WO2011007795A1 - Composition pour inhiber l'expression d'un gène cible - Google Patents

Composition pour inhiber l'expression d'un gène cible Download PDF

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Publication number
WO2011007795A1
WO2011007795A1 PCT/JP2010/061884 JP2010061884W WO2011007795A1 WO 2011007795 A1 WO2011007795 A1 WO 2011007795A1 JP 2010061884 W JP2010061884 W JP 2010061884W WO 2011007795 A1 WO2011007795 A1 WO 2011007795A1
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sequence
ribose
bases
deoxyribose
substituted
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PCT/JP2010/061884
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English (en)
Japanese (ja)
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史一 篠原
哲郎 吉田
寛子 杉下
智幸 直井
俊彦 石井
淳一 榎園
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協和発酵キリン株式会社
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Priority to JP2011522828A priority Critical patent/JP5872898B2/ja
Publication of WO2011007795A1 publication Critical patent/WO2011007795A1/fr
Priority to US13/348,959 priority patent/US20120207818A1/en
Priority to US13/437,428 priority patent/US20120244210A1/en

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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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar

Definitions

  • the present invention relates to a composition for suppressing the expression of a target gene.
  • RNAi RNA interference
  • RNAi has been extensively verified in in vivo tests, and reports on fetal animal effects using siRNAs of 50 base pairs or less (see Patent Document 1) and adult mice (see Patent Document 2) are reported. Has been. Further, when siRNA is intravenously administered to mouse fetuses, the effect of suppressing the expression of specific genes has been confirmed in each organ of kidney, spleen, lung, pancreas and liver (see Non-Patent Document 1). Furthermore, it has been reported that expression of a specific gene is also suppressed in brain cells by directly administering siRNA (see Non-Patent Document 2).
  • a method using a cationic liposome or a cationic polymer is known as a means for delivering a nucleic acid into a cell.
  • the nucleic acid is quickly removed from the blood, and when the target tissue is other than the liver or lung, for example, a tumor site, etc. In this case, the nucleic acid cannot be delivered to the target tissue, and sufficient expression of the action has not been made possible.
  • nucleic acid-encapsulated liposomes liposomes in which nucleic acids are encapsulated in liposomes
  • Patent Documents 3 to 6 and Non-patent Document 3 As a method for producing a liposome encapsulating nucleic acid or the like, for example, a cationic lipid is dissolved in chloroform in advance, then mixed with an oligodeoxynucleotide (ODN) aqueous solution and methanol, and then centrifuged.
  • ODN oligodeoxynucleotide
  • Patent Documents 5 and 6 report that a liposome encapsulating an active ingredient such as a nucleic acid is produced by a method of coating fine particles with a lipid bilayer in a liquid.
  • this method by reducing the concentration of the polar organic solvent in the aqueous solution containing the polar organic solvent in which the fine particles are dispersed and the lipid is dissolved, the fine particles are coated with the lipid bilayer membrane in the liquid.
  • fine particles (coated fine particles) coated with a lipid bilayer having a size suitable for fine particles for intravenous injection, for example, are produced with excellent efficiency.
  • Patent Documents 5 and 6 exemplify, for example, a complex formed by electrostatic interaction comprising ODN or siRNA and a cationic lipid as an example of fine particles to be coated.
  • the coated microparticles coated with the microparticles have a small particle size and can be used as an injection, and the coated microparticles exhibit high blood retention when administered intravenously and accumulate in tumor tissues. It has been reported.
  • the surface of the liposome is generally modified with a water-soluble polymer such as polyethylene glycol (PEG), and the surface-modified liposome is less likely to interact with serum proteins such as opsonin, and further, macrophages.
  • PEG polyethylene glycol
  • the liposome has a long residence time in blood.
  • Nucleic acid-encapsulated liposomes have also been reported to show higher blood retention and to accumulate in tumor tissues by using PEG-modified liposomes.
  • PEG-modified liposomes are known to be significantly reduced in blood retention of PEG-modified liposomes in the second dose when administered twice at 3-7 day intervals, and were induced by the first dose of PEG-modified liposomes.
  • anti-PEG-IgM antibody binds to PEG of the second administration liposome and then activates the complement system to enhance uptake by hepatic macrophages and significantly reduce the blood retention of the second administration PEG-modified liposome. (See Non-Patent Document 4).
  • An object of the present invention is to provide a composition for suppressing the expression of a target gene.
  • a composition containing liposomes whose surface is modified with a water-soluble polymer such as polyethylene glycol (PEG) it is possible to suppress a significant decrease in blood retention during the second administration, and to achieve higher blood retention.
  • PEG polyethylene glycol
  • the present invention relates to the following (1) to (56).
  • a composition comprising a liposome encapsulating a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand,
  • the antisense strand has a base sequence complementary to the 17-base sequence of the target gene mRNA in which the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end (sequence a)
  • a polynucleotide having a length of 17 to 30 bases, wherein the sugar in the antisense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • composition 50% to 70% of the sugar that binds to a base other than sequence b of the sense strand is deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group, Composition.
  • the double-stranded nucleic acid molecule is a double-stranded nucleic acid molecule having an action of suppressing the expression of the target gene using RNA interference (RNAi).
  • RNAi RNA interference
  • the target gene is vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor, hepatocyte growth factor,
  • the composition according to any one of (1) to (4) above which is a gene of any one of a hepatocyte growth factor receptor, a Kruppel-like factor, an Ets transcription factor, a nuclear factor and a hypoxia-inducing factor.
  • a liposome encapsulating a double-stranded nucleic acid molecule is a liposome comprising a lead particle, a composite particle comprising the double-stranded nucleic acid molecule as a constituent component, and a lipid bilayer coating the composite particle,
  • the components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration.
  • the composition according to any one of (1) to (7).
  • the composition according to (8), wherein the polar organic solvent is an alcohol.
  • the composition according to (8) above, wherein the polar organic solvent is ethanol.
  • the lead particle is a lead particle containing a cationic substance
  • the lipid bilayer coating the composite particle is a constituent component of a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance.
  • the composition according to any one of (8) to (10), wherein the composition is a lipid bilayer.
  • a liposome encapsulating a double-stranded nucleic acid molecule is a liposome comprising a lead particle containing a cationic substance, a composite particle comprising the double-stranded nucleic acid molecule as a constituent component, and a lipid bilayer coating the composite particle.
  • the lipid bilayer coating the composite particles is a lipid bilayer comprising a neutral lipid and a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component (1) to (7 ).
  • Cancer or inflammation containing a composite particle comprising a lead particle and a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand, and a liposome containing a lipid bilayer coating the composite particle
  • a therapeutic agent for a disease The antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is of a size that can be administered intravenously;
  • the components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles are dispersible in a liquid containing the polar organic solvent at a specific concentration.
  • a therapeutic agent for cancer or inflammatory diseases wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative as a constituent component.
  • the polar organic solvent is alcohol.
  • the lead particles are lead particles containing a cationic substance
  • the lipid bilayer membrane is a lipid double substance comprising a neutral lipid and a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component
  • the therapeutic agent for cancer or inflammatory disease according to any one of (14) to (16), which is a membrane.
  • a composite particle comprising a lead particle containing a cationic substance and a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand as constituent components, and a liposome comprising a lipid bilayer coating the composite particle
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is of a size that can be administered intravenously;
  • a therapeutic agent for cancer or inflammatory diseases wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance.
  • Tumor or inflammation-related target genes are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor
  • a therapeutic agent for cancer or inflammatory diseases is human or mouse mRNA.
  • composition comprising a lead particle, a composite particle comprising a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand as a constituent, and a liposome comprising a lipid bilayer coating the composite particle
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is of a size that can be administered intravenously;
  • the components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles are dispersible in a liquid containing the polar organic solvent at a specific concentration.
  • a method for treating cancer or inflammatory disease wherein the composition is administered to a mammal, wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative as a water-soluble substance. .
  • the polar organic solvent is alcohol.
  • the polar organic solvent is ethanol.
  • the lead particle is a lead particle containing a cationic substance
  • the lipid bilayer membrane is a lipid double substance comprising a neutral lipid and a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component.
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • the liposome is of a size that can be administered intravenously; Cancer or inflammation in which the composition is administered to a mammal, wherein the lipid bilayer is a lipid bilayer comprising a neutral lipid and a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component How to treat the disease.
  • Tumor or inflammation-related target genes are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor , Any of the genes for hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor, Ets transcription factor, nuclear factor and hypoxia-inducing factor according to any one of (24) to (30) above Methods for treating cancer or inflammatory diseases. (33) The method for treating cancer or inflammatory disease according to any one of (24) to (32), wherein the mRNA is human or mouse mRNA.
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is of a size that can be administered intravenously;
  • the components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles are dispersible in a liquid containing the polar organic solvent at a specific concentration.
  • the lipid bilayer membrane is a lipid bilayer membrane comprising a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative as a constituent, for producing a therapeutic agent for cancer or inflammatory disease of the composition use.
  • the polar organic solvent is an alcohol.
  • the polar organic solvent is ethanol.
  • the lead particle is a lead particle containing a cationic substance
  • the lipid bilayer membrane is a lipid double substance comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance.
  • (38) Contains a lead particle containing a cationic substance, a composite particle comprising a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand, and a liposome containing a lipid bilayer covering the composite particle
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is of a size that can be administered intravenously;
  • the lipid bilayer is a lipid bilayer comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent of the therapeutic agent for cancer or inflammatory diseases of the composition Use for manufacturing.
  • Target genes related to tumor or inflammation are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor , Any of the genes for hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor, Ets transcription factor, nuclear factor and hypoxia-inducing factor according to any one of (34) to (40) above use. (43) The use according to any one of (34) to (42), wherein the mRNA is human or mouse mRNA.
  • a method for suppressing the expression of a target gene comprising a liposome encapsulating a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand, wherein the composition is administered to a mammal
  • the antisense strand has a base sequence complementary to the 17-base sequence of the target gene mRNA in which the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end (sequence a)
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • the liposome is a liposome of a size that can be administered intravenously, and the liposome is a liposome having a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative as a water-soluble substance.
  • a method for inhibiting the expression of a target gene comprising administering the composition to a mammal.
  • (45) (v) 50.-70% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group.
  • the method for suppressing the expression of the target gene (46)
  • the expression of the target gene according to (44) or (45), wherein the double-stranded nucleic acid molecule is a double-stranded nucleic acid molecule having an action of suppressing expression of the target gene using RNA interference (RNAi). Suppression method.
  • RNAi RNA interference
  • the target gene is vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, platelet-derived growth factor, platelet-derived growth factor receptor, hepatocyte growth factor, Inhibition of expression of the target gene according to any of (44) to (47) above, which is a gene of any one of a hepatocyte growth factor receptor, a Kruppel-like factor, an Ets transcription factor, a nuclear factor and a hypoxia-inducing factor Method.
