) containing conventional auxiliaries such as buffers and/or stabilizers, and ointments containing conventional pharmaceutical carriers. , creams, solutions or salves.
• compositions of the present invention can be used to introduce active ingredients into many types of cells, tissues or organs.
• Cells to which the composition of the present invention can be applied include, for example, splenocytes, nerve cells, glial cells, pancreatic B cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, and fibroblasts.
• muscle cells e.g., skeletal muscle cells, cardiomyocytes, myoblasts, muscle satellite cells
• adipocytes immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, leukocytes, basophils, eosinophils, monocytes, megakaryocytes), synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, egg cells, sperm cells , or progenitor cells that can be induced to differentiate into these cells, stem cells (including, for example, induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells)), blood cells, oocytes, and fertilized eggs.
• stem cells including, for example, induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells)
• iPS cells induced pluripotent stem cells
• ES cells embryonic stem cells
• tissue or organ to which the composition of the present invention can be applied includes any tissue or organ in which the above cells are present, such as the brain, each part of the brain (e.g., olfactory bulb, amygdaloid nucleus, cerebral basal ball, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, corpus callosum, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g.
• intestine large intestine, small intestine
• blood vessel heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, prostate, Testicles, testes, ovaries, placenta, uterus, bones, joints and skeletal muscle.
• cells, tissues, or organs may be cancerous cancer cells, cancer tissues, or the like.
• compositions of the present invention are stable, low-toxic and safe to use.
• administration subjects e.g., humans or non-human mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys)
• the composition may be administered such that an effective amount of the nucleic acid is delivered to the targeted cells.
• composition of the present invention when used in vivo or used as a medicine, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), powders, granules, capsules (soft capsules, microcapsules), liquids, lozenges, syrups, emulsions, suspensions, injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), external preparations (e.g., , nasal preparations, transdermal preparations, ointments) suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), drips, etc. can be safely administered orally or parenterally (eg, topically, rectally, intravenously, etc.).
• These formulations may be controlled release formulations such as immediate release formulations or sustained release formulations (eg,
• the raw materials and reagents used in each step of the manufacturing method below, as well as the resulting compound, may each form a salt.
• Such salts include, for example, those similar to the above-described salts of the compound of the present invention.
• the compound obtained in each step is a free compound, it can be converted into the desired salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted into the free form or other desired salt by known methods.
• the compound obtained in each step can be used in the next reaction after being obtained as a reaction solution or as a crude product, or the compound obtained in each step can be concentrated from the reaction mixture in accordance with a conventional method. , crystallization, recrystallization, distillation, solvent extraction, fractionation, chromatography, and the like.
• the raw materials and reagent compounds for each process are commercially available, the commercially available products can be used as they are.
• the reaction time may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually 1 minute to 72 hours, preferably 10 minutes to 48 hours.
• the reaction temperature may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually -78°C to 300°C, preferably -78°C to 150°C.
• the pressure may vary depending on the reagents and solvents used, but unless otherwise specified, it is usually 1 to 20 atmospheres, preferably 1 to 3 atmospheres.
• a Microwave synthesizer such as Initiator manufactured by Biotage may be used.
• the reaction temperature may vary depending on the reagents and solvents used, but is generally room temperature to 300°C, preferably room temperature to 250°C, more preferably 50°C to 250°C, unless otherwise specified.
• the reaction time may vary depending on the reagents and solvents used, and is generally 1 minute to 48 hours, preferably 1 minute to 8 hours, unless otherwise specified.
• the reagent is used in an amount of 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, relative to the substrate unless otherwise specified.
• the reagent is used in an amount of 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalent, relative to the substrate.
• the solvent amount is used for the reagent.
