WO2010110314A1 - Therapeutic agent for pulmonary hypertension comprising nucleic acid - Google Patents

Therapeutic agent for pulmonary hypertension comprising nucleic acid Download PDF

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WO2010110314A1
WO2010110314A1 PCT/JP2010/055080 JP2010055080W WO2010110314A1 WO 2010110314 A1 WO2010110314 A1 WO 2010110314A1 JP 2010055080 W JP2010055080 W JP 2010055080W WO 2010110314 A1 WO2010110314 A1 WO 2010110314A1
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rna
lipid
lipid bilayer
liposome
pulmonary hypertension
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PCT/JP2010/055080
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French (fr)
Japanese (ja)
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信宏 八木
智幸 直井
憲歳 永谷
裕明 小幡
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協和発酵キリン株式会社
独立行政法人国立循環器病研究センター
中村 篤宏
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Priority to JP2011506084A priority Critical patent/JPWO2010110314A1/en
Publication of WO2010110314A1 publication Critical patent/WO2010110314A1/en

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    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/12—Dual-specificity kinases (2.7.12)
    • C12Y207/12002—Mitogen-activated protein kinase kinase (2.7.12.2), i.e. MAPKK or MEK1 or MEK2
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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.

Definitions

  • the present invention relates to a therapeutic agent for pulmonary hypertension, a composition for treating pulmonary hypertension, and the like.
  • Pulmonary hypertension is an increase in vascular resistance in the pulmonary vasculature. It refers to a pathological condition that causes a continuous increase in pulmonary artery pressure. Pulmonary hypertension originates from primary pulmonary hypertension and atrial septal defect, pulmonary embolism, patent ductus arteriosus, mitral stenosis, pulmonary fibrosis, emphysema, collagen disease or cirrhosis There is secondary pulmonary hypertension.
  • Non-Patent Documents 1 and 2 it is known that an excessive factor for cell proliferation or growth exists (see Non-Patent Documents 1 and 2). It is expected to suppress pulmonary vascular wall tissue hypertrophy or sclerosis and suppress pulmonary hypertension by suppressing the expression of a gene encoding a factor that proliferates or grows these cells.
  • a nucleic acid is used as a drug.
  • there is no report of such delivery means until now, although it has to be delivered with high selectivity to enlarged pulmonary vessel wall tissue.
  • nucleic acid-encapsulated liposomes liposomes in which nucleic acids are encapsulated in liposomes
  • a cationic lipid is dissolved in chloroform in advance, and then an oligodeoxynucleotide (ODN) aqueous solution and methanol are added and mixed, followed by centrifugation.
  • ODN oligodeoxynucleotide
  • the ODN-encapsulated liposomes were prepared by reducing the ethanol concentration to 20 v / v%, sizing filtered, excess ethanol was removed by dialysis, and the sample was further adjusted to pH 7.5. Dialysed to remove ODN adhering to the liposome surface is reported a method of producing the ODN encapsulated liposomes, liposomes were each encapsulating active ingredients such as nucleic acid has been produced.
  • Patent Document 3 reports that a liposome in which an active ingredient such as a nucleic acid is encapsulated 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 microparticles are dispersed and the lipid is dissolved, the microparticles are coated with the lipid bilayer membrane, and the coating is not performed in the liquid.
  • fine particles (coated fine particles) coated with a lipid bilayer having a size suitable for fine particles for intravenous injection or the like are produced with excellent efficiency.
  • Patent Document 3 exemplifies a complex formed by electrostatic interaction composed of, for example, a water-soluble drug and a cationic lipid as an example of fine particles.
  • the particle size of the coated fine particles coated with the composite particles varies depending on the coated composite particles, but the coated fine particles obtained by coating the ODN-lipid complex have a small particle size and can be used as an injection.
  • the coated microparticles show a high blood retention when administered intravenously and accumulate in tumor tissues in large amounts.
  • Patent Documents 1 to 3 and Non-Patent Document 3 have reports on selective delivery of nucleic acids to enlarged pulmonary vascular wall tissues.
  • An object of the present invention is to provide a therapeutic agent for pulmonary hypertension and the like containing a nucleic acid.
  • the present invention relates to the following (1) to (60).
  • (1) (i) RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside A composition containing the prepared liposomes.
  • (2) The composition according to (1), wherein the liposome is a liposome having a size that can be administered intravenously.
  • RNAi RNA interference
  • the gene related to hypertrophy of pulmonary vascular wall tissue is a gene for any of cell growth-related factor, mitogen-activated protein kinase (MAP kinase; MAPK) signaling-related factor and apoptosis-related factor.
  • MAP kinase mitogen-activated protein kinase
  • MAPK mitogen-activated protein kinase
  • the composition according to any one of (1) to (3), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signaling-related factor.
  • MEK MAPK / ERK kinase
  • the liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a constituent, 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.
  • (1) to (7). The composition according to (8), wherein the polar organic solvent is an alcohol.
  • the composition according to (8), wherein the polar organic solvent is ethanol.
  • 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, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component.
  • the liposome encapsulating RNA is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent component, and a lipid bilayer coating the composite particle,
  • the lipid bilayer membrane according to any one of (1) to (7), wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative. Composition.
  • the cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- (11) one or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3 ⁇ - [N-[
  • the neutral lipid is egg yolk phosphatidylcholine.