  • a liposome encapsulating a double-stranded nucleic acid molecule is a liposome comprising a lead particle, a composite particle comprising the double-stranded nucleic acid molecule as a constituent component, and a lipid bilayer coating the composite particle,
  • the components of the lipid bilayer coating the composite particles are soluble in a specific polar organic solvent, and the components of the lipid bilayer coating the composite particles and the composite particles are polar at a specific concentration
  • the lead particle is a lead particle containing a cationic substance, and the lipid bilayer coating the composite particle is a constituent component of a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative.
  • a liposome encapsulating a double-stranded nucleic acid molecule is a liposome comprising a lead particle containing a cationic substance, a composite particle comprising the double-stranded nucleic acid molecule as a constituent component, and a lipid bilayer covering the composite particle.
  • the lipid bilayer coating the composite particle is a lipid bilayer comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as constituent components.
  • the method for suppressing expression of the target gene according to any one of the above.
  • the expression of the target gene can be suppressed by administering the composition of the present invention to mammals and the like.
  • the vertical axis represents the BCL-2 mRNA expression inhibition rate (ratio).
  • the preparations obtained in Examples 1-2 and Comparative Examples 1-9 were administered to mice, and then the preparation obtained in Comparative Example 1 was administered as a second-administered PEG-modified liposome at an interval of 7 days. It shows the concentration of double-stranded nucleic acid molecules in blood 3 hours after administration.
  • the vertical axis represents the concentration ( ⁇ mol / L) of double-stranded nucleic acid molecules in blood. This shows the siRNA activity of the double-stranded nucleic acid molecules used in Example 5 and Comparative Examples 10-13.
  • the vertical axis represents the BCL2 mRNA expression level ratio (ratio).
  • the preparations obtained in Example 5 and Comparative Examples 10 to 13 were administered to mice, and then obtained in the same Example 5 and Comparative Examples 10 to 13 as PEG-modified liposomes for the second administration at intervals of 7 days, respectively. It shows the concentration of double-stranded nucleic acid molecules in the blood 3 hours after administration of the obtained preparation.
  • the vertical axis represents the concentration ( ⁇ mol / L) of double-stranded nucleic acid molecules in blood.
  • the target gene used in the present invention is not particularly limited as long as it is a gene that produces and expresses mRNA in mammals.
  • a gene related to tumor or inflammation is preferable, and a gene involved in angiogenesis is more preferable.
  • vascular endothelial growth factor hereinafter abbreviated as VEGF
  • VEGFR vascular endothelial growth factor receptor
  • fibroblast growth factor fibroblast growth factor receptor
  • Platelet-derived growth factor platelet-derived growth factor receptor
  • hepatocyte growth factor hepatocyte growth factor receptor
  • Kruppel-like factor KLF
  • Ets transcription factor nuclear factor
  • hypoxia induction examples include genes encoding proteins such as factors, specifically VEGF gene, VEGFR gene, fibroblast growth factor gene, fibroblast growth factor reception Body gene, platelet-derived growth factor gene, platelet-derived growth factor receptor gene, hepatocyte growth factor gene, hepatocyte growth factor receptor gene, KLF gene, Ets transcription factor
  • VEGF gene, VEGFR gene, KLF gene, etc. are mentioned, More preferably, KLF gene is mentioned, More preferably, KLF5 gene is mentioned.
  • the KLF family is a family of transcription factors characterized by a zinc finger motif on the C-terminal side. KLF12, KLF13, KLF14, KLF15, KLF16, etc. are known.
  • the KLF family is important for the differentiation of various tissues and cells such as red blood cells, vascular endothelial cells, smooth muscle, skin, lymphocytes, etc., and cancer, cardiovascular disease, cirrhosis, kidney disease, immune disease It has been reported to play an important role in the pathogenesis of various diseases such as [The Journal of Biological Chemistry, 2001, 276, 37, p. 34355-34358, Genome Biology, 2003, Vol. 4, No. 2, p. 206].
  • KLF5 in the KLF family is also called BTEB2 (basic transcriptional element binding protein 2) or IKLF (intestinal-enriched Kruppel-like factor). Expression of KLF5 in vascular smooth muscle is controlled at the developmental stage, and high expression is observed in fetal vascular smooth muscle, whereas expression is not observed in normal adult vascular smooth muscle. In addition, KLF5 is highly expressed in intimal smooth muscle that has been born after exfoliation with a balloon catheter, and KLF5 is also expressed in smooth muscle in lesions of arteriosclerosis and restenosis [Circulation, 2000, Vol. 102, No. 20, p.2528-2534].
  • VEGF is a growth factor specific for vascular endothelial cells discovered by Ferrara et al. In 1983. In the same year, a factor with vascular permeability was discovered by Senger, Dvorak et al. And named VPF (vascular permeability factor). Analysis of the amino acid sequence of the protein revealed that the two were identical. VEGF binds to endothelial cell receptors inside blood vessels to promote proliferation. VEGF not only creates blood vessels during fetal life, but also acts when creating pathological blood vessels. For example, if the cancer grows to some extent and becomes deficient in oxygen, VEGF and its receptors increase and angiogenesis occurs. It is also thought to cause cancerous ascites due to the vascular permeability enhancing action.
  • VPF vascular permeability factor
  • VEGF As diabetes progresses, new blood vessels form in the retina, and VEGF also works there. In other words, it is a protein that creates new blood vessels. It can be said that it plays an important role in angiogenesis by its expression being induced by hypoxia. Moreover, the involvement of this factor is strongly suggested in explaining not only angiogenesis but also the mechanism of edema observed in tumors or inflammatory lesions.
  • VEGFR is possessed by vascular endothelial cells and cancer cells themselves, and when VEGF binds to VEGFR, the receptor itself is phosphorylated (activated), and as a result, various commands such as proliferation and migration are transmitted to the cells. Is done. It is known that by inhibiting phosphorylation of this receptor, intracellular transmission is inhibited and angiogenesis is inhibited.
  • BCL2 is a mitochondrial inner membrane protein that shows cell death inhibition by apoptosis in several cell types. Inhibition of apoptosis due to the large expression of bcl2 gene is thought to cause cancer and hematological malignancies. Indeed, BCL2 is produced in large quantities in various solid cancers such as lymphosarcoma, prostate cancer, breast cancer, lung cancer, colon cancer and rectal cancer (TJMcDonnell et al., “Cancer Research”, December 15, 1992, 52 Vol.24, p.6940-6944).
  • a gene expressed in the liver, lung, kidney or spleen is preferable.
  • the above-mentioned gene related to tumor or inflammation hepatitis B virus genome, hepatitis C virus genome , Apolipoprotein (APO), hydroxymethylglutaryl (HMG) CoA reductase, kexin 9 type serine protease (PCSK9), factor 12, glucagon receptor, glucocorticoid receptor, leukotriene, thromboxane A2 receptor, histamine H1
  • APO hydroxymethylglutaryl
  • PCSK9 hydroxymethylglutaryl
  • factor 12 glucagon receptor
  • glucocorticoid receptor glucocorticoid receptor
  • leukotriene thromboxane A2 receptor
  • histamine H1 examples thereof include genes encoding proteins such as receptors, carbonic anhydrase, angiotensin converting enzyme, renin, p53, tyrosine
  • the double-stranded nucleic acid molecule used in the present invention is a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand
  • the antisense strand has a base sequence complementary to the 17-base sequence of the target gene mRNA in which the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end (sequence a)
  • a ribose having a length of 17 to 30 bases, preferably 19 to 25 bases, wherein the sugar in the antisense strand is ribose, deoxyribose or the hydroxyl group at the 2 ′ position is substituted with a modifying group, respectively.
  • the sense strand includes 17 to 30 bases, preferably including a base sequence (sequence b) complementary to the 1st to 17th base sequence from the 5 ′ end to the 3 ′ end of the antisense strand.
  • a base sequence sequence b
  • the sense strand includes 17 to 30 bases, preferably including a base sequence (sequence b) complementary to the 1st to 17th base sequence from the 5 ′ end to the 3 ′ end of the antisense strand.
  • a polynucleotide having a length of 19 to 25 bases wherein the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group, (i) 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2
  • 0% of the sugars bonded to the 9th to 11th bases are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group, (iii) 30 to 100%, preferably 40% or more, of deoxyribose or 2 ′ of the sugar that binds to the 17th to 3 ′ terminal bases from the 5 ′ end to the 3 ′ end of the antisense strand Ribose in which the hydroxyl group at the position is substituted with a modifying group, (iv) 10 to 70%, preferably 30 to 60% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the sequence b are deoxyribose or 2 ′ position, respectively.
  • Ribose in which the hydroxyl group is substituted with a modifying group provided that 0% of the sugar bonded to the 9th to 11th bases is deoxyribose or ribose in which the 2′-position hydroxyl group is substituted with a modifying group, (v) 30 to 100%, preferably 40% or more of the sugars that bind to the base other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group A strand nucleic acid molecule.
  • 0% of the sugar bonded to the m to n-th base is deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group is:
  • the sugar that binds to the m to n-th base does not include deoxyribose and ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group, that is, all sugars that bind to the m to n-th base are ribose.
  • the double-stranded nucleic acid molecule used in the present invention is preferably a double-stranded nucleic acid molecule having an action of suppressing the expression of the target gene using RNA interference (RNAi).
  • RNAi RNA interference
  • the nucleotide sequence of the nucleotide added adjacent to the 3 'end of the antisense strand sequence a is used as the base sequence complementary to the base sequence adjacent to sequence a in the target gene mRNA. This structure is more preferable from the viewpoint of the suppressive action of target gene expression using RNA interference (RNAi).
  • the antisense strand is a base sequence in which at least the 1st to 17th base sequence from the 5 ′ end to the 3 ′ end is complementary to the 17 base sequence of the mRNA of the target gene.
  • the antisense strand has a base sequence in which the sequence of the 1st to 19th bases from the 5 ′ end to the 3 ′ end is complementary to the sequence of 19 bases continuous in the mRNA of the target gene
  • the sequence of the 1st to 21st bases is a base sequence complementary to the sequence of 21 consecutive bases of the target gene mRNA, or the sequence of the 1st to 25th bases is the target gene mRNA. This is a base sequence complementary to the continuous 25 base sequence.
  • the base other than the sequence b of the sense strand and the base of the antisense strand are complementary to each other. More preferably, it is a base pair.
  • the double-stranded nucleic acid molecule used in the present invention is modified at the 2 ′ position by 10 to 70%, preferably 15 to 60%, more preferably 20 to 50% of the sugar in the double-stranded nucleic acid molecule. Ribose substituted with a group.
  • substituted with a modifying group at the 2′-position of ribose means that the hydroxyl group at the 2′-position is substituted with the modifying group, and the configuration is the same as the hydroxyl group at the 2′-position of ribose. However, the configuration is preferably the same as that of the hydroxyl group at the 2 ′ position of ribose.
  • the double-stranded nucleic acid molecule 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 a nucleic acid structure is substituted with another atom such as a sulfur atom.