• Alcohols methanol, ethanol, isopropanol, isobutanol, tert-butyl alcohol, 2-methoxyethanol, etc.; Ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether and the like; Aromatic hydrocarbons: chlorobenzene, toluene, xylene, etc.; saturated hydrocarbons: cyclohexane, hexane, heptane, etc.; Amides: N,N-dimethylformamide, N-methylpyrrolidone, etc.; Halogenated hydrocarbons: dichloromethane, carbon tetrachloride, etc.; Nitriles: acetonitrile and the like; Sulfoxides: dimethylsulfoxide and the like; aromatic organic bases: pyridine and the like; Acid anhydrides: acetic an
• Inorganic bases sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.
• Basic salts sodium carbonate, calcium carbonate, sodium bicarbonate, etc.
• Organic bases triethylamine, diethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[ 5.4.0]-7-undecene, imidazole, piperidine and the like
• metal alkoxides sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide and the like
• Alkali metal hydrides sodium hydride and the like
• Metal amides sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, etc.
• Organic lithiums n-butyllithium, sec-butyllith
• an acid or acidic catalyst is used in the reaction of each step, for example, the acids and acidic catalysts shown below, or the acids and acidic catalysts described in Examples are used.
• Inorganic acids hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, etc.
• Organic acids acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, etc.
• Lewis acid boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous iron chloride, etc.
• the reaction in each step is performed by a known method, for example, 5th Edition Jikken Kagaku Koza, Vol. 13-19 (edited by The Chemical Society of Japan); Association); Fine Organic Chemistry Revised 2nd Edition (L. F. Tietze, Th. Eicher, Nankodo); Revised Organic Personal Name Reaction Mechanism and Points (by Hideo Togo, Kodansha); ORGANIC SYNTHESES Collective Volume I-VII (John Wiley & Sons Inc); Modern Organic Synthesis in the Laboratory A Collection of Standard Experimental Procedures (written by Jie Jack Li, published by OXFORD UNIVERSITY); Comprehensive Heterocyclic Chemistry III, Vol. 1 to Vol.
• the protection or deprotection reaction of the functional group is carried out by a known method, for example, Wiley-Interscience, 2007, “Protective Groups in Organic Synthesis, 4th Ed.” (Theodora W. Greene, Peter G.M. Wuts) the method described in "Protecting Groups 3rd Ed.” (by PJ Kocienski) published by Thieme in 2004, or according to the method described in Examples.
• Protective groups for hydroxyl groups such as alcohols and phenolic hydroxyl groups include, for example, ether types such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, and tetrahydropyranyl ether. protective groups; carboxylic acid ester-type protective groups such as acetate; sulfonic acid ester-type protective groups such as methanesulfonate; carbonate ester-type protective groups such as t-butyl carbonate;
• protective groups for carbonyl groups of aldehydes include acetal-type protective groups such as dimethylacetal; and cyclic acetal-type protective groups such as cyclic 1,3-dioxane.
• protective groups for carbonyl groups of ketones include, for example, ketal-type protective groups such as dimethyl ketal; cyclic ketal-type protective groups such as cyclic 1,3-dioxane; oxime-type protective groups such as O-methyloxime; hydrazone-type protecting groups such as dimethylhydrazone;
• protective groups for carboxyl groups include ester-type protective groups such as methyl ester and benzyl ester; and amide-type protective groups such as N,N-dimethylamide.
• thiol-protecting groups include ether-type protecting groups such as benzylthioether; and ester-type protecting groups such as thioacetate, thiocarbonate, and thiocarbamate.
• Removal of protecting groups is carried out by known methods such as acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halides (e.g. , trimethylsilyl iodide, trimethylsilyl bromide) or a reduction method.
• acids acids, bases, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, trialkylsilyl halides (e.g. , trimethylsilyl iodide, trimethylsilyl bromide) or a reduction method.
• the reducing agent used includes lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBAL-H), and sodium borohydride.
• metal hydrides such as tetramethylammonium triacetoxyborohydride; boranes such as borane tetrahydrofuran complex; Raney nickel; Raney cobalt; hydrogen;
• catalysts such as palladium-carbon, Raney nickel and Raney cobalt can be used in the presence of hydrogen or formic acid.