  • RNAi RNA interference
  • the gene associated with hypertrophy of the pulmonary vascular wall tissue is a gene for any one of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor, according to any one of (16) to (18) The therapeutic agent for pulmonary hypertension as described.
  • a liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a 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 therapeutic agent for pulmonary hypertension according to any one of (16) to (22).
  • the therapeutic agent for pulmonary hypertension according to (23), wherein the polar organic solvent is alcohol.
  • the therapeutic agent for pulmonary hypertension according to (23), wherein the polar organic solvent is ethanol.
  • a lipid particle comprising lead particles containing a cationic substance and a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance.
  • the therapeutic agent for pulmonary hypertension according to any one of (23) to (25), which is a membrane.
  • the RNA encapsulated liposome is a liposome composed of a lead particle containing a cationic substance, a composite particle containing the RNA as a constituent component, and a lipid bilayer coating the composite particle,
  • the lipid bilayer membrane according to any one of (16) to (22), 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.
  • the cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- One or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3 ⁇ - [N- (N-
  • RNA interference RNA interference
  • the gene associated with hypertrophy of pulmonary vascular wall tissue is a gene for any of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor, and any of (31) to (33) The method for treating pulmonary hypertension as described.
  • RNA encapsulated liposome is a liposome composed of a lead particle, a composite particle comprising the RNA as a constituent, 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 liposome encapsulating RNA is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent component, and a lipid bilayer coating the composite particle,
  • the lipid bilayer membrane according to any one of (31) to (37), 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.
  • Cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- (41) one or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3 ⁇ - [N- (
  • RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside Use of the composition for the manufacture of a therapeutic agent for pulmonary hypertension, comprising the prepared liposomes.
  • RNA RNA having an action of suppressing the expression of the gene using RNA interference (RNAi).
  • RNAi RNA interference
  • the gene related to hypertrophy of the pulmonary vascular wall tissue is a gene for any of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor, according to any of (46) to (48) Use of description.
  • the liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising the RNA as a constituent, 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 use according to (53), wherein the polar organic solvent is an alcohol.
  • the use according to (53), wherein the polar organic solvent is ethanol.
  • a lipid particle comprising lead particles containing a cationic substance and a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance.
  • RNA encapsulated liposome is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent, and a lipid bilayer coating the composite particle,
  • the lipid bilayer membrane according to any one of (46) to (52), 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.
  • Cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- One or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3 ⁇ - [N- (N- (
  • Administration to the pulmonary vascular wall tissue with enlarged bronchi or lung can suppress the expression of genes related to pulmonary vascular wall tissue hypertrophy.
  • pulmonary hypertension can be treated by suppressing the expression of the gene associated with hypertrophy of pulmonary vascular wall tissue in bronchi or lung.
  • RNA is accumulated at a high concentration specifically in the MCT rat, which is a model causing pulmonary hypertension.
  • the horizontal axis represents the number of days after the start of administration, and the vertical axis represents the concentration (nM) of Cy5-labeled MEK siRNA in the lung.
  • ⁇ indicates the mean and standard deviation of the control group, and ⁇ indicates the monocrotaline-induced pulmonary hypertension rat (MCT rat) group.
  • * shows the point by which significant difference p value ⁇ 0.05 was recognized with respect to the base line as a result of statistical analysis (student t test).
  • ⁇ indicates a point where a significant difference between groups was found as a result of statistical analysis (student t test) ⁇ 0.05. It was shown that when the composition obtained in Example 1 was administered, RNA was selectively accumulated in the lung, which is a disease site of pulmonary hypertension.
  • the horizontal axis represents the organ or tissue name, and the vertical axis represents the concentration (nM) of Cy5-labeled MEK siRNA in each organ or tissue.
  • the white column represents the mean and standard deviation of the control group, and the black column represents the MCT rat group.
  • * shows the point by which significant difference p value ⁇ 0.05 was recognized between groups as a result of the statistical analysis (student
  • FIG. 1 Shown are immunostained sections of the pulmonary artery and its vicinity in the control group (left figure) and MCT rat group (right figure).
  • the white part indicates vascular endothelial cells stained in green
  • the arrow indicates the accumulation of Cy5-labeled MEK siRNA in which strong red fluorescence was observed. It was shown that when the composition obtained in Example 2 was administered, gene expression was suppressed in a dose-dependent manner in the lungs in which pulmonary hypertension occurred.
  • the vertical axis represents the ratio of the expression intensity of MEK1 (left figure) and MEK2 (right figure) mRNA in the lung to that of GAPDH.
  • 0.2 and 2 represent groups in which the composition obtained in Example 2 was intravenously administered at doses of 0.2 mg / kg and 2 mg / kg, and Control administered the composition obtained in Comparative Example 1.
  • the vertical axis represents the ratio of the expression intensity of MEK1 (left figure) and MEK2 (right figure) mRNA in the lung to that of GAPDH.
  • 24, 48, and 96 represent 24, 48, and 96 hours after administration of the composition obtained in Example 2, respectively, and Control represents a group that was administered with the composition obtained in Comparative Example 1.
  • MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. * Indicates a significant difference between the group administered with the composition obtained in Example 2 and the group administered with the composition obtained in Comparative Example 1 as a result of statistical analysis (student t test).
  • MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. * Indicates a significant difference between the group administered with the composition obtained in Example 2 and the group administered with the composition obtained in Comparative Example 1 as a result of statistical analysis (student t test).
  • MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. It was shown that when the composition obtained in Example 2 was administered, enlargement of the pulmonary artery wall was suppressed. The vertical axis shows the degree of enlargement of the pulmonary artery wall.
  • MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously.
  • Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously.
  • the gene associated with hypertrophy of pulmonary vascular wall tissue used in the present invention is not particularly limited as long as it is a gene associated with hypertrophy of pulmonary vascular wall tissue that is produced and expressed in mammalian bronchi or lung, for example, Examples include genes related to proliferation or growth of pulmonary blood vessels, preferably genes related to cell proliferation-related factors, MAP kinase signal transduction-related factors, apoptosis-related factors and the like.
  • Cell growth-related factors include, for example, fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor receptor, vascular endothelial growth factor (VGEF), vascular endothelial growth factor receptor, platelet-derived Growth factor, platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor, Ets transcription factor, hypoxia inducer, nuclear factor, etc.
  • VGEF vascular endothelial growth factor
  • mitogen-activated protein kinase MAPK kinase (MAPKK), MAPKK kinase (MAPKKK), c-Jun N-terminal kinase (JNK), p38MAPK and the like
  • apoptosis-related factors for example, Survivin, FLICE inhibitory protein (FLIP), apoptosis inhibitor related proteins Bcl-2, Bcl-xL, Mcl-1, and microtubule-binding proteins are preferred, preferably fibroblast growth factor Fibroblast growth factor receptor, MAP kinase signaling-related factor, Kruppel-like factor, Bcl-2 and Bcl-xL, and the like, more preferably MAP kinase signaling associated factors, Bcl-2 and the like.
  • FLIP FLICE inhibitory protein
  • Bcl-2, Bcl-xL, Mcl-1, and microtubule-binding proteins are preferred, preferably fibroblast growth factor Fibroblast growth factor receptor, MAP
  • Mitogen-activated protein kinase is one of serine / threonine kinases and is activated by receiving some kind of stimulation (oxidative stress, cytokine, etc.). It is widely expressed in cells throughout the body and plays an important role in the functional expression of various cells. Often referred to simply as MAP kinase. When a stimulus from outside the cell enters, Ras, which is a low molecular weight G protein, is activated, and further activation of the signal cascade downstream thereof is caused. In addition, dephosphorylation by MAPK phosphatase (MAPK Phosphatase: MKP) inactivates MAPK and acts on this mechanism in a suppressive manner.
  • MAPK Phosphatase MKP
  • MAPK refers only to extracellular signal-regulated kinase (ERK) 1/2, but in a broad sense, c-Jun N-terminal kinase (JNK) ), Including molecules such as p38 MAPK, ERK5 and ERK7, and is also referred to as the MAPK family.
  • ERK extracellular signal-regulated kinase
  • JNK c-Jun N-terminal kinase
  • ERK1 / 2 was first identified in the MAPK family and is also referred to as classical MAPK.
  • ERK1 / 2 consists of ERK1 with a molecular weight of 44 kDa and ERK2 with a 42 kDa molecular weight, and the amino acid sequences of these proteins are 85% homologous to each other.
  • the activation of ERK1 / 2 occurs by the following mechanism. When a ligand binds to a tyrosine kinase-related receptor such as epidermal growth factor receptor (EGFR), phosphorylation of the receptor intracellular domain occurs.
  • EGFR epidermal growth factor receptor
  • Grb2 When an adapter protein containing an SH2 domain such as Grb2 binds to phosphorylated tyrosine of the receptor, Grb2 binds to Sos through the SH3 domain and activates Sos. Activated Sos activates Ras by the GDP-GTP exchange reaction of Ras. Ras then transmits signals to the MAP kinase cascade. Causes activation of ERK.
  • the MEK for ERK1 is MEK1
  • the MEK for ERK2 is MEK2.
  • Thr183 and Tyr185 play an important role in the activation of ERK. Normally, ERK1 / 2 exists predominantly in the cytoplasm, but when activated, it translocates into the nucleus and interacts with transcription factors to control transcription.
  • JNK was identified as a kinase with the activity to phosphorylate Ser63 and Ser73 of c-Jun. JNK is activated by stress such as radiation, lipopolysaccharide, IL-1, osmotic pressure and heat shock, and is also called stress-responsive MAPK (Stress-activated Protein Kinase, SAPK). There are JNK1 to JNK3 in the JNK gene. JNK1 is involved in processes such as apoptosis and neurodegeneration, cell differentiation and proliferation, and production of inflammatory cytokines. JNK phosphorylates various intracellular proteins and modifies their functions.
  • stress-responsive MAPK Stress-activated Protein Kinase
  • the RNA used in the present invention includes a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of the mRNA of the gene, and a base sequence complementary to the sequence. RNA.
  • the RNA used in the present invention also includes DNA in which part or all of ribose is substituted with deoxyribose, that is, DNA.
  • ribonucleotides and deoxyribonucleotides in RNA used in the present invention may be modified, for example, sugar-modified nucleotide analogs, phosphodiester bond-modified nucleotide analogs, and the like.
  • the RNA used in the present invention also includes derivatives in which an oxygen atom or the like contained in a phosphate part, an ester part, or the like in the RNA is substituted with another atom such as a sulfur atom.