  • Examples of the modifying group in the present invention include 2′-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-alkenyl, 2′-substituted alkenyl, 2′-halogen, 2′-O-cyano, 2 '-O-alkyl, 2'-O-substituted alkyl, 2'-O-alkenyl, 2'-O-substituted alkenyl, 2'-S-alkyl, 2'-S-substituted alkyl, 2'-S-alkenyl 2'-S-substituted alkenyl, 2'-amino, 2'-NH-alkyl, 2'-NH-substituted alkyl, 2'-NH-alkenyl, 2'-NH-substituted alkenyl, 2'-SO-alkyl 2'-SO-substituted alkyl, 2'-carboxy, 2'-
  • Oxypeptide nucleic acid [J. Am. Chem. Soc., 123, 4653 (2001)]
  • peptide ribonucleic acid PRNA
  • a crosslinked structure-type artificial nucleic acid having a structure in which the modifying group at the 2 ′ position is crosslinked to the carbon atom at the 4 ′ position, more specifically, the oxygen atom at the 2 ′ position and the 4 ′ position Locked Nucleic Acid (LNA) in which the carbon atom at the position is cross-linked via methylene, and Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004) ] And the like are also included in the ribose substituted with a modifying group at the 2 ′ position in the present invention.
  • BNA crosslinked structure-type artificial nucleic acid
  • 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 are preferred, 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,
  • the modifying group in the present invention can also define a preferable range from its size, and from the size of fluoro, the one corresponding to the size of -O-butyl is preferable, and from the size of -O-methyl A size corresponding to the size of -O-ethyl is more preferable.
  • alkyl in the modifying group examples include linear or branched carbon atoms having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- Pentyl, hexyl and the like can be mentioned, and methyl, ethyl, propyl, isopropyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, tert-pentyl and the like are preferable.
  • alkenyl in the modifying group examples include linear or branched carbon atoms having 1 to 6 carbon atoms such as vinyl, allyl, isopropenyl and the like.
  • halogen examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • amino acids include aliphatic amino acids (specifically, glycine, alanine, valine, leucine, isoleucine, etc.), hydroxy amino acids (specifically, serine, threonine, etc.), acidic amino acids (specifically, aspartic acid).
  • acidic amino acid amide specifically, asparagine, glutamine, etc.
  • basic amino acid specifically, lysine, hydroxylysine, arginine, ornithine, etc.
  • sulfur-containing amino acid specifically, cysteine , Cystine, methionine, etc.
  • imino acid specifically, proline, 4-hydroxyproline, etc.
  • substituent of substituted alkyl and substituted alkenyl examples include halogen (as defined above for halogen), hydroxy, sulfanyl, amino, oxo, -O-alkyl (wherein the alkyl portion of -O-alkyl is as defined above for alkyl),- S-alkyl (the alkyl part of the -S-alkyl has the same meaning as the alkyl), -NH-alkyl (the alkyl part of the -NH-alkyl has the same meaning as the alkyl), dialkylaminooxy (the dialkylaminooxy Alkyl is the same or different and is synonymous with the alkyl), dialkylamino (two alkyls of the dialkylamino are the same or different and synonymous with the alkyl), dialkylaminoalkyleneoxy (the two alkyls of the dialkylaminoalkyleneoxy are the same or Differently, it is synonymous with said alkyl, and said alkylene is
  • the ribose substituted with the modifying group at the 2′-position of the sugar in the double-stranded nucleic acid molecule has the same structure as the final one, as long as the structure is the same. Even if the raw material or intermediate is DNA or deoxyribose, it is included in the double-stranded nucleic acid molecule used in the present invention as long as the structure is finally the same. That is, ribose substituted with a modifying group at the 2 ′ position of ribose in the present invention includes deoxyribose in which hydrogen is substituted with a modifying group at the 2 ′ position.
  • ribose substituted with a modifying group at the 2 ′ position is distributed so as to minimize adjacentity.
  • the sugar that binds to the 3 ′ end of the antisense strand, the 5 ′ end of the sense strand and the 2-7 bases of the 3 ′ end is adjacent to deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group. It is preferable. It is more preferable that ribose is substituted with a modifying group on only one of the opposing complementary base pairs.
  • either the antisense strand or the 5′-end or 3′-end of the sense strand is either deoxyribose or a ribose in which the hydroxyl group at the 2′-position is substituted with a modifying group at both opposing base pairs.
  • the double-stranded nucleic acid molecule used in the present invention comprises (A) the 5 ′ end of the antisense strand and the 3 ′ end of the sense strand, and the 3 ′ end of the antisense strand and the 5 ′ end of the sense strand facing each other and complementary. (B) 1 to 6, preferably 2 to 4 nucleotides on the 3 ′ end side of the antisense strand and the sense strand are the same or different and face each other. It may be an overhang that is added, or (C) a blunt end and an overhang may be combined.
  • the base of the added nucleotide may be one or more of guanine, adenine, cytosine, thymine and uracil, and the sugar bonded to each base is ribose, deoxyribose or a hydroxyl group at the 2 ′ position. May be any of ribose substituted with a modifying group, but as the added nucleotide, one or two of uridylic acid (U) and deoxythymidylic acid (dT) are more preferable. Further, the nucleotide base sequence added adjacent to the 3 ′ end side of the sense strand may be the same base sequence as the base sequence adjacent to sequence a in mRNA, and this structure is more preferred.
  • the nucleotide sequence of the nucleotide added adjacent to the 3 ′ end of the antisense strand may be a base sequence complementary to the corresponding base sequence in the mRNA of the target gene. More preferred.
  • the base sequence of the antisense strand is most preferably a base sequence that is completely complementary to the base sequence corresponding to the mRNA of the target gene.
  • the sugar that binds to the 5 ′ terminal base of the antisense strand and the sense strand has a phosphate group or the above-mentioned modifying group, or a nucleolytic enzyme in vivo, etc. Or you may modify by the group used as the said modification group.
  • the sugar that binds to the 3 ′ terminal base of the antisense strand and the sense strand has a phosphate group or the above-mentioned modifying group, or a nucleolytic enzyme in vivo, etc. Or you may modify by the group used as the said modification group.
  • the double-stranded nucleic acid molecule of the present invention may be produced after being degraded by a nucleolytic enzyme or the like in a living body, and the double-stranded nucleic acid molecule before being degraded is a double-stranded nucleic acid molecule of the present invention.
  • a prodrug of a strand nucleic acid molecule As a prodrug of a double-stranded nucleic acid molecule, for example, 4 to 8, preferably 5 to 6 nucleotides, which are the same or different, are added to the 5 ′ end side of the sequence a of the antisense strand.
  • the same number of base sequences complementary to the base sequence of the antisense strand is added to the 3 ′ end side of the sequence b of the sequence b, and two target genes are added to the 3 ′ end side of the sequence a of the antisense strand.
  • the same sequence as the base sequence corresponding to the mRNA is added, and two base sequences complementary to the base sequence of the antisense strand are added to the 5 ′ end side of the sense strand sequence b.
  • the sugars that bind to the 'terminal base are two that have a 5' hydroxyl group phosphorylated
  • This prodrug is added by Dicer to all nucleotides added to the 5 ′ end of the antisense strand sequence a and the first and second nucleotides added to the 3 ′ end of the sense strand sequence b. Nucleotides other than are removed to form the double-stranded nucleic acid molecule of the present invention.
  • 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 double-stranded nucleic acid molecule having the sequence UUCAAGAGA. Either of the two double-stranded nucleic acid molecules connected by the spacer oligonucleotide may be on the 5 'side.
  • the double-stranded nucleic acid molecule used in the present invention may be produced using a known RNA or DNA synthesis method and RNA or DNA modification method. For example, it can be obtained by requesting chemical synthesis from Hokkaido System Science Co., Ltd.
  • the liposome (hereinafter referred to as liposome A) in the composition of the present invention includes a liposome encapsulating a double-stranded nucleic acid molecule containing a sequence of 17 to 30 bases of mRNA of a target gene and a base sequence complementary to the sequence.
  • the liposome A is not particularly limited as long as it is a liposome that reaches the tissue or organ containing the target gene expression site.
  • the liposome A may be a lipid derivative comprising a water-soluble substance, a fatty acid derivative, or an aliphatic hydrocarbon derivative.
  • Liposomes having a multilayer film specifically, liposomes whose surface is modified with a water-soluble polymer such as polyethylene glycol (PEG).
  • a cationic lipid is previously dissolved in chloroform, and then the aqueous solution of the double-stranded nucleic acid molecule and methanol are added and mixed to form a complex of the cationic lipid / double-stranded nucleic acid molecule, Furthermore, the chloroform layer was taken out, polyethylene glycolated phospholipid, neutral lipid and water were added to this to form a water-in-oil (W / O) emulsion, and liposomes produced by processing by the reverse phase evaporation method (See JP-T-2002-508765), the double-stranded nucleic acid molecule is dissolved in an acidic electrolyte aqueous solution, lipid (in ethanol) is added, and the ethanol concentration is reduced to 20 v / v% to reduce the double-stranded nucleic acid molecule.
  • Encapsulated liposomes are prepared, sizing-filtered, and excess ethanol is removed by dialysis. The sample is further dialyzed at a higher pH to remove the double-stranded nucleic acid molecules attached to the liposome surface.
  • Liposomes see Japanese Patent Publication No. 2002-501511 and Biochimica et Biophysica Acta, 2001, Vol. 1510, p.152-166
  • lead particles and the double-stranded nucleic acid Liposomes composed of composite particles containing molecules and lipid bilayers encapsulating the composite particles (see WO02 / 28367 pamphlet and WO2006 / 080118 pamphlet), etc.
  • Liposomes composed of composite particles containing double-stranded nucleic acid molecules and lipid bilayer membranes encapsulating the composite particles are preferred, and the components of the lipid bilayer membranes are soluble in a specific polar organic solvent, and the lipids More preferably, the components of the double membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration.
  • the liposome A is preferably composed of a lead particle containing a cationic substance, a composite particle comprising the double-stranded nucleic acid molecule as a constituent component, and a lipid bilayer membrane covering the composite particle.
  • Liposomes whose membranes are composed of lipids, fatty acid derivatives or aliphatic hydrocarbon derivatives of neutral lipids and water-soluble substances are also included, and the components of the lipid bilayer membrane are soluble in specific polar organic solvents. More preferably, the components of the lipid bilayer membrane and the composite particles are dispersible in a liquid containing the polar organic solvent at a specific concentration.
  • the term “dispersed” means to be dispersed without dissolving.
  • exemplified liposomes have been reported to be delivered to tumors or inflamed tissues or organs, specifically solid tumors and solid cancers, blood vessels or inflammatory sites near blood vessels, etc.
  • the target gene is a target gene
  • it can be exemplified as a liposome that can be used more preferably.
  • these exemplified liposomes have been reported as liposomes with increased retention in blood, and the possibility of being delivered to any tissue or organ via systemic circulation is increasing.
  • the genes that can be made are not limited.
  • Examples of the lead particles in the present invention include fine particles such as lipid aggregates, liposomes (hereinafter referred to as “liposome B”), polymer micelles, and the like, preferably fine particles that are liposome B.
  • the lead particle in the present invention is a complex obtained by combining two or more lipid aggregates, liposome B, polymer micelles, and the like, for example, polymer micelles as a complex including lipid aggregates and lipids that are constituents of liposome B, It may be a lipid aggregate or a liposome B as a complex containing a polymer that is a constituent component of the polymer micelle.