• the oxidizing agent used includes peracids such as m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, and t-butyl hydroperoxide; perchlorates; chlorates such as sodium chlorate; chlorites such as sodium chlorite; periodic acids such as sodium periodate; high-valent iodine reagents such as iodosylbenzene; reagents containing manganese such as potassium manganate; lead compounds such as lead tetraacetate; reagents containing chromium such as pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), Jones reagent; N-bromosuccinimide (NBS) sulfur trioxide/pyridine complex; osmium tetroxide; zelene dioxide; 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and the like.
• MCPBA m-chloroperbenz
• the radical initiator used includes azo compounds such as azobisisobutyronitrile (AIBN); 4-4'-azobis-4-cyanopentanoic acid (ACPA) water-soluble radical initiators such as; triethylboron in the presence of air or oxygen; and benzoyl peroxide.
• azo compounds such as azobisisobutyronitrile (AIBN); 4-4'-azobis-4-cyanopentanoic acid (ACPA) water-soluble radical initiators such as; triethylboron in the presence of air or oxygen; and benzoyl peroxide.
• the radical reactant used include tributylstannane, trimethylsilylsilane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, and samarium iodide.
• the Wittig reagents used include alkylidene phosphoranes and the like.
• Alkylidenephospholanes can be prepared by known methods, for example, by reacting a phosphonium salt with a strong base.
• the reagents used include phosphonoacetic esters such as methyl dimethylphosphonoacetate and ethyl diethylphosphonoacetate; bases such as alkali metal hydrides and organic lithiums. mentioned.
• the reagents used include Lewis acids, acid chlorides or alkylating agents (eg, alkyl halides, alcohols, olefins, etc.).
• an organic acid or an inorganic acid can be used instead of a Lewis acid, and an acid anhydride such as acetic anhydride can be used instead of an acid chloride.
• nucleophiles eg, amines, imidazole, etc.
• bases eg, basic salts, organic bases, etc.
• a base used to generate a carbanion examples include organic lithiums, metal alkoxides, inorganic bases, organic bases and the like.
• examples of the Grignard reagent include arylmagnesium halides such as phenylmagnesium bromide; and alkylmagnesium halides such as methylmagnesium bromide and isopropylmagnesium bromide.
• the Grignard reagent can be prepared by a known method, for example, by reacting an alkyl halide or aryl halide with metallic magnesium using ether or tetrahydrofuran as a solvent.
• reagents include active methylene compounds sandwiched between two electron-withdrawing groups (e.g., malonic acid, diethyl malonate, malononitrile, etc.) and bases (e.g., organic bases, metal alkoxides, inorganic bases) are used.
• two electron-withdrawing groups e.g., malonic acid, diethyl malonate, malononitrile, etc.
• bases e.g., organic bases, metal alkoxides, inorganic bases
• phosphoryl chloride and amide derivatives are used as reagents.
• the azidation agent used includes diphenylphosphoryl azide (DPPA), trimethylsilyl azide, sodium azide, and the like.
• DPPA diphenylphosphoryl azide
• DBU 1,8-diazabicyclo[5,4,0]undec-7-ene
• the reducing agent used includes sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen, and formic acid.
• carbonyl compounds used include paraformaldehyde, aldehydes such as acetaldehyde, and ketones such as cyclohexanone.
• amines to be used include primary amines such as ammonia and methylamine; secondary amines such as dimethylamine.
• azodicarboxylic acid esters eg, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), etc.
• triphenylphosphine eg, triphenylphosphine
• the reagents used include esters, acyl halides such as acid chlorides and acid bromides; acid anhydrides, active esters, Examples thereof include activated carboxylic acids such as sulfate esters.