  • the ribonucleotide in the RNA used in the present invention is deoxyribonucleotide
  • the ribonucleotide and deoxyribonucleotide in the RNA used in the present invention are modified
  • the phosphate in the RNA used in the present invention Substituting oxygen atoms, etc., contained in the ester part, ester part, etc., with other atoms, such as sulfur atoms, improves the nuclease resistance compared to RNA or DNA, and stabilizes it. It may be formed for any purpose such as increasing affinity, increasing cell permeability, or visualizing.
  • the sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to part or all of the chemical structure of the sugar of the nucleotide.
  • any chemical structural substance for example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged Nucleic Acid (BNA), more specifically, 2′-position oxygen atom
  • PNA Nucleic acid
  • OPNA oxypeptide nucleic acid
  • PRNA peptide ribonucleic acid
  • the phosphodiester bond-modified nucleotide analogue may be any one in which any chemical substance is added or substituted to a part or all of the chemical structure of the phosphodiester bond of a nucleotide.
  • Examples include nucleotide analogues substituted with thioate linkages, nucleotide analogues substituted with N3'-P5 'phosphoramidate linkages [Cell engineering, 16, 1463-1473 (1997)] [RNAi method And Antisense, Kodansha (2005)].
  • RNA used in the present invention is preferably an RNA having an action of suppressing the expression of the gene using RNA interference (RNAi).
  • RNAi RNA that suppresses gene expression using RNA interference
  • Other genes have similar structures and can be obtained by similar operations.
  • RNA that suppresses the expression of the MEK gene is a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of MEK ⁇ ⁇ mRNA (hereinafter referred to as sequence X) and complementary to the sequence. It contains a base sequence (hereinafter referred to as complementary sequence X ′).
  • RNA includes: (A) double-stranded RNA consisting of the strand of sequence X (sense strand) and complementary strand X ′ (antisense strand); (B) the strand of sequence X (sense strand) and complementary sequence X ′ 1 to 6, preferably 2 to 4 nucleotides are the same at the 3 ′ end of the strand of the sequence X or the complementary sequence X ′ of the double-stranded RNA comprising the strands (antisense strands) RNA that consists of differently added double-stranded RNAs that suppress the expression of the MEK gene (hereinafter referred to as RNA with a structure like (A) and (B) is called MEKsiRNA), and (C) RNA consisting of sequence X And RNA consisting of the complementary sequence X ′ is an RNA having a hairpin structure that is connected by a spacer oligonucleotide and suppresses the expression of the MEK gene, (D) RNA consist
  • the nucleotide base added to these RNAs may be one or more of guanine, adenine, cytosine, thymine and uracil, and may be RNA or DNA, but uridylic acid (U) and deoxythymidylic acid ( Any one or two of dT) are preferred.
  • the spacer oligonucleotide is preferably RNA of 6 to 12 bases, and the sequence at the 5 'end is preferably 2 U.
  • An example of the spacer oligonucleotide is RNA having the sequence UUCAAGAGA. Either of the two RNAs connected by the spacer oligonucleotide may be on the 5 'side.
  • the nucleotide sequence of the nucleotide added adjacent to the 3 ′ end side of the complementary sequence X ′ may be the base sequence complementary to the sequence of the nucleotide adjacent to the sequence X in the mRNA.
  • the sequence X may be any sequence as long as it is a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of MEK mRNA.
  • the partial sequence of 21 bases beginning with AA is extracted from the base sequence of MEK cDNA. More preferable is a sequence designed by calculating the GC content of the extracted sequence and selecting a plurality of sequences having a GC content of 20 to 80%, preferably 30% to 70%, more preferably 40 to 60%.
  • RNA that suppresses MEK gene expression has different strength of suppression of MEK gene expression depending on the sequence X, and there are cases where the suppression is weak.
  • the RNA of the present invention is prepared by introducing RNA into a cell in which the MEK gene is expressed, measuring the expression of the MEK gene, and selecting an RNA that strongly suppresses the expression of the MEK gene. Can be obtained.
  • Examples of RNA that suppresses the expression of the MEK1 gene include No. 1 to No. 40 RNA shown in Table 1.
  • Examples of RNA that suppresses the expression of the MEK2 gene include RNA No. 41 to No. 74 shown in Table 2.
  • the method for synthesizing the RNA used in the present invention is not particularly limited, and it can be synthesized by a method using a known chemical synthesis, an enzymatic transcription method or the like.
  • known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, etc., for example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do.
  • transcription or synthesis can be performed using a plasmid or DNA having a target base sequence as a template and using a typical phage RNA polymerase, for example, T7 polymerase, T3 polymerase, SP6 RNA polymerase, or the like.
  • MEKsiRNA No. 1 in Table 1 can be prepared by, for example, requesting Nippon Bio Service Co., Ltd., chemical synthesis, and annealing.
  • MEK siRNA Nos. 2 to 74 in Tables 1 and 2 can be prepared by in vitro transcription using a silencer siRNA preparation kit (Silencer (registered trademark) siRNA-Construction-Kit, manufactured by Ambion).
  • the DNA used for template production for in vitro transcription can be obtained, for example, by requesting chemical synthesis from Hokkaido System Science Co., Ltd.
  • the liposome in the composition of the present invention (hereinafter referred to as liposome A) is not particularly limited as long as it is a liposome encapsulating RNA used in the present invention.
  • a cationic lipid / RNA complex is a hydrophobic organic solvent.