  • the lipid aggregates or liposomes B as lead particles are preferably composed of polar lipids or the like that have amphiphilic properties that have both hydrophilic and hydrophobic properties and have a lipid bilayer structure in water.
  • the lipid may be any of simple lipids, complex lipids or derived lipids, and examples include, but are not limited to, phospholipids, glyceroglycolipids, glycosphingolipids, sphingoids, sterols or cationic lipids. Not. Preferable examples include phospholipids and cationic lipids.
  • Examples of the phospholipid in the lipid constituting the lead particles include phosphatidylcholine (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine, Oleoylphosphatidylcholine), phosphatidylethanolamine (specifically distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DORE), dimyristoylphosphoethanolamine (DMPE)) 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidyl ethanol (POPE), 1-stearoyl-2-oleo
  • Examples of the glyceroglycolipid in the lipid constituting the lead particles include sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
  • glycosphingolipid in the lipid constituting the lead particle examples include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.
  • Examples of the sphingoid in the lipid constituting the lead particle include sphingan, icosasphingan, sphingosine, and derivatives thereof.
  • Derivatives include, for example, —NH 2 such as sphingan, icosasphingan or sphingosine —NHCO (CH 2 ) xCH 3 (wherein x represents an integer of 0 to 18, among which 6, 12 or 18 is preferred) And the like converted to.
  • Examples of the sterol in the lipid constituting the lead particles include cholesterol, dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucosterol, or 3 ⁇ - [N- (N ′, N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like.
  • the cationic lipid in the lipid constituting the lead particle among the polar lipids having amphipathic properties that have both hydrophilic and hydrophobic properties and having a lipid bilayer structure in water, It has a structure having a primary amine, secondary amine, tertiary amine, quaternary ammonium, a heterocyclic ring containing a nitrogen atom, etc., for example, N- [1- (2,3-dioleoyl Propyl)]-N, N, N-trimethylammonium chloride (DOTAP), N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine (DODAP), N- [1- ( 2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-di
  • Liposomes B may also contain a membrane stabilizer such as sterol such as cholesterol, for example, an antioxidant such as tocopherol, if necessary. These stabilizers may be used alone or in combination of two or more.
  • lipid aggregates include spherical micelles, spherical reverse micelles, sausage-like micelles, sausage-like reverse micelles, plate-like micelles, plate-like reverse micelles, hexagonal I, hexagonal II, and aggregates composed of two or more lipid molecules. .
  • polymer micelle examples include protein, albumin, dextran, polyfect, chitosan, dextran sulfate, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acrylpyrrolidone
  • micelles composed of one or more polymers such as polymers, polyethylene glycol-modified dendrimers, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or salts thereof.
  • the salts in the polymer include, for example, metal salts, ammonium salts, acid addition salts, organic amine addition salts, amino acid addition salts and the like.
  • the metal salt include 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.
  • the ammonium salt include salts such as ammonium and tetramethylammonium.
  • 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.
  • organic amine addition salts include addition salts such as morpholine and piperidine.
  • amino acid addition salts include addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid or lysine.
  • the lead particles in the present invention can 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.
  • 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.
  • One or more lipid derivatives or fatty acid derivatives or surfactants selected from sugars, peptides, nucleic acids and water-soluble polymers may be contained as lead particles, or may be used in addition to lead particles.
  • the lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers is preferably a glycolipid or a lipid derivative or fatty acid derivative of a water-soluble polymer. More preferred are water-soluble polymer lipid derivatives or fatty acid derivatives.
  • Lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers are those in which part of the molecule and other components of the lead particle, such as hydrophobic affinity, electrostatic It is a substance with a two-sided property that has the property of binding due to mechanical interaction, etc., and the other part has the property of binding to the solvent at the time of lead particle production, for example, hydrophilic affinity, electrostatic interaction, etc. Is preferred.
  • lipid derivatives or fatty acid derivatives selected from sugars, peptides and 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
  • nucleic acids such as DNA, RNA, plasmid, siRNA, ODN, etc. and lipids mentioned in the definition of the lead particles or fatty acids such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. Can be given.
  • sugar lipid derivative or fatty acid derivative examples include glyceroglycolipid and glycosphingolipid mentioned in the definition of the lead particles.
  • water-soluble polymer lipid derivative or fatty acid derivative examples include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Water-soluble polymers such as chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol, etc.
  • lipid derivative or fatty acid derivatives such as polyglycerol derivative can be mentioned, more preferably, a lipid derivative or a fatty acid derivative of a polyethylene glycol derivative.
  • Examples of lipid derivatives or fatty acid derivatives of polyethylene glycol derivatives include polyethylene glycolated lipids (specifically, polyethylene glycol-phosphatidylethanolamine (more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine). -N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), etc.), polyoxyethylene hydrogenated castor oil 60, Cremophor EL, etc.), polyethylene glycol sorbitan fatty acid esters (specifically mono Oleic acid polyoxyethylene sorbitan, etc.) or polyethylene glycol fatty acid esters, and the like, more preferably polyethylene glycolated lipids.
  • polyethylene glycolated lipids specifically, polyethylene glycol-phosphatidylethanolamine (more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine).
  • lipid derivatives or fatty acid derivatives of polyglycerin derivatives include polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine) and polyglycerin fatty acid esters, and more preferably polyglycerinized lipids. can give.
  • surfactant examples include polyoxyethylene sorbitan monooleate (specifically, polysorbate 80), polyoxyethylene polyoxypropylene glycol (specifically, Pluronic F68), 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. are preferred, Examples thereof include polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether.
  • the above-described lead particles preferably have a positive charge.
  • the positive charge described here includes charges in the double-stranded nucleic acid molecule, charges that generate an electrostatic attraction with respect to intramolecular polarization, surface polarization, and the like.
  • the lead particles preferably contain a cationic substance.
  • the cationic substance contained in the lead particles is a substance exhibiting a cationic property, but even if it is an amphoteric substance having both a cationic group and an anionic group, it binds to pH and other substances. Since the relative negative degree changes by etc., what can be classified into a cationic substance according to the time is also included.
  • These cationic substances may be contained as lead particles, or may be used in addition to the lead particles.
  • a cationic substance for example, a cationic substance (specifically, a cationic substance in a lipid, a cationic polymer, etc.) among those exemplified in the definition of the lead particle, at a pH value equal to or lower than the isoelectric point.
  • a cationic substance specifically, a cationic substance in a lipid, a cationic polymer, etc.
  • examples thereof include proteins or peptides exhibiting cationic properties, preferably cationic substances in lipids, and more preferably N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethyl.
  • Ammonium chloride N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine, N- [1- (2,3-dioleoyloxypropyl)]-N, N, N- Trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl)]-N, N-dimethyl-N-hydroxyethylammonium bromide and 3 ⁇ - [N- (N ', N'dimethylaminoethyl) ) Carbamoyl] one or more selected from cholesterol.
  • Examples of the cationic substance in the lipid include cationic lipids (DOTAP, DODAP, DOTMA, DOSPA, DMRIE, DORIE, etc.) and DC-Chol.
  • Examples of the cationic polymer include poly-L-lysine, polyethyleneimine, polyfect, and chitosan.
  • the protein or peptide exhibiting a cationic property at a pH below the isoelectric point is not particularly limited as long as it is a protein or a peptide exhibiting a cationic property at a pH below the isoelectric point of the substance.
  • Examples of the protein or peptide include albumin, orosomucoid, globulin, fibrinogen, pepsin, and ribonuclease T1.
  • the lead particles in the present invention can be produced according to a known production method or similar methods, and may be produced by any production method.
  • a known liposome preparation method can be applied to the production of liposome B, which is one of the lead particles.
  • 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 ["Journal of Cell Biology", 1975, Vol. 66, pp. 621-634], French press method ["FBS. Letters (FEBS Lett.), 1979, Vol. 99, p.210-214], freeze-thaw method [“Arch. Biochem.
  • liposome B for example, 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. It is.
  • liposomes B 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.
  • surface modification of the lead particles such as liposome B with a cationic substance, polymer, polyoxyethylene derivative, etc. can be arbitrarily performed [Radics, edited by F. Martin, “Stealth” • Liposomes ”(USA), CRC Press Inc., 1995, p. 93-102].
  • the polymer that can be used for the surface modification include dextran, pullulan, mannan, amylopectin, and hydroxyethyl starch.
  • the polyoxyethylene derivative include polysorbate 80, Pluronic F68, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, and PEG-DSPE.
  • Lead particles such as liposome B are one of the methods in which lead particles contain lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers. It is.
  • the average particle size of liposome B can be freely selected as desired, but the following particle size is preferred.
  • Examples of the method for adjusting the average particle size of liposome B include an extrusion method and a method of mechanically crushing large multilamellar liposomes (MLV) (specifically, using a manton gourin, a microfluidizer, etc.) [Muller (RHMuller), S. Benita, B. Bohm, “Emulsion and Nanosuspensions” for Emulsionsusand Nanosuspensions for the "Formulation" of "Poorly” Soluble “Drugs)", Germany, Scientific Publishers Stuttgart, 1998, p.267-294].
  • MMV multilamellar liposomes
  • a method for producing a complex that combines two or more selected from, for example, lipid aggregates, liposome B, polymer micelles, and the like, which are lead particles may be, for example, simply mixing lipids, polymers, etc. in water, If desired, a sizing step, a sterilization step, and the like can be further added.
  • the complex can be produced in various solvents such as acetone or ether.
  • the lead particles preferably have an average particle diameter of several nm to several tens of ⁇ m, more preferably about 10 nm to 1000 nm, and further preferably about 50 nm to 300 nm.
  • the components other than the lipid derivatives, fatty acid derivatives or aliphatic hydrocarbon derivatives of water-soluble substances include, for example, Examples thereof include the lipids mentioned in the definition of the lead particles, preferably neutral lipids among the lipids.
  • the neutral lipid refers to a cationic substance in the lipid mentioned in the cationic substance in the case where the lead particle has a positive charge, and an anion mentioned in the adhesion competitor described below.
  • the neutral lipid is more preferably phospholipid, glyceroglycolipid, or sphingoglycolipid. More preferred are phospholipids, and more preferred is EPC. These lipids can be used alone or in combination of two or more.
  • the constituent component of the lipid bilayer membrane covering the composite particles is preferably soluble in a polar organic solvent, and preferably dispersible in a liquid containing the polar organic solvent at a specific concentration.
  • the concentration of the polar solvent in the liquid containing the polar solvent at a specific concentration is preferably a concentration at which the constituent components of the lipid bilayer membrane can be dispersed and the composite particles can be dispersed.
  • the polar organic solvent include alcohols such as methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol and tert-butanol, glycols such as glycerin, ethylene glycol and propylene glycol, and polyethylene glycol.
  • Examples thereof include polyalkylene glycols, among which alcohol is preferable and ethanol is more preferable.