• Carboxylic acid activators include carbodiimide condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N,N'-dicyclohexylcarbodiimide (DCC); ,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-n-hydrate (DMT-MM) and other triazine condensing agents; 1,1-carbonyldiimidazole ( Carbonate condensing agent such as CDI); diphenyl phosphate azide (DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent) thionyl chloride; lower alkyl haloformate such as ethyl chloroformate; O-(7
• the metal catalysts used include palladium (II) acetate, tetrakis(triphenylphosphine)palladium (0), dichlorobis(triphenylphosphine)palladium (II), dichlorobis(triethyl palladium compounds such as phosphine)palladium(II), tris(dibenzylideneacetone)dipalladium(0), 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) chloride, palladium(II) acetate; phenylphosphine) nickel compounds such as nickel (0); rhodium compounds such as tris(triphenylphosphine)chloride rhodium (III); cobalt compounds; copper compounds such as copper oxide and copper iodide (I); be done.
• a base may be added to the reaction, and examples of such bases include inorganic bases,
• phosphorus pentasulfide is typically used as the thiocarbonylating agent.
• phosphorus pentasulfide is typically used as the thiocarbonylating agent.
• -1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lowesson's reagent) and other reagents having a 1,3,2,4-dithiadiphosphetane-2,4-disulfide structure may be used.
• the halogenating agent used includes N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, sulfuryl chloride, and the like. is mentioned. Furthermore, the reaction can be accelerated by adding heat, light, radical initiators such as benzoyl peroxide, azobisisobutyronitrile, etc. to the reaction.
• the halogenating agents used include hydrohalic acids and acid halides of inorganic acids. Phosphorus chloride and the like, and for bromination, 48% hydrobromic acid and the like.
• a method of obtaining an alkyl halide from an alcohol by the action of triphenylphosphine and carbon tetrachloride or carbon tetrabromide may be used.
• a method of synthesizing an alkyl halide through a two-step reaction such as reaction with lithium bromide, lithium chloride or sodium iodide after converting an alcohol to a sulfonate ester may be used.
• reagents used include alkyl halides such as ethyl bromoacetate; and phosphites such as triethylphosphite and tri(isopropyl)phosphite.
• sulfonating agents used include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride, p-toluenesulfonic anhydride, and trifluoromethanesulfonic anhydride. things, etc.
• an acid or base is used as a reagent.
• formic acid, triethylsilane, or the like may be added in order to reductively trap the t-butyl cation produced as a by-product.
• dehydrating agents used include sulfuric acid, diphosphorus pentoxide, phosphorus oxychloride, N,N'-dicyclohexylcarbodiimide, alumina, and polyphosphoric acid.
• an acid may be used when performing a decarboxylation reaction.
• acids include inorganic acids and organic acids.
• a base may be used when performing a nucleophilic substitution reaction in each step.
• Examples of the base include metal alkoxides, inorganic bases, organic bases and the like.
• Compound (I) can be produced, for example, by the following method.
• a salt of compound (I) can be obtained by appropriate mixing with an inorganic base, an organic base, an organic acid, or a basic or acidic amino acid.
• Scheme 1 shows an example of a method for producing compound (I).
• P1 represents a protecting group, and other symbols are the same as in Formula (I) (the same applies to other schemes related to Scheme 1).
• L represents a leaving group, and R A , R B and R C may be interchanged with each other as appropriate.
• R A and/or R B of compound (I) in Scheme 1 is R A/B Group 1 (optionally substituted C 1-17 alkyl group, optionally substituted C 3-17 alkenyl group, optionally substituted C 15-17 alkadienyl group), that is, R A and/or R B are R A1 represented by formula (II) or formula (III) and/or R 1 B1 , the method for synthesizing the compound used for esterification (R 1 A1 or R 1 B1 -COOH).
• P 2 , P 3 , P 4 and P 5 represent protecting groups
• R a , R b , R c , R d and R g are H, an optionally substituted alkyl group
• a substituted represents an optionally substituted alkenyl group or an optionally substituted alkadienyl group, and constitutes a part of RA and/or RB .
• the number of carbon atoms, substituents and structures of R a , R b , R c , R d and R g are appropriately adjusted according to the target structure of R A and/or R B .