  • Liposomes produced by dispersing in layers, adding polyethylene glycolated lipids, neutral lipids and water to form water-in-oil (W / O) emulsions and processing by reverse phase evaporation see Patent Document 1
  • RNA is dissolved in an acidic electrolyte aqueous solution
  • lipid (in ethanol) is added
  • RNA-encapsulated liposomes are prepared by lowering the ethanol concentration, and then the RNA attached to the liposome surface by dialysis by raising the pH of the sample
  • composite particles composed of lead particles and RNA, and liposomes composed of lipid bilayers encapsulating the composite particles special Reference 3 and International Publication No.
  • a liposome composed of a composite particle composed of a lead particle and the RNA and a lipid bilayer membrane encapsulating the composite particle is preferred. More preferably, the components of the 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. preferable.
  • the liposome A is preferably composed of a lead particle containing a cationic substance, a composite particle containing the RNA as a constituent component, and a lipid bilayer covering the composite particle.
  • liposomes comprising lipid derivatives, fatty acid derivatives or aliphatic hydrocarbon derivatives of water-soluble substances as a constituent, and the lipid bilayer constituent is soluble in a specific polar organic solvent. 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 term “dispersing” means dispersing without dissolving.
  • the lead particles in the present invention include, for example, fine particles comprising lipid aggregates, liposomes (hereinafter referred to as liposome B), emulsion particles, polymer micelles, metal colloids, etc., preferably liposome B as a constituent component. Fine particles.
  • the lead particles in the present invention may be composed of a complex comprising a combination of two or more lipid aggregates, liposome B, emulsion particles, polymer micelles, metal colloids, etc., and lipid aggregates, liposome B, emulsion particles, A complex formed by combining polymer micelles, metal colloids, and the like with other compounds (for example, sugars, lipids, inorganic compounds, etc.) may be used as a constituent component.
  • Lipid aggregates or liposomes B as constituents of lead particles are composed of, for example, polar lipids that have a lipid bilayer structure in water with amphiphilic properties that combine both hydrophilic and hydrophobic properties.
  • the lipid may be any of simple lipids, complex lipids or derived lipids, such as phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids, sterols, and cationic lipids, but are not limited thereto. 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)) , Palmitoyl oleoyl-phosphatidylethanolamine (POPE), 1 -stearoyl- 2 -oleoyl-phosphine Glycidylphosphoamine (specific
  • 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 particles include sphingan, icosasphingan, sphingosine, and derivatives thereof.
  • the derivative for example, —NH 2 such as sphingan, icosasphingan or sphingosine —NHCO (CH 2 ) x CH 3 (wherein x represents an integer of 0 to 18, among which 6, 12 or 18 is preferable. ) And the like.
  • Examples of the sterol in the lipid constituting the lead particle include cholesterol, dihydrocholesterol, lanosterol, ⁇ -sitosterol, campesterol, stigmasterol, brush casterol, ergocasterol, fucostosterol 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
  • these lipids are used singly or in combination of two or more, preferably in combination of two or more.
  • a combination when used in combination of two or more, for example, hydrogenated soybean phosphatidylcholine, polyethyleneglycolized lipid (synonymous with polyethyleneglycolized lipid described later) and cholesterol, a combination of two or more components, distearoylphosphatidylcholine, polyethyleneglycolated Combination of two or more components selected from lipid and cholesterol, combination of EPC and DOTAP, combination of DOTAP and polyethylene glycolated lipid, combination of EPC, DOTAP and polyethylene glycolated lipid, combination of EPC, DOTAP, cholesterol and polyethylene glycolated lipid Etc.
  • Liposomes B may contain a film stabilizer such as sterol such as cholesterol, for example, and a stabilizer such as 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 or aggregates composed of two or more lipid molecules. .
  • emulsion particles include fat emulsions, emulsions composed of nonionic surfactants and oils such as soybean oil, oil-in-water (O / W) emulsions such as lipid emulsions and lipid nanospheres, and water-in-oil-in-water (W / O / W) emulsion particles and the like.
  • nonionic surfactant in the emulsion particles constituting the lead particles examples include polyoxyethylene sorbitan monooleate (specifically polysorbate 80), polyoxyethylene polyoxypropylene glycol (specifically Pluronic F68). ), Sorbitan fatty acid esters (specifically sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.) or glycerin fatty acid Examples include esters.
  • polymer micelle examples include natural polymers such as albumin, dextran, polyfect, chitosan, dextran sulfate or DNA, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropyl
  • examples include micelles composed of one or more polymers such as acrylamide-acrylpyrrolidone copolymer, polyethylene glycol-modified dendrimer, 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.
  • metal colloid examples include metal colloids containing gold, silver, platinum, copper, rhodium, silica, calcium, aluminum, iron, indium, cadmium, barium or lead.
  • the lead particles in the present invention preferably contain a lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers. It is more preferable to contain a lipid derivative or fatty acid derivative of a water-soluble polymer, and it is further preferable to contain a lipid derivative or fatty acid derivative of a water-soluble polymer.
  • 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 with the solvent at the time of lead particle production, for example, hydrophilic affinity, electrostatic interaction, etc.
  • the lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugar, peptide, nucleic acid and water-soluble polymer may be contained as a component of the lead particle, and in addition to the component of the lead particle It may be used.
  • lipid derivatives or fatty acid derivatives of one or more substances 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, and glutathione peptides.
  • a nucleic acid such as DNA, RNA, plasmid, siRNA, or ODN and a lipid listed in the definition of the lead particle or a fatty acid such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. And the like.