  • Examples of the solvent other than the polar organic solvent in the liquid containing the polar organic solvent in the present invention include water, liquid carbon dioxide, liquid hydrocarbon, halogenated carbon or halogenated hydrocarbon, and preferably water. Can be mentioned. Moreover, an ion or a buffer component may be included. One or more solvents can be used, but when two or more solvents are used, a compatible combination is preferred.
  • the lipid bilayer of the liposome and the lipid bilayer covering the composite particles in the composition of the present invention are composed of a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance or the above surfactant as a constituent component. It is preferable that a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance is contained as a constituent component.
  • the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance include one or more lipid derivatives or fatty acid derivatives, or sugars, peptides selected from the aforementioned sugars, peptides, nucleic acids and water-soluble polymers.
  • aliphatic hydrocarbon derivatives of one or more substances selected from nucleic acids and water-soluble polymers are more preferably the lipid derivative or fatty acid derivative of the water-soluble polymer, more preferably the polyethylene glycolated phospholipid, and polyethylene glycol-phosphatidylethanolamine. Most preferred.
  • aliphatic hydrocarbon derivative of the water-soluble substance in the present invention a substance obtained by binding a water-soluble substance and, for example, an alcoholic residue of a long-chain aliphatic alcohol, polyoxypropylene alkyl or glycerin fatty acid ester, etc. Can be given.
  • aliphatic hydrocarbon derivatives of sugars, peptides or nucleic acids include sugars such as sucrose, sorbitol or lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides or peptides such as glutathione, or DNA, RNA, plasmids, etc. , Aliphatic hydrocarbon derivatives of nucleic acids such as siRNA or ODN.
  • Examples of the aliphatic hydrocarbon derivative of the water-soluble polymer include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Examples thereof include aliphatic hydrocarbon derivatives of chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol, etc. or their derivatives, more preferably polyethylene glycol derivatives or polyglycerin derivatives, etc.
  • An aliphatic hydrocarbon derivative is mentioned, More preferably, the aliphatic hydrocarbon derivative of a polyethylene glycol derivative is mentioned.
  • liposome A is a composite particle containing liposome B and the double-stranded nucleic acid molecule and a lipid bilayer coating the composite particle. Even when the lead particles are other than the fine particles of liposome B, they are classified as liposomes in a broad sense because they are covered with a lipid bilayer membrane. In the present invention, it is more preferable that the lead particles are liposome B.
  • the composite particle comprising the lead particle and the double-stranded nucleic acid molecule as a constituent component in the present invention attaches or encloses the double-stranded nucleic acid molecule to the lead particle after the lead particle is produced or simultaneously with the production of the lead particle.
  • the composite particles can be produced, and liposome A can be produced by coating the composite particles with a lipid bilayer after the production of the composite particles or simultaneously with the production of the composite particles.
  • Liposome A is, for example, JP 2002-508765 JP, JP 2002-501511 JP, “Biochimica et Biophysica Acta”, 2001, Vol. 1510, p.152-.
  • the lead particle is produced in water, and the double-stranded nucleic acid molecule is produced after the lead particle is produced or simultaneously with the production of the lead particle.
  • a composite particle is produced by dispersing or dissolving in water and mixing and adhering or encapsulating the double-stranded nucleic acid molecule to the lead particle, or after producing the lead particle in an arbitrary solvent, It is preferably dispersed by dispersing in water, mixing or dissolving the double-stranded nucleic acid molecule in water, and attaching the double-stranded nucleic acid molecule to the lead particle, and producing the lead particle in water, More preferably, after producing the lead particles, the double-stranded nucleic acid molecules are dispersed or dissolved in water and mixed, and the double-stranded nucleic acid molecules are attached to the lead particles.
  • step 1 As a preferred method for producing liposome A in the composition of the present invention, the following steps of producing composite particles comprising the following lead particles and the double-stranded nucleic acid molecule (step 1) and the composite particles as lipid bilayer membranes are used. And a production method including a step of coating with (step 2 or step 3).
  • Step 1) Step of producing a composite particle comprising lead particles and the double-stranded nucleic acid molecule as constituent components
  • Lead particles are dispersed in a solvent such as water, and the double-stranded particles are dispersed in a liquid in which the lead particles are dispersed. It is preferable that the nucleic acid molecules are dispersed or dissolved and mixed to be mixed, and the double-stranded nucleic acid molecules are attached to the lead particles.
  • the lead particles are preferably lead particles containing an aggregation inhibitor.
  • Preferred examples of the aggregation inhibitor include lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from the sugars, peptides, nucleic acids, and water-soluble polymers.
  • the lead particle has a positive charge
  • the double-stranded nucleic acid molecule and the adhesion competitor are allowed to coexist in the liquid in which the lead particle is dispersed, and the adhesion competitor is read together with the double-stranded nucleic acid molecule.
  • An adhesion competitor may be used to further suppress the aggregation of the lead particles even when the lead particles are the lead particles containing the aggregation inhibitor.
  • the composite particle can be dispersed in a liquid containing a polar organic solvent, and the solubility of the composite particle in the polar organic solvent is It is more preferable that it is lower than that of the components of the lipid bilayer membrane used in step 2 or 3, and the components of the lipid bilayer membrane can be dispersed in the liquid containing the polar organic solvent. It is more preferable to select a combination in which a liquid containing the polar organic solvent is present at such a concentration that particles can be dispersed.
  • adhesion competitors include anionic substances.
  • the anionic substance includes a substance that adheres electrostatically to the lead particles due to electrostatic attraction due to intramolecular charge, intramolecular polarization, and the like.
  • Anionic substances as adhesion competitors are substances that exhibit anionic properties, but even if they are amphoteric substances that have both an anionic group and a cationic group, pH, binding to other substances, etc. Since the relative negative degree changes depending on the situation, it can be classified as an anionic substance depending on the occasion.
  • anionic substance examples include anionic lipids, anionic surfactants, anionic polymers, and the like, and proteins, peptides, or nucleic acids that exhibit anionic properties at a pH higher than the isoelectric point, preferably dextran sulfate, dextran.
  • examples thereof include sodium sulfate, chondroitin sulfate, sodium chondroitin sulfate, hyaluronic acid, chondroitin, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and dextran fluorescein anionic. These anionic substances can be used alone or in combination of two or more.
  • anionic lipid examples include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid.
  • anionic surfactant examples include acyl sarcosine, sodium alkyl sulfate, alkyl benzene sulfonate, and fatty acid sodium having 7 to 22 carbon atoms. Specific examples include sodium dodecyl sulfate, sodium lauryl sulfate, sodium cholate, sodium deoxycholate, or sodium taurodeoxycholate.
  • anionic polymer examples include polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acryl pyrrolidone copolymer, polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid, polyethylene glycolated polylactic acid, dextran sulfate. Dextran sodium sulfate, chondroitin sulfate, chondroitin sulfate sodium, hyaluronic acid, chondroitin, dermatan sulfate, heparan sulfate, heparin, keratan sulfate or dextran fluorescein anionic.
  • the protein or peptide exhibiting anionic property at a pH value equal to or higher than the isoelectric point is not particularly limited as long as it is a protein or peptide exhibiting anionic property at a pH value equal to or higher than the isoelectric point of the substance.
  • examples include albumin, orosomucoid, globulin, fibrinogen, histone, protamine, ribonuclease or lysozyme.
  • nucleic acid as the anionic substance examples include DNA, RNA, plasmid, siRNA, and ODN, and any nucleic acid having any length and sequence may be used as long as it does not exhibit physiological activity.
  • the adhesion competing agent is preferably electrostatically attached to the lead particles, and is a substance that does not form a cross-link that agglomerates the lead particles even if it adheres to the lead particles, or a part that adheres in the molecule. And a substance having a portion that repels adhesion and suppresses aggregation of lead particles.
  • step 1 includes, for example, an operation for producing a liquid in which lead particles containing an aggregation inhibitor are dispersed, and the double-stranded nucleic acid molecule is dispersed or dissolved in the liquid in which the lead particles are dispersed.
  • the operation of containing for example, the operation of adding and dispersing or dissolving the double-stranded nucleic acid molecule in the liquid in which the lead particles are dispersed; the double-stranded nucleic acid molecule being dispersed or dissolved in the liquid in which the lead particles are dispersed; For example, an operation of adding the prepared liquid).
  • the composite particle obtained by the step of dispersing or dissolving the double-stranded nucleic acid molecule in the liquid in which the lead particles are dispersed is specifically a liposome B containing a cationic substance.
  • Composite particles formed by attaching the double-stranded nucleic acid molecules to fine particles composite particles formed by attaching the double-stranded nucleic acid molecules to fine particles which are lipid aggregates containing a cationic substance, poly-L -Composite particles formed by adhering the double-stranded nucleic acid molecule to fine particles that are a polymer containing a cationic polymer such as lysine.
  • the operation of dispersing or dissolving the double-stranded nucleic acid molecule in the liquid in which the lead particles are dispersed includes adding an adhesion competitor to the liquid in which the double-stranded nucleic acid molecule is dispersed or dissolved. In this case, it is preferable to add this to the liquid in which the lead particles are dispersed. In this case, the double-stranded nucleic acid molecule and the adhesion competitor are attached to the lead particles to produce composite particles. Aggregation of the lead particles during the production of the composite particles and the aggregation of the composite particles after the production can be further suppressed.
  • the ratio of the lead particles to the liquid in which the lead particles are dispersed is not particularly limited as long as the double-stranded nucleic acid molecule can be attached to the lead particles, but is preferably about 1 ⁇ g / mL to 1 g / mL, about 0.1 ⁇ g / mL. More preferably, it is ⁇ 500 mg / mL.
  • Step 2 Step of coating composite particles with lipid bilayer membrane
  • Part 1 Operation for preparing a liquid (liquid A) containing the polar organic solvent in which the composite particles obtained in step 1 are dispersed and the lipid bilayer components are dissolved, and then the concentration of the polar organic solvent in the liquid A
  • liposome A can be produced by a production method including an operation of coating composite particles with a lipid bilayer membrane.
  • liposome A is obtained in the form of a dispersion (liquid B).
  • the solvent in the liquid A is a solvent containing the polar organic solvent at a concentration of the polar organic solvent in which the components of the lipid bilayer membrane are soluble and the composite particles can be dispersed.
  • the constituent components of the lipid bilayer membrane can be dispersed and the composite particles can also be dispersed.
  • the solvent in the liquid A is a mixed liquid of a polar organic solvent and a solvent other than the polar organic solvent, for example, a solvent (liquid C) containing a solvent other than the polar organic solvent that can be mixed with the polar organic solvent is added.
  • the concentration of the polar organic solvent can be reduced by selectively removing the polar organic solvent by evaporative distillation, semipermeable membrane separation, fractional distillation, or the like.
  • the liquid C is preferably a liquid containing a solvent other than the polar organic solvent, but the polar organic solvent may be included as long as it is lower than the concentration of the polar organic solvent in the liquid A.
  • Examples of the solvent other than the polar organic solvent in Step 2 include water, liquid carbon dioxide, liquid hydrocarbon, halogenated carbon, halogenated hydrocarbon, and the like, and preferably water.