• R A and/or R B of compound (I) in Scheme 1 is R A/B Group 2 (-R -C (O)O-R 4 or -R -OC ( O)—R 4 ), i.e. when R A and/or R B are R A2 and/or R B2 represented by formula (II) or formula (III), for esterification (R A2 or R B2 -COOH) used for and a compound (R C -COOH) used for esterification of R C of compound (I).
• P6 and P7 represent protecting groups.
• Scheme 4 shows a method for synthesizing the compound (R 4 —OH) used for esterification in Scheme 3.
• P 4 represents a protecting group
• R a , R b , R c and R d are H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or a substituted represents a good alkadienyl group and constitutes part of R4 .
• the number of carbon atoms, substituents and structures of R a , R b , R c and R d are appropriately adjusted according to the desired structure of R 4 .
• Scheme 5 shows a method for synthesizing the compound (R 4 —COOH) used for the esterification in Scheme 3.
• P 4 and P 5 represent protecting groups
• R a , R b , R c and R d are H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or a substituted represents an alkadienyl group which may be substituted and constitutes part of R 4 .
• the number of carbon atoms, substituents and structures of R a , R b , R c and R d are appropriately adjusted according to the desired structure of R 4 .
• Scheme 6 shows a method for synthesizing the compound (W—X—COOH) used for esterification in Scheme 1.
• R e represents an optionally substituted alkylene group
• L represents a leaving group
• P 8 represents a protecting group.
• the number of carbon atoms, substituents and structure of R e are appropriately adjusted according to the desired structure of X.
• Scheme 7 shows a method for synthesizing compound (I).
• R f represents an optionally substituted alkylene group
• P 9 represents a protecting group.
• the number of carbon atoms, substituents and structure of Rf are appropriately adjusted according to the target structure of W.
• a method for producing lipid particles containing the compound of the present invention and a composition for nucleic acid introduction (transfection) containing the lipid particles and a nucleic acid as an active ingredient are described below.
• the lipid particles of the present invention are obtained by mixing the compound of the present invention (compound (I) or a salt thereof) as a cationic lipid with other lipid components, if necessary, and then using a known method for preparing lipid particles from the lipid components.
• a lipid particle dispersion can be produced by dissolving the above (mixed) lipid component in an organic solvent and mixing the resulting organic solvent solution with water or a buffer solution (eg, emulsification method).
• the mixing can be performed using a microfluidic mixing system (for example, NanoAssemblr device (Precision NanoSystems)).
• the resulting lipid particles may be subjected to desalting or dialysis and sterile filtration.
• pH adjustment and osmotic pressure adjustment may be performed as necessary.
• the compound of the present invention can have multiple structures depending on the combination of definitions of the symbols (substituents, etc.) in formula (I).
• one type of compound (I) having a specific structure or a salt thereof may be used alone, or a plurality of types of compounds having different structures (I) or The salts may be mixed and used.
• “Other lipid components” include the structural lipids described above, such as sterols, phospholipids, and polyethylene glycol lipids. “Other lipid components” are used, for example, in an amount of 0.008 to 4 mol per 1 mol of the compound of the present invention.
• the compounds of the present invention are preferably used in admixture with other lipid components (particularly cholesterol, phosphatidylcholine and polyethylene glycol lipids).
• a preferred embodiment when the compound of the present invention is used in combination with other lipid components is 1 to 4 mol of the compound of the present invention, 0 to 3 mol of sterols, 0 to 2 mol of phospholipid, and 0 to 1 mol of polyethylene glycol lipid.
• a more preferred embodiment when the compound of the present invention is used in combination with other lipid components is 1 to 1.5 mol of the compound of the present invention, 0 to 1.25 mol of sterols, and 0 to 0.5 mol of phospholipid. and polyethylene glycol lipid 0-0.125 moles.
• the concentration of the compound of the present invention or the mixture of the compound of the present invention and other lipid components in the organic solvent solution described above is preferably 0.5-100 mg/mL.