  • sugar lipid derivatives or fatty acid derivatives include, for example, glyceroglycolipids or sphingoglycolipids 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, Chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like or their derivatives and the lipids mentioned in the definition of lead particles, for example, stearic acid, palmitic acid, Examples include those formed by bonding with fatty acids such as myristic acid or lauric acid, and more preferred are lipid derivatives such as polyethylene glycol derivatives and polyglycerin derivatives, or fatty acid derivatives. Is, 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 a charge in RNA used in the present invention, a charge that generates an electrostatic attraction with respect to intramolecular polarization, a surface polarization, and the like.
  • the lead particles preferably contain a cationic substance, and the lead particles more preferably contain a cationic lipid.
  • 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 a constituent component of lead particles, or may be used in addition to the constituent components of lead particles.
  • a cationic substance for example, a cationic substance [specifically, a cationic lipid (as defined above), a cationic polymer, etc.] among those exemplified in the definition of the lead particle, a value below the isoelectric point Examples thereof include proteins or peptides capable of forming a complex at a pH of, preferably cationic lipids.
  • Examples of the cationic polymer include poly-L-lysine, polyethyleneimine, polyfect, and chitosan.
  • the protein or peptide capable of forming a complex at a pH below the isoelectric point is not particularly limited as long as it is a protein or peptide capable of forming a complex at a pH below the isoelectric point of the substance.
  • the protein or peptide include albumin, orosomucoid, globulin, fibrinogen, pepsin, and ribonuclease T1.
  • the lead particles in the present invention can be produced by a known production method or a method according thereto, and may be produced by any production method.
  • a known liposome preparation method can be applied to the production of lead particles containing liposome B, which is one of the lead particles, as a constituent component.
  • 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.
  • 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 composite comprising a combination of two or more selected from lipid aggregates, liposome B, emulsion particles, polymer micelles, metal colloids and the like constituting the lead particles, for example, lipids, polymers, etc. in water May be mixed, and a granulation step, a sterilization step, and the like may be added if desired.
  • the complex can be produced in various solvents such as acetone or ether.
  • the average size of the lead particles in the present invention is preferably about 10 nm to 1000 nm, more preferably about 30 nm to 300 nm, and further preferably about 50 nm to 200 nm.
  • Examples of the component of the lipid bilayer membrane covering the composite particles containing lead particles and RNA in the present invention include the lipids and surfactants mentioned in the definition of the lead particles.
  • Our neutral lipids refers to the cationic lipids mentioned in the cationic substance and the anionic lipids mentioned in the adhesion competitor described later when the lead particles have a positive charge.
  • neutral lipids include phospholipids, glyceroglycolipids or sphingoglycolipids. More preferred are phospholipids, and more preferred is EPC. These lipids can be used alone or in combination of two or more.
  • the components of the lipid bilayer membrane covering the composite particles are preferably soluble in a specific 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 give. Moreover, an ion or a buffer component etc. 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 coating the composite particles preferably contains a lipid derivative of a water-soluble substance, a fatty acid derivative or an aliphatic hydrocarbon derivative, polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether, More preferably, it contains a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance.
  • 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.
  • An aliphatic hydrocarbon derivative of one or more substances selected from nucleic acids and water-soluble polymers preferably lipid derivatives or fatty acid derivatives of the water-soluble polymers, more preferably the polyethylene glycolated lipids. More preferred is polyethylene glycol-phosphatidylethanolamine.
  • 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 also be raised.
  • 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 derivatives of water-soluble polymers include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, chitosan, polyvinyl
  • Examples thereof include aliphatic hydrocarbon derivatives of pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like or derivatives thereof, more preferably aliphatic carbonization such as polyethylene glycol derivatives or polyglycerin derivatives.
  • Examples thereof include hydrogen derivatives, and more preferable examples include aliphatic hydrocarbon derivatives of polyethylene glycol derivatives.
  • the liposome A is composed of a composite particle comprising liposome B and RNA used in the present invention and a lipid bilayer coating the composite particle.
  • the lead particle is classified as a liposome in a narrow sense based on its configuration, and even when the lead particle is other than a fine particle containing liposome B as a constituent component, it is classified as a liposome in a broad sense because it is covered with a lipid bilayer membrane.
  • the lead particles are more preferably fine particles containing liposome B as a constituent component.
  • the composite particles comprising the lead particles in the present invention and the RNA used in the present invention are prepared by attaching or enclosing the RNA used in the present invention to the lead particles after the lead particles are produced or simultaneously with the production of the lead particles. Further, 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 produced by, for example, a known production method described in Patent Documents 1, 2, 3 and Non-Patent Document 3 or a method similar thereto, or, for example, RNA used in the present invention is attached to or encapsulated in lead particles.
  • the composite particles and the coating layer component contain a polar organic solvent in which the coating layer component is soluble, the composite particles do not dissolve, and the coating layer component exists in a dispersed state. It can be produced by a production method including a step of dispersing in a liquid having a possible concentration and a step of coating the composite particles with the coating layer component.
  • 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 RNA used in the present invention (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 composite particles comprising lead particles and RNA used in the present invention as constituent components
  • Lead particles are dispersed in a solvent such as water, and used in the present invention in a liquid in which the lead particles are dispersed. It is preferable to disperse or dissolve and mix the RNA to be used, and to attach the RNA used in the present invention to the lead particles.
  • the lead particles are preferably lead particles containing an aggregation inhibitor.