  • the liquid A and the liquid C may contain an ion or a buffer component. These solvents can be used alone or in combination of two or more.
  • the combination of the polar organic solvent and the solvent other than the polar organic solvent is preferably a combination that can be mixed with each other, and the components of the composite particles and the lipid bilayer membrane with respect to the solvent in liquid A and liquid B and liquid C
  • the solubility can be selected in consideration of the solubility.
  • the solubility in any of the solvent in liquid A and liquid B and liquid C is low, and the solubility in any solvent other than polar organic solvent and polar organic solvent is also low.
  • the components of the lipid bilayer membrane are preferably low in solubility in the solvent in liquid B and in liquid C, preferably high in solubility in the solvent in liquid A, and polar organic
  • the solubility in a solvent is preferably high, and the solubility in a solvent other than the polar organic solvent is preferably low.
  • “the solubility of the composite particles is low” means that the components such as lead particles, double-stranded nucleic acid molecules and adhesion competitors contained in the composite particles have low elution properties in the solvent. Even if the solubility of each component is high, it is only necessary that the elution of each component is reduced due to the bond between the components.
  • the lead particle even when the solubility of any of the components contained in the lead particle in the solvent in the liquid A is high, if the lead particle has a positive charge, the charge in the double-stranded nucleic acid molecule, the intramolecular polarization, etc. By binding to, elution of the components in the composite particles is suppressed, and the solubility of the composite particles in the solvent in the liquid A can be lowered. That is, the fact that the lead particles have a positive charge also has the effect of suppressing the elution of the components of the composite particles in the production of liposome A and improving the productivity and yield.
  • the concentration of the polar organic solvent in the liquid A is not particularly limited as long as the components of the lipid bilayer membrane are soluble and the composite particles can be dispersed.
  • the solvent, the composite particles, and the configuration of the lipid bilayer membrane to be used Although it varies depending on the type of component, etc., it is preferably about 30 v / v% or more, more preferably about 60 to 90 v / v%.
  • the concentration of the polar organic solvent in the liquid B is particularly limited as long as it contains the polar organic solvent at a lower concentration than the liquid A, the constituent components of the lipid bilayer membrane can be dispersed, and the composite particles can also be dispersed. Although it is not a thing, Preferably it is about 50 v / v% or less.
  • the step of preparing the liquid A includes a step of preparing the liquid A by mixing polar organic solvents, composite particles and components of the lipid bilayer membrane, and if necessary, a solvent other than the polar organic solvent.
  • the components of the polar organic solvent, the composite particle and the lipid bilayer membrane, and optionally the solvent other than the polar organic solvent are not particularly limited in the order of adding them unless the composite particles are dissolved.
  • a liquid (liquid D) containing a polar organic solvent in which particles are dispersed is prepared, and the components of the lipid bilayer membrane are dissolved in a solvent containing a polar organic solvent that is the same as or different from the polar organic solvent in liquid D (Liquid E) is prepared, and liquid D and liquid E are mixed and prepared.
  • liquid D and liquid E it is preferable to mix gradually.
  • Step 3) Step of coating composite particles with lipid bilayer (Part 2)
  • the component of the composite particle and lipid bilayer membrane obtained in step 1 includes a polar organic solvent in which the component of the lipid bilayer membrane is soluble, and the component of the lipid bilayer membrane is present in a dispersed state
  • Liposome A can be produced by a production method including an operation of dispersing in a liquid (liquid F) containing the polar organic solvent at a concentration capable of being obtained. In this case, liposome A is obtained in the state of a dispersion.
  • Liquid F contains a polar organic solvent in which the constituent components of the lipid bilayer membrane are soluble, but the polar organic solvent at a specific concentration at which both the constituent components of the lipid bilayer membrane and the composite particles can be dispersed. It is a liquid containing.
  • liquid F can take any form.
  • liquid F may be prepared by mixing both solutions.
  • Liquid F may be prepared by preparing a dispersion of either one of the components, and adding and dispersing one of the remaining components of the composite particles in the solid state or the lipid bilayer membrane to the dispersion.
  • the composite particle dispersion medium may contain a polar organic solvent in advance.
  • the component solvent or dispersion medium may be a liquid containing a polar organic solvent or a liquid composed only of a polar organic solvent.
  • the dispersion is preferably a liquid containing a polar organic solvent.
  • the polar organic particles are not dissolved and the components of the lipid bilayer are dispersed.
  • a polar organic solvent may be added, the polar organic solvent may be removed, or the concentration may be decreased within the range of the solvent concentration.
  • the composite particles are not dissolved after preparing the liquid F.
  • the composite particles are not dissolved and the components of the lipid bilayer membrane are dispersed.
  • the polar organic solvent may be removed or the concentration reduced within the range of the polar organic solvent concentration.
  • the components of the composite particles and lipid bilayer membrane are mixed in advance in a solvent other than the polar organic solvent, and the range of polar organic solvent concentration in which the composite particles do not dissolve and the components of the lipid bilayer membrane are dispersed
  • a polar organic solvent may be added.
  • each of the components of the composite particle and the lipid bilayer membrane may be dispersed in a solvent other than the polar organic solvent, and after mixing both dispersions, the polar organic solvent may be added.
  • Either one of the components of the lipid bilayer membrane was dispersed in a solvent other than the polar organic solvent, and the remaining one of the solid-state composite particles or the components of the lipid bilayer membrane was added to the dispersion and dispersed. Later, a polar organic solvent may be added.
  • the component of the composite particles and the lipid bilayer membrane is dispersed, and a liquid containing a polar organic solvent is allowed to stand or mix for a time sufficient for the composite particles to be coated with the lipid bilayer membrane. Is preferred.
  • the time for standing or mixing the components of the composite particles and the lipid bilayer membrane with the polar organic solvent There is no limitation unless it is instantaneously terminated after being dispersed in the liquid containing, but can be arbitrarily set according to the components of the lipid bilayer membrane and the type of liquid containing the polar organic solvent, It is preferable to set a time during which the yield of the obtained liposome A is a steady amount, for example, about 3 seconds to 30 minutes.
  • the coating of the lipid bilayer on the composite particle is started, and the lipid bilayer on the composite particle is quickly
  • the coating of the membrane may be completed. For example, after preparing a solution of lipid bilayer components, mix the composite particle dispersion and the solution of lipid bilayer components.
  • preparing F if the solubility of the components of lipid bilayer membrane in liquid F is low, the components of lipid bilayer membrane are dispersed almost simultaneously with the liquid containing a specific polar organic solvent. In some cases, the coating of the lipid bilayer on the composite particles may be completed.
  • Examples of the solvent other than the polar organic solvent in the liquid F include those exemplified for the solvent other than the polar organic solvent in Step 2, and preferably water.
  • the concentration of the polar organic solvent in the liquid F is not particularly limited as long as the composite particles and the components of the lipid bilayer membrane are both dispersed.
  • the solvent, the composite particles, and the lipid bilayer to be used are not limited. Although it varies depending on the type of membrane constituents, etc., it is preferably about 1-80 v / v%, more preferably about 10-60 v / v%, more preferably about 20-50 v / v%, most preferably about 30-40 v. / v%.
  • the component of the lipid bilayer membrane is soluble in a specific polar organic solvent means that the component of the lipid bilayer membrane is soluble in a specific polar organic solvent
  • the components of the lipid bilayer membrane form aggregates or micelles in the specific polar organic solvent.
  • the components of the lipid bilayer membrane are dispersed means that all of the components of the lipid bilayer membrane are aggregated or micelles and are emulsified or emulsified.
  • part of the constituents forms aggregates or micelles to become an emulsion or emulsion, and the remaining part is dissolved, part of the constituents of the lipid bilayer membrane forms aggregates or micelles, etc. It includes a state in which it is emulsified or emulsified and the remaining part is precipitated. Note that “the constituents of the lipid bilayer membrane dissolve” does not include a state in which all of the constituent components of the lipid bilayer membrane form an aggregate or micelle to be emulsified or emulsified.
  • composite particles are dispersed means a state in which the composite particles are suspended, emulsified or emulsified, and a part of the composite particles are suspended, emulsified or emulsified, and the remaining part. And a state where a part of the composite particle is emulsified or emulsified and a remaining part is precipitated. “Composite particles do not dissolve” has the same meaning as “composite particles are dispersed”.
  • the concentration of the composite particles in the polar organic solvent-containing aqueous solution used in the method for producing liposome A in the present invention is not particularly limited as long as the composite particles can be covered with a lipid bilayer membrane, but is about 1 ⁇ g / mL to 1 g. / mL, preferably about 0.1 to 500 mg / mL.
  • the concentration of the constituent components of the lipid bilayer membrane used is not particularly limited as long as the composite particles can be coated, but is preferably about 1 ⁇ g / mL to 1 g / mL, preferably about 0.1 to 400 mg / mL. More preferably.
  • the size of the liposome A in the present invention is more preferably an average particle diameter of about 30 nm to 300 nm, more preferably about 50 nm to 200 nm, and specifically, for example, an injectable size. Is preferred.
  • the liposome A obtained above can be modified with substances such as proteins such as antibodies, saccharides, glycolipids, amino acids, nucleic acids, various low molecular compounds or high molecular compounds, and the coated composite particles obtained from these Also included in liposome A.
  • the liposome A obtained above can be further subjected to surface modification of the lipid bilayer with proteins such as antibodies, peptides or fatty acids [D. D. Lasic ), Edited by F. Martin, "Stealth Liposomes" (USA), CRC Press Inc, 1995, p. 93-102].
  • the liposome A can be optionally subjected to surface modification with, for example, a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative, and the water-soluble substance lipid derivative, fatty acid derivative or
  • the aliphatic hydrocarbon derivative is synonymous with a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component of the lipid bilayer membrane.
  • the composition of the present invention By administering the composition of the present invention to mammals including humans and delivering the double-stranded nucleic acid molecule to the target gene expression site, the gene expression is suppressed in mammalian cells in vivo. RNA and the like can be introduced, and the expression of genes and the like can be suppressed.
  • the nucleic acid in the composition of the present invention is delivered into cells of the delivered organ or site, for example, by being delivered to an organ or site in which cancer or inflammation has occurred. Can be introduced.
  • the organ or site where cancer or inflammation has occurred is not particularly limited, and examples thereof include stomach, large intestine, liver, lung, spleen, pancreas, kidney, bladder, skin, blood vessel, and eyeball.
  • the composition of the present invention is intravenously administered to mammals including humans, so that it is delivered to, for example, blood vessels, liver, lungs, spleen and / or kidney, and the composition of the present invention is contained in cells of a delivery organ or site.
  • the nucleic acid in can be introduced.
  • Liver, lung, spleen and / or kidney cells may be normal cells, cells associated with cancer or inflammation or cells associated with other diseases. That is, the present invention also provides a method for suppressing the expression of a target gene, wherein the composition of the present invention described above is administered to a mammal.
  • the administration subject is preferably a human.