• organic solvents examples include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, and mixtures thereof.
• Organic solvents may contain 0-20% water or buffers.
• buffers include acidic buffers (eg, acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, phosphate buffer) and neutral buffers (eg, 4-(2 -hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, phosphate buffered saline (PBS)).
• acidic buffers eg, acetate buffer, citrate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, phosphate buffer
• neutral buffers eg, 4-(2 -hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer, phosphate buffered saline (PBS)
• the mixed solution (mixed solution of organic solvent solution and water or buffer solution) flow rate is, for example, 0.01 to 20 mL/min, preferably 0.1 to 10 mL/min, and the temperature is, for example, 5 to 60°C, preferably 15 to 45°C.
• composition of the present invention can be produced as a nucleic acid-containing lipid particle dispersion by adding the nucleic acid to water or a buffer when producing the lipid particles or the lipid particle dispersion.
• the nucleic acid is preferably added so that the concentration of the nucleic acid in water or buffer solution is, for example, 0.01-20 mg/mL, preferably 0.05-2.0 mg/mL.
• composition of the present invention can also be produced as a lipid particle dispersion containing an active ingredient by mixing lipid particles or a lipid particle dispersion with a nucleic acid or an aqueous solution thereof by a known method.
• a lipid particle dispersion can be prepared by dispersing lipid particles in a suitable dispersion medium.
• an aqueous solution of the active ingredient can be prepared by dissolving the active ingredient in a suitable solvent.
• the content of the compound of the present invention in the composition of the present invention excluding the dispersion medium and solvent is usually 10-70% by weight, preferably 40-70% by weight.
• the content of the nucleic acid in the composition of the present invention excluding the dispersion medium and solvent is usually 0.1-25% by weight, preferably 1-20% by weight.
• the dispersion medium of the lipid particle dispersion or the dispersion containing the composition can be replaced with water or a buffer solution by dialysis.
• Dialysis is performed at 4° C. to room temperature using an ultrafiltration membrane with a molecular weight cutoff of 10 to 20K. Repeat dialysis may be performed. Tangential flow filtration (TFF) may be used to displace the dispersion medium.
• pH adjustment and osmotic pressure adjustment may be performed as necessary.
• pH adjusters include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate.
• osmotic pressure regulators include inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate, and polyols such as glycerol, mannitol, and sorbitol. and sugars such as glucose, fructose, lactose and sucrose.
• the pH is usually adjusted to 6.5-8.0, preferably 7.0-7.8.
• Osmotic pressure is preferably adjusted to 250-350 Osm/kg.
• composition of the present invention may contain components other than lipid particles and nucleic acids, if necessary.
• Such ingredients include, for example, appropriate amounts of stabilizers and antioxidants.
• stabilizers include, but are not limited to, sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose.
• Antioxidants include, for example, ascorbic acid, uric acid, cysteine, tocopherol congeners (vitamin E, four isomers of tocopherol ⁇ , ⁇ , ⁇ , ⁇ , etc.), EDTA, cysteine, and the like.
• a method for analyzing lipid particles containing the compound of the present invention and a composition containing the lipid particles and a nucleic acid as an active ingredient will be described below.
• the particle size of the lipid particles (in the composition) can be measured by known means.
• the Z-average particle size can be calculated by cumulant analysis of the autocorrelation function using a particle size measuring device based on dynamic light scattering measurement technology, Zetasizer Nano ZS (Malvern Instruments).
• the particle size (average particle size) of the lipid particles (in the composition) is, for example, 10 to 200 nm, preferably 60 to 170 nm.
• the concentration and encapsulation rate of nucleic acids (eg, siRNA, mRNA) in the composition of the present invention can be measured by known means.
• nucleic acids eg, siRNA, mRNA
• Quant-iT TM RiboGreen registered trademark
• Invitrogen is used to fluorescently label nucleic acids, and the concentration and encapsulation rate can be determined by measuring the fluorescence intensity.