  • 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 RNA used in the present invention and the adhesion competitor are coexisted in the liquid in which the lead particle is dispersed, and the adhesion competitor is attached to the lead particle together with the RNA.
  • an adhesion competitor may be used to further suppress the aggregation of the lead particles.
  • the solubility 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 a concentration capable of dispersing the composite particles.
  • adhesion competitors include anionic substances.
  • the anionic substance includes a substance that adheres electrostatically to the constituent components of the lead particles by electrostatic attraction due to intramolecular charge, intramolecular polarization, and the like.
  • An anionic substance as an adhesion competing agent is an anionic substance, but even an amphoteric substance having both an anionic group and a cationic group is affected by pH, binding to other substances, etc. Since the relative negative degree changes, it can be classified into anionic substances depending on the occasion.
  • anionic substance examples include anionic lipids, anionic surfactants, anionic polymers, etc., and proteins, peptides, or nucleic acids that can form a complex at a pH higher than the isoelectric point, and preferably dextran sulfate. Dextran sodium sulfate, chondroitin sulfate, chondroitin sulfate sodium, hyaluronic acid, chondroitin, dermatan sulfate, heparan sulfate, heparin, keratan sulfate or dextran fluorescein anionic. These anionic substances can be used alone or in combination of two or more.
  • anionic lipid examples include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, and the like.
  • 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-acrylpyrrolidone 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 capable of forming a complex 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 capable of forming a complex 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 preferably adheres electrostatically to the constituents of the lead particles, and is a substance having a size that does not form a crosslink that causes the constituents of the lead particles to aggregate even if attached to the constituents of the lead particles. It is preferable that the substance has a part that adheres in the molecule and a part that repels the adhesion and suppresses the aggregation of the lead particles.
  • step 1 includes, for example, an operation for producing a liquid in which lead particles containing an aggregation-inhibiting substance are dispersed, and RNA used in the present invention is dispersed or dissolved in the liquid in which the lead particles are dispersed.
  • Operation of containing for example, an operation of adding and dispersing or dissolving RNA used in the present invention in a liquid in which the lead particles are dispersed, and RNA used in the present invention being dispersed or dissolved in a liquid in which the lead particles are dispersed
  • an operation of adding the prepared liquid For example, an operation of adding the prepared liquid).
  • the composite particles obtained by the step of dispersing or dissolving the RNA used in the present invention in the liquid in which the lead particles are dispersed specifically, for example, liposome B containing a cationic lipid.
  • Composite particles formed by adhering RNA used in the present invention to fine particles as constituent components formed by adhering RNA used in the present invention to fine particles containing lipid aggregates containing cationic lipids
  • the operation of dispersing or dissolving the RNA used in the present invention in the liquid in which the lead particles are dispersed includes adding an adhesion competitor to the liquid in which the RNA used in the present invention is dispersed or dissolved.
  • the lead particles are preferably added to a liquid in which the lead particles are dispersed.
  • the RNA used in the present invention and the adhesion competitor are both 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 RNA used in the present invention can adhere to the lead particles, but it is preferably about 1 ⁇ g / mL to 1 g / mL, and about 0.1 More preferably, it is ⁇ 500 mg / mL.
  • Step 2) Step of coating composite particles with lipid bilayer (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 all or part of the components of the lipid bilayer are dissolved, and then the polarity in the liquid A
  • liposome A can be produced by a production method including an operation of coating the 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.
  • the solvent in the liquid A and the liquid B and the components of the composite particles and the lipid bilayer membrane for the liquid C It can be selected in consideration of solubility.
  • the lipid bilayer component is preferably low in solubility in the solvent in solution B and in solution C, preferably high in solubility in the solvent in solution A, and
  • the solubility in a polar organic 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 each component such as the lead particles contained in the composite particles, the RNA used in the present invention, and the adhesion competing agent has low elution in a solvent, Even if the individual solubility of each component is high, it is sufficient that the elution property of each component is reduced by the binding between the components.
  • the lead particle even if 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 RNA used in the present invention, the intramolecular polarization, etc. Thus, the 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)
  • a 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, the composite particle does not dissolve, and the lipid bilayer membrane Liposome A can be produced by a production method including an operation of dispersing in a liquid having a concentration that allows the constituent components to exist in a dispersed state (the liquid obtained is liquid F). Obtained in the state.
  • the solvent in the liquid F is a solvent containing a polar organic solvent in which the components of the lipid bilayer membrane are soluble, and the liquid F at a specific concentration at which both the components of the lipid bilayer membrane and the composite particles can be dispersed. Included.
  • 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 within the solvent concentration range, the polar organic solvent may be removed, or the concentration may be decreased.
  • 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 lipid bilayer components in liquid F is low, the lipid bilayer components are complexed almost simultaneously with the dispersion in the liquid containing the polar organic solvent.
  • the coating of the lipid bilayer on the particles is complete.
  • 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 the polar organic solvent means that when the component of the lipid bilayer membrane has the property of being dissolved in the polar organic solvent, a solubilizer or the like is used.
  • the components of the lipid bilayer membrane can form emulsions or micelles in the polar organic solvent and become emulsion or emulsion The case where it has is included.
  • 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 where the emulsion is emulsified or emulsified, and the remaining part is precipitated, and does not include a state where all the components of the lipid bilayer are dissolved.