  • the composition of the present invention is treated with a therapeutic or preventive agent for cancer or inflammatory disease, preferably a solid cancer or blood vessel or blood vessel. It can be used as a therapeutic or prophylactic agent for nearby inflammation.
  • a therapeutic or preventive agent for cancer or inflammatory disease preferably a solid cancer or blood vessel or blood vessel. It can be used as a therapeutic or prophylactic agent for nearby inflammation.
  • the target gene in the composition of the present invention is a gene related to angiogenesis
  • the proliferation of vascular smooth muscle, angiogenesis, etc. can be suppressed.
  • It can be used as a therapeutic or prophylactic agent for cancer or inflammatory diseases accompanied by muscle growth and angiogenesis. That is, the present invention also provides a method for treating cancer or inflammatory disease, wherein the composition of the present invention described above is administered to a mammal.
  • the administration subject is preferably a person, more preferably a person suffering from cancer or an inflammatory disease.
  • the composition of the present invention can also be used as a tool for obtaining POC (Proof of concept) in an in vivo screening system for a therapeutic or prophylactic agent for cancer or inflammatory diseases.
  • composition of the present invention can be used to stabilize a double-stranded nucleic acid molecule in a biological component such as a blood component (for example, blood, digestive tract, etc.), 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.), 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.
  • composition of the present invention When the composition of the present invention is used as a therapeutic agent or preventive agent for pharmaceutical cancer or inflammatory disease, it is desirable to use the most effective administration route for treatment.
  • Oral administration such as rectal, subcutaneous, intramuscular or intravenous, or oral administration can be mentioned, preferably intravenous administration or intramuscular administration can be mentioned, and intravenous administration is more preferred.
  • the dose varies depending on the disease state, age, administration route, etc. of the administration subject, but for example, the dose may be administered so that the daily dose converted to RNA is about 0.1 ⁇ g to 1000 mg.
  • Examples of the preparation suitable for intravenous administration or intramuscular administration include injections, and the dispersion of liposome A prepared by the above-described method can be used as it is in the form of injections, for example.
  • the dispersion can be used after removing the solvent, for example, by filtration, centrifugation, etc., or the dispersion can be used after lyophilization, or an excipient such as mannitol, lactose, trehalose, maltose or glycine is added.
  • the dispersion can also be lyophilized for use.
  • injections for example, water, acid, alkali, various buffer solutions, physiological saline, amino acid infusion, etc.
  • 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. Moreover, it can also be cryopreserved by adding a cryopreservation agent such as glycerin.
  • an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA or an isotonic agent such as glycerin, glucose or sodium chloride.
  • an isotonic agent such as glycerin, glucose or sodium chloride.
  • it can also be cryopreserved by adding a cryopreservation agent such as glycerin.
  • the double-stranded nucleic acid molecule is a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) The double-stranded nucleic acid molecule, wherein 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group.
  • Liposome A is a liposome comprising (1) a composite particle comprising a lead particle and the double-stranded nucleic acid molecule as a constituent component and a lipid bilayer membrane covering the composite particle, and the constituent component of the lipid bilayer membrane Is soluble in a specific polar organic solvent, the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration, and the lipid bilayer membrane is Lipids that are lipid bilayer membranes comprising lipid derivatives, fatty acid derivatives or aliphatic hydrocarbon derivatives of water-soluble substances, or (2) lead particles containing cationic substances and the double-stranded nucleic acid molecules as constituents Composite particles and lipids that coat the composite particles A liposome containing a bilayer, wherein the lipid bilayer is a lipid bilayer comprising a lipid, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble
  • the present invention is a double-stranded nucleic acid molecule in which the double-stranded nucleic acid molecule is composed of a sense strand and an antisense strand in the composition of the present invention
  • the antisense strand has a sequence of bases 1 to 17 (sequence a) from the 5 ′ end to the 3 ′ end, and a sequence of 17 bases of mRNA of a target gene related to tumor or inflammation.
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) The double-stranded nucleic acid molecule, wherein 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group.
  • Liposome A is a liposome comprising (1) a composite particle comprising a lead particle and the double-stranded nucleic acid molecule as a constituent component and a lipid bilayer membrane covering the composite particle, and the constituent component of the lipid bilayer membrane Is soluble in a specific polar organic solvent, the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration, and the lipid bilayer membrane is Lipids that are lipid bilayer membranes comprising lipid derivatives, fatty acid derivatives or aliphatic hydrocarbon derivatives of water-soluble substances, or (2) lead particles containing cationic substances and the double-stranded nucleic acid molecules as constituents Composite particles and lipids that coat the composite particles A liposome containing a bilayer, wherein the lipid bilayer is a lipid bilayer comprising a lipid, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble
  • the present invention will be specifically described with reference to examples and test examples.
  • the present invention is not limited to these examples and test examples. It consists of the sense (sense) chain and the antisense (antsense) chain shown in Table 1 (the sugar that binds to the base marked with d in the table is deoxyribose, and the sugar that binds to the base marked with m Is a ribose substituted with 2'-O-methyl), a double-stranded nucleic acid molecule comprising the sequence of 19 bases 5'-GUG AAG UCA ACA UGC CUG C-3 'of the BCL2 gene mRNA Used in Examples 1 to 4 and Comparative Examples 1 to 9. Those double-stranded nucleic acid molecules were prepared by obtaining the respective sense strand and antisense strand from Hokkaido System Science Co., Ltd. and annealing them.
  • DOTAP (manufactured by Avanti Polar Lipds)
  • PEG-DSPE (manufactured by NOF Corporation)
  • distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.)
  • This suspension was applied to a 0.4 ⁇ m polycarbonate membrane filter (manufactured by Coster) at 70 ° C 10 times, to a 0.2 ⁇ m polycarbonate membrane filter (manufactured by Whatman) three times, and to a 0.1 ⁇ m polycarbonate membrane filter (manufactured by Corning).
  • the sample was passed 10 times and further 20 times through a 0.05 ⁇ m polycarbonate membrane filter (Whatman).
  • the average particle diameter of the lead particles obtained by dynamic light scattering (DLS) was measured and found to be 70.71 nm.
  • EPC manufactured by NOF Corporation
  • PEG-DSPE manufactured by NOF Corporation
  • ethanol manufactured by Wako Pure Chemical Industries, Ltd.
  • Water is mixed to 15 mg / 3.125 mg / 0.625 mL / 0.375 mL, and lipid A solution of the components of the bilayer membrane was prepared.
  • the resulting dispersion of lead particles (0.0125 mL) was mixed with 0.00417 mL of an aqueous solution obtained by mixing BCL2siRNA-Exp.1 / water as shown in Table 1 to a concentration of 24 mg / 1 mL to obtain composite particles.
  • the resulting composite particle dispersion was added to 0.06667 mL of the lipid bilayer component solution, followed by 0.02083 mL of distilled water.
  • liposomes were prepared.
  • the resulting liposome suspension was made isotonic with saline.
  • the final solution volume was adjusted to 1 mL with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) to adjust the BCL2 siRNA-Exp.1 concentration to 0.1 mg / mL, thereby obtaining a preparation.
  • physiological saline manufactured by Otsuka Pharmaceutical Co., Ltd.
  • an average particle size of the liposome in the preparation was measured by DLS, it was 82.59 nm.
  • a preparation was obtained in the same manner as in Example 1 except that BCL2siRNA-Exp.1 was changed to BCL2siRNA-Exp.2.
  • an average particle size of the liposome in the preparation was measured by DLS, it was 83.94 nm.
  • Comparative Examples 1-9 A preparation was obtained in the same manner as in Example 1 except that BCL2siRNA-Exp.1 was changed to BCL2siRNA-Com.1-9, respectively.
  • the average particle size of liposomes in each preparation was measured by DLS. Table 1 shows the average particle size of liposomes in each preparation.
  • a preparation was obtained in the same manner as in Example 1 except that BCL2siRNA-Exp.1 was changed to BCL2siRNA-Exp.3.
  • an average particle size of the liposome in the preparation was measured by DLS, it was 82.42 nm.
  • a preparation was obtained in the same manner as in Example 1 except that BCL2siRNA-Exp.1 was changed to BCL2siRNA-Exp.4. It was 83.47 nm when the average particle diameter of the liposome in a formulation was measured by DLS.
  • Test example 1 The RNAi activities of BCL2siRNA-Exp.1-4 and BCL2siRNA-Com.1-9 were evaluated by measuring the Bcl2 mRNA expression inhibitory effect as shown below.
  • Human prostate cancer cells PC-3 were seeded at a density of 2 ⁇ 10 5 cells / dish in a 6 cm diameter culture dish, in F-12 Kaighn's medium (GIBCO, 21127) containing 10% fetal calf serum, 37 ° C., 5% Incubate overnight under CO 2 conditions. The next day, the medium was aspirated from the culture dish and replaced with 0.8 mL of low serum basal medium OPTI-MEM (GIBCO, 31985).
  • the siRNA was introduced into PC-3 by adding 0.2 mL of siRNA-oligofectamine complex solution mixed in OPTI-MEM. The final concentration of siRNA was 2 points of 3 nM and 30 nM.
  • Human prostate cancer cells PC-3 introduced with siRNA were cultured in a 5% CO 2 incubator at 37 ° C. for 48 hours, washed twice with PBS, and transferred to a 1.5 mL tube using a cell scraper. After centrifugation at 1000 xg for 2 minutes and removing the supernatant, the cells are lysed and collected in RLT buffer (attached to Qiagen RNA Recovery Kit ⁇ RNeasy ''), and all cells are collected according to the instructions attached to the kit. RNA was recovered.
  • a reverse transcription reaction was performed using 1 ⁇ g of total RNA as a template and Superscript VILO (Invitrogen) to prepare cDNA.
  • cDNA as a template for PCR reaction, specific to bcl-2 gene and GADPH (D-glyceraldehyde-3-phosphate dehydrogenase) gene which is a constitutive expression gene by Taqman probe method using ABI7900HT Fast (ABI) PCR amplification was performed, and the amount of mRNA was quantified.
  • 250 ng of total RNA-derived cDNA was used as a template.
  • the amount of mRNA in the sample was expressed as a relative ratio when the amount of bcl-2 mRNA or GADPH mRNA was 1 in the siRNA non-introduced group (untreated).
  • the expression level ratio of each specimen subtracted from 1 is expressed as the expression suppression rate and is shown in FIG.
  • Test example 2 As shown below, the preparations obtained in Examples 1-2 and Comparative Examples 1-9 as PEG-modified liposomes for the first administration were administered to mice, and then administered for the second time with an interval of 7 days. It was obtained in Examples 1-2 and Comparative Examples 1-9 by administering the preparation obtained in Comparative Example 1 as a PEG-modified liposome and measuring the concentration of BCL2 siRNA-Com.1 in the blood 3 hours after administration. The effect of the obtained preparation on the blood retention of the second dose PEG-modified liposome was evaluated.
  • the preparations obtained in Examples 1 and 2 and Comparative Examples 1 to 9 were administered to male Balb / c mice (6 weeks old, Claire, Japan) with 100 ⁇ L of drug solution (siRNA concentration 50 ⁇ g / mL) from the tail vein ( The dose is 5 ⁇ g / mouse). At intervals of 7 days, 100 ⁇ L of the drug solution of the preparation obtained in Comparative Example 1 (siRNA concentration 50 ⁇ g / mL) was administered from the tail vein (dose was 5 ⁇ g / mouse).