• concentration of nucleic acid in the composition can be calculated using a standard curve prepared from aqueous nucleic acid solutions of known concentration, and the encapsulation rate is Triton-X100 (surfactant for disintegrating lipid particles).
• the concentration of nucleic acids in the composition refers to the total concentration of nucleic acids encapsulated in lipid particles and nucleic acids that are not encapsulated, and the encapsulation ratio is the total concentration of nucleic acids in the composition that is contained in lipid particles. Refers to the percentage of things that are sealed.
• Root temperature in the following examples usually indicates about 10°C to about 35°C.
• the ratios shown for mixed solvents are volume ratios unless otherwise specified. % indicates % by weight unless otherwise specified.
• Elution in column chromatography in the Examples was performed under observation by TLC (Thin Layer Chromatography) unless otherwise specified.
• TLC Thin Layer Chromatography
• the solvent used as the elution solvent in the column chromatography was used as the developing solvent.
• a UV detector was employed for detection, and observation was performed using a TLC coloring reagent as necessary.
• silica gel column chromatography aminopropylsilane-bonded silica gel was used when NH was used, and 3-(2,3-dihydroxypropoxy)propylsilane-bonded silica gel was used when Diol was used.
• preparative HPLC high performance liquid chromatography
• octadecyl-bonded silica gel was used when C18 was indicated.
• the ratios shown for the elution solvents are volume ratios unless otherwise specified.
• 1 H NMR was measured by Fourier transform NMR.
• ACD/SpecManager (trade name) software or the like was used for 1 H NMR analysis. Peaks with very slow proton peaks, such as hydroxyl groups and amino groups, are sometimes not described.
• MS was measured by LC/MS or MALDI/TOFMS.
• ESI method, APCI method, or MALDI method was used as the ionization method.
• CHCA was used as the matrix. The data described the measured value (found).
• Molecular ion peaks are usually observed, but they may be observed as multiply charged ions or fragment ions. In the case of salts, a molecular ion peak, cationic species, anionic species, or fragment ion peak of the free form is usually observed.
• MS mass spectrum M: molar concentration N: normality CDCl 3 : deuterated chloroform DMSO-d 6 : deuterated dimethylsulfoxide 1 H NMR: proton nuclear magnetic resonance LC/MS: liquid chromatograph mass spectrometer ESI: electrospray ionization, electrospray ionization APCI: atmospheric pressure chemical ionization, atmospheric pressure chemical ionization MALDI: Matrix-assisted laser desorption/ionization, Matrix Assisted laser desorption ionization TOFMS: Time-of-flight mass spectrometry, time-of-flight mass spectrometry CHCA: ⁇ -cyano-4-hydroxycinnamic acid DCM: dichloromethane DMA: N,N-dimethylacetamide DMF: N,N-dimethyl Formamide THF: tetrahydrofuran MeOH: methanol EtOH: ethanol DMAP: 4-d
• A was extracted with a mixed solvent of ethyl acetate and heptane, washed with 20% aqueous citric acid solution, 5% aqueous sodium hydrogencarbonate solution, saturated brine and water, and concentrated under reduced pressure (C).
• B was extracted with a mixed solvent of water and DMA, and the resulting aqueous layer was extracted with a mixed solvent of ethyl acetate and heptane, then washed with 20% aqueous citric acid solution, 5% aqueous sodium bicarbonate solution, saturated brine and water. and concentrated under reduced pressure (D).
• C and D were mixed to give the title compound (78g).
• Acetic acid was added to the mixture to neutralize the solution and stirred for 10 minutes. It was poured into water and extracted with ethyl acetate/petroleum ether. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether) to give the title compound (3.8 g).
• A) 5-Hexyl-2,2-dimethyl-1,3-dioxane-4,6-dione A mixture of 2,2-dimethyl-1,3-dioxane-4,6-dione (1 g) and THF (15 mL) was added 60% sodium hydride (305 mg) at 0° C. over 30 minutes. After stirring for 30 minutes, 1-iodohexane (1.47 g) was added dropwise and the mixture was stirred at room temperature for 14 hours. Aqueous ammonium chloride solution was added to the mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure.