  • 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. Including a state in which a part of the composite particles is emulsified or emulsified and a remaining part is precipitated, and does not include a state in which all of the composite particles are dissolved. “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 ratio of the lipid bilayer membrane to the liposome A of the present invention is preferably about 1: 0.1 to 1: 1000, more preferably about 1: 1 to 1:10 by weight.
  • the size of the liposome A in the present invention is preferably an injectable size, for example.
  • the average particle size is preferably about 10 nm to 1000 nm, more preferably about 50 nm to 300 nm, and further preferably about 70 nm to 200 nm.
  • 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 RNA used in the present invention can be used to enlarge the pulmonary vascular wall tissue.
  • the gene can be delivered to the enlarged pulmonary vascular wall tissue in the bronchus or lung, which is the site of expression of the related gene, and the expression of the gene is suppressed.
  • hypertrophy of pulmonary vascular wall tissue is suppressed, thereby treating or preventing pulmonary hypertension.
  • the present invention also provides a method for treating pulmonary hypertension, wherein the above-described composition of the present invention is administered to a mammal.
  • the administration subject is preferably a person suffering from pulmonary hypertension or a person having an enlarged pulmonary vascular wall tissue in the bronchi or lung, more preferably a person suffering from pulmonary hypertension.
  • RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, and nucleic acids such as oligonucleotides and plasmids is also delivered to the enlarged pulmonary vascular wall tissue in the bronchi or lung. And can be used as a therapeutic or prophylactic agent for diseases in the bronchi or lungs.
  • composition of the present invention and the composition in which the RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, etc., are delivered directly or indirectly. It can also be used as a diagnostic to measure whether it suffers from pulmonary hypertension or has enlarged pulmonary vascular wall tissue in the bronchi or lung.
  • composition of the present invention and the composition in which the RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, and the like, for example, biological components such as blood components (for example, it can also be used as a preparation for the purpose of stabilizing the RNA, peptide, protein or nucleic acid in blood, gastrointestinal tract, etc., reducing side effects or increasing drug accumulation in bronchi or lung.
  • peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, and the like
  • biological components such as blood components
  • it can also be used as a preparation for the purpose of stabilizing the RNA, peptide, protein or nucleic acid in blood, gastrointestinal tract, etc., reducing side effects or increasing drug accumulation in bronchi or lung.
  • RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, etc.
  • Oral administration or oral administration can be exemplified, preferably intravenous administration or intramuscular administration can be exemplified, and more preferably intravenous administration is exemplified.
  • the dose varies depending on the disease state, age, route of administration, etc. of the administration subject, but for example, it may be administered so that the daily dose converted to RNA is about 0.1 ⁇ g to 1000 mg.
  • suitable dosage forms 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, for example, in the form of injections.
  • the dispersion can be used after removing the solvent by, for example, filtration, centrifugation, etc., or the dispersion can be used by lyophilization, or an excipient such as mannitol, lactose, trehalose, maltose or glycine can be used.
  • the added dispersion can be lyophilized for use.
  • injections for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, etc.
  • an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA, or an isotonic agent such as glycerin, glucose or sodium chloride can be added.
  • an isotonic agent such as glycerin, glucose or sodium chloride
  • it can also be cryopreserved by adding a cryopreservation agent such as glycerin.
  • Examples of the therapeutic agent for pulmonary hypertension of the present invention include the composition of the present invention intended to be used for the treatment or prevention of pulmonary hypertension.
  • liposome A is a lead particle.
  • a lipid bilayer coating the composite particle the component of the lipid bilayer is soluble in a specific polar organic solvent, and the lipid bilayer And a composite particle comprising a lead particle containing a cationic substance and the RNA as constituents, and the composite particle, wherein the component and the composite particle are dispersible in a liquid containing the polar organic solvent at a specific concentration
  • a component of the lipid bilayer membrane is soluble in a polar organic solvent, and the component of the lipid bilayer membrane and the composite particle are mixed with the polar organic membrane at a specific concentration. Dispersible in liquid containing solvent It is preferable to contain a certain liposome.
  • the present invention also provides use of the composition of the present invention described above for the manufacture of a therapeutic agent for pulmonary hypertension.
  • RNA used in Example 1 is an RNA containing a 19-base sequence homologous to the rat MEK1 / 2 gene and a base sequence complementary to the sequence, and dTdT is added to each 3 ′ end.
  • RNA [5'-GCAUCUGCAUGGAGCACAUdTdT-3 '(sequence a, SEQ ID NO: 149), 5'-AUGUGCUCCAUGCAGAUGCdTdT-3' (sequence b, SEQ ID NO: 150)]
  • the 5 'end of the single-stranded RNA of sequence a RNA modified with Cyanine5 (Cy5) [5′-Cy5-GCAUCUGCAUGGAGCACAUdTdT-3 ′ (sequence c, SEQ ID NO: 151)] was obtained from Eurogenetec and prepared by annealing sequence c and sequence b (hereinafter, "Cy5-labeled MEKsiRNA").
  • RNA used in Example 2 was similarly prepared using RNAs of sequence a and sequence b (hereinafter referred to as “MEKsiRNA”).
  • the RNA used in Comparative Example 1 is an RNA [5′-UACACGAGGUACGUCUACGdTdT-3 ′ (SEQ ID NO: 152) containing a 19-base sequence not homologous to the rat MEK1 / 2 gene and a base sequence complementary to the sequence. 5′-CGUAGACGUACCUCGUGUAdTdT-