  • diethylpyrocarbonate aqueous solution diethylpyrocarbonate was added to ultrapure water in a volume of 0.1 v / v% and mixed
  • IS solution as IS
  • GenTLE precipitation carrier (Takara Bio) 15-fold diluted with the above-mentioned diethyl pyrocarbonate aqueous solution) to 65 ⁇ L of the supernatant and mixing, add ethanol and mix and centrifuge Discard the supernatant, add 75 v / v% ethanol to the precipitate, centrifuge, discard the supernatant, air dry the precipitate, and re-dissolve the solution (diethylpyrocarbonate / triethylamine / hexafluoroisopropanol / water 0.1 / It was dissolved in 50 ⁇ L (mixed at a volume ratio of 0.4 / 30/1000) and quantified by HPLC.
  • the double-stranded nucleic acid molecules (Exp.1 to Exp.4) used in Examples 1 to 4 showed siRNA activity comparable to that of the double-stranded nucleic acid molecule used in Comparative Example 1, The siRNA activity is higher than the double-stranded nucleic acid molecules used in Comparative Examples 2 to 9.
  • the preparation obtained in Comparative Example 1 was administered, and then two mice were administered with the preparation obtained in Comparative Example 1 as the second administration PEG-modified liposome at an interval of 7 days.
  • composition comprising a liposome encapsulating a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand
  • the antisense strand has a base sequence complementary to the 17-base sequence of the target gene mRNA in which the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end (sequence a)
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is a liposome having a size that can be administered intravenously, and the liposome is a liposome having a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component.
  • composition of the present invention can reduce the side effects due to the suppression of the blood retention in the second administration of PEG-modified liposomes, and can also reduce the side effects and the tissue containing the target gene expression site or It was revealed that drug accumulation in organs can be increased.
  • DOTAP (manufactured by Avanti Polar Lipds)
  • PEG-DSPE (manufactured by NOF Corporation)
  • distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.)
  • This suspension was applied to a 0.4 ⁇ m polycarbonate membrane filter (manufactured by Coster) at 70 ° C 10 times, to a 0.2 ⁇ m polycarbonate membrane filter (manufactured by Whatman) three times, and to a 0.1 ⁇ m polycarbonate membrane filter (manufactured by Corning).
  • the sample was passed 10 times and further 20 times through a 0.05 ⁇ m polycarbonate membrane filter (Whatman).
  • the average particle diameter of the lead particles obtained by dynamic light scattering (DLS) was measured and found to be 71.44 nm.
  • EPC manufactured by NOF Corporation
  • PEG-DSPE manufactured by NOF Corporation
  • ethanol manufactured by Wako Pure Chemical Industries, Ltd.
  • Water is mixed to 15 mg / 3.125 mg / 0.625 mL / 0.375 mL, and lipid A solution of the components of the bilayer membrane was prepared.
  • Composite particles were prepared by mixing 0.00833 mL of a 24 mg / mL aqueous solution of BCL2siRNA-Exp.5 with 0.025 mL of the obtained lead particle dispersion. The resulting composite particle dispersion was added to 0.13334 mL of the lipid bilayer component solution followed by 0.04166 mL of distilled water. Add 0.01334 mL of a solution of EPC / PEG-DSPE dissolved in 40 vol% ethanol to 62.5 mg / 62.5 mg / mL, then gradually add 1.5517 mL of distilled water to bring the ethanol concentration to 5% or less. As described above, liposomes were prepared.
  • the resulting liposome suspension was made isotonic with saline. Furthermore, the final solution volume was adjusted to 2 mL with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) to adjust the BCL2 siRNA-Exp.5 concentration to 0.1 mg / mL to obtain a preparation. When an average particle size of the liposome in the preparation was measured by DLS, it was 77.26 nm.
  • Comparative Examples 10-13 A preparation was obtained in the same manner as in Example 5 except that BCL2siRNA-Exp.5 was changed to BCL2siRNA-Com.10 to 13 respectively.
  • the average particle size of liposomes in each preparation was measured by DLS.
  • Table 2 shows the average particle size of the liposomes of each preparation.
  • Test example 3 The RNAi activities of BCL2siRNA-Exp.5 and BCL2siRNA-Com.10-13 were evaluated by measuring the Bcl2 mRNA expression inhibitory effect as shown below.
  • PC-3 is seeded at a density of 2 ⁇ 10 5 cells / dish in a 6 cm diameter culture dish, in F-12 Kaighn's medium (GIBCO, 21127) containing 10% fetal calf serum, at 37 ° C. and 5% CO 2 Incubated overnight. The next day, the medium was aspirated from the culture dish and replaced with 0.8 mL of low serum basal medium OPTI-MEM (GIBCO, 31985).
  • the siRNA was introduced into PC-3 by adding 0.2 mL of siRNA-oligofectamine complex solution mixed in OPTI-MEM. The final siRNA concentration was 10 nM.
  • the cells into which the siRNA had been introduced were cultured in a 5% CO 2 incubator at 37 ° C. for 48 hours, washed twice with PBS, and transferred to a 1.5 mL tube using a cell scraper. After centrifugation at 1000 xg for 2 minutes and removing the supernatant, the cells are lysed and collected in RLT buffer (attached to Qiagen RNA Recovery Kit ⁇ RNeasy ''), and all cells are collected according to the instructions attached to the kit. RNA was recovered.
  • a reverse transcription reaction was performed using 1 ⁇ g of total RNA as a template and Superscript VILO (Invitrogen) to prepare cDNA.
  • cDNA as a template for PCR reaction, specific to bcl-2 gene and GADPH (D-glyceraldehyde-3-phosphate dehydrogenase) gene which is a constitutive expression gene by Taqman probe method using ABI7900HT Fast (ABI) PCR amplification was performed, and the amount of mRNA was quantified.
  • 250 ng of total RNA-derived cDNA was used as a template.
  • the amount of mRNA in the sample was expressed as a relative ratio when the amount of bcl-2 mRNA or the amount of GADPH mRNA in the siRNA non-introduced group (untreated) was 1, and is shown in FIG.
  • Test example 4 The preparations obtained in Example 5 and Comparative Examples 10 to 13 were used as the first-administered PEG-modified liposomes, while the same Example 5 and Comparative Examples 10 to 13 were used as the second-administration PEG-modified liposomes.
  • the effects of the preparations obtained in Example 5 and Comparative Examples 10 to 13 on the blood retention of PEG-modified liposomes administered for the second time were evaluated.
  • the internal standard is 5'-GmUG mAAmG UmCA mACmA UmGC mCUmG CdT-3 '(2, 4, 6, 8, 10, 12, 14, 16, 18th m from the 5'-side)
  • the sugar that binds to the attached base is ribose substituted with 2'-O-methyl, and the sugar that binds to the base attached with d is deoxyribose) (SEQ ID NO: 39) and 5 ' -GCA GGC AUG UUG ACU UCA CdT-3 '(the sugar bonded to the base to which d is attached is deoxyribose) (SEQ ID NO: 40) was used.
  • FIG. 3 shows that the double-stranded nucleic acid molecule used in Example 5 shows siRNA activity equivalent to that of the double-stranded nucleic acid molecule used in Comparative Examples 10-13.
  • FIG. 4 shows that in the mice administered with the preparations obtained in Comparative Examples 10 to 13 twice at an interval of 7 days, double-stranded nucleic acid molecules were not found in the blood, and the second administration of PEG-modified liposomes. In the mice in which the preparation obtained in Example 5 was administered twice with a 7-day interval, the concentration of double-stranded nucleic acid molecules in the blood was high, It shows that the decrease in the blood retention in the second administration of the modified liposome is suppressed.
  • composition comprising a liposome encapsulating a double-stranded nucleic acid molecule composed of a sense strand and an antisense strand
  • the antisense strand has a base sequence complementary to the 17-base sequence of the target gene mRNA in which the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end (sequence a)
  • the sense strand has a length of 17 to 30 bases including a base sequence (sequence b) complementary to the sequence of the 1st to 17th bases from the 5 ′ end to the 3 ′ end of the antisense strand.
  • the sugar in the sense strand is ribose, deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group
  • a modifying group 0-30% of the sugars that bind to the 1st to 8th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • 0-20% of the sugars that bind to the 9th to 16th bases from the 5 ′ end to the 3 ′ end of sequence a are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose
  • iii) 30-100% of the sugars bound to the 17th to 3 'terminal bases from the 5' end side to the 3 'end side of the antisense strand are deoxyribose or the hydroxyl
  • Ribose substituted with (iv) 10-70% of the sugars that bind to the 1st to 17th bases from the 5 ′ end to the 3 ′ end of sequence b are each substituted with deoxyribose or the hydroxyl group at the 2 ′ position with a modifying group Ribose, (v) 30-100% of the sugars that bind to bases other than sequence b of the sense strand are deoxyribose or ribose in which the hydroxyl group at the 2 ′ position is substituted with a modifying group,
  • the liposome is a liposome having a size that can be administered intravenously, and the liposome is a liposome having a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component.
  • composition of the present invention can reduce the side effects due to the suppression of the blood retention in the second administration of PEG-modified liposomes, and can also reduce the side effects and the tissue containing the target gene expression site or It was revealed that drug accumulation in organs can be increased.
  • the expression of the target gene can be suppressed by administering the composition of the present invention to mammals or the like.

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Abstract

L'invention porte sur : une composition comprenant une molécule d'acide nucléique double brin et un liposome dans lequel est encapsulée la molécule d'acide nucléique double brin ; et d'autres éléments. Dans la composition, la molécule d'acide nucléique double brin comprend un brin non codant et un brin codant, le brin non codant étant un polynucléotide qui contient une séquence de nucléotides complémentaire à une séquence composée de 17 nucléotides contigües contenus dans de l'ARNm pour un gène cible, et qui a une longueur de 17 à 30 nucléotides, le brin codant étant un polynucléotide qui contient une séquence de nucléotides complémentaire à une séquence située entre le 1er nucléotide et le 17ième nucléotide compté à partir de la terminaison 5' vers la terminaison 3' dans le brin non codant, et qui a une longueur de 17 à 30 nucléotides, chacun des sucres liés respectivement à un nucléotide se situant en une position spécifique dans le brin non codant et un nucléotide se situant en une position spécifique dans le brin codant étant un désoxyribose ou un ribose dans lequel un groupe hydroxy en position 2' est substitué par un groupe modifiant, et le désoxyribose et le ribose étant contenus sous un rapport spécifique. Dans la composition, le liposome a une membrane bicouche lipidique comprenant, comme composant, un dérivé de lipide, un dérivé d'acide gras ou un dérivé hydrocarboné aliphatique d'une substance soluble dans l'eau.
PCT/JP2010/061884 2009-07-14 2010-07-14 Composition pour inhiber l'expression d'un gène cible WO2011007795A1 (fr)

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