• Methyl octylpropanedioate tert-butyl Methyl propanedioate tert-butyl (8g) was added to a mixture of 60% sodium hydride (2.8g) and DMF (300mL) at 0°C. After stirring for 1 hour, 1-iodooctane (7.7 g) was added and stirred at room temperature for 12 hours. Water was added to the mixture and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure.
• the resulting dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4° C. for 23 hours using a Slyde-A-Lyzer (20K molecular weight cutoff, Thermo scientific). Subsequently, filtration was performed using a 0.2 ⁇ m syringe filter (Iwaki), 60% sucrose/PBS solution was added, and the mixture was stored at ⁇ 80° C. as a 20% sucrose/PBS solution. Table 2 also shows the results of analyzing the particle size and encapsulation rate of the DNA-encapsulating lipid nanoparticles.
• Test Example 1 Test Example for Evaluation of Mouse Bioluminescence Imaging
• ICrl:CD1(ICR) mice via the tail vein (10 mL/kg iv).
• luciferin 15 mg/mL was administered to the mice (10 mL/kg ip) and set in an IVIS (In Vivo Imaging System) device (manufactured by PerkinElmer) under isoflurane inhalation anesthesia.
• Luminescent images of the ventral side (supine) of the mice were taken 15 minutes after administration of luciferin. The amount of luminescence was quantified using IVIS software and evaluated using the value of Total Flux.
• Table 2 also shows the measurement results.
• the light emission amount is divided into three levels of A, B, and C in descending order.
• A indicates a light emission amount of 3.0E+07 or more
• B indicates a light emission amount of 1.0E+0.7 or more and less than 3.0E+07
• C indicates a light emission amount of less than 1.0E+07.
• a high amount of luminescence was observed when using any of the cationic lipids of the Examples.
• the compounds, lipid particles or compositions of the present invention enable efficient introduction of nucleic acids into various cells, tissues or organs. Therefore, the compounds, lipid particles or compositions of the present invention can be used as DDS technology in nucleic acid medicine. In addition, the compounds, lipid particles or compositions of the present invention can also be used as nucleic acid introduction reagents for research.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Pharmacology & Pharmacy (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nanotechnology (AREA)
• Optics & Photonics (AREA)
• Emergency Medicine (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (1)

Family

ID=86335736

Family Applications (1)

Country Status (13)

Citations (7)

• 2022
  • 2022-11-09 MX MX2024005608A patent/MX2024005608A/es unknown
  • 2022-11-09 US US18/709,440 patent/US20250197343A1/en active Pending
  • 2022-11-09 AR ARP220103070A patent/AR127615A1/es unknown
  • 2022-11-09 CN CN202280074121.3A patent/CN118201905A/zh active Pending
  • 2022-11-09 AU AU2022388185A patent/AU2022388185A1/en active Pending
  • 2022-11-09 CA CA3237659A patent/CA3237659A1/en active Pending
  • 2022-11-09 KR KR1020247015085A patent/KR20240109991A/ko active Pending
  • 2022-11-09 TW TW111142815A patent/TW202340137A/zh unknown
  • 2022-11-09 EP EP22892797.6A patent/EP4431489A4/en active Pending
  • 2022-11-09 WO PCT/JP2022/041651 patent/WO2023085299A1/ja not_active Ceased
  • 2022-11-09 IL IL312605A patent/IL312605A/en unknown
  • 2022-11-09 JP JP2023559658A patent/JPWO2023085299A1/ja active Pending
• 2024
  • 2024-05-03 CO CONC2024/0005797A patent/CO2024005797A2/es unknown

Patent Citations (7)

Non-Patent Citations (10)

Also Published As

Similar Documents

Legal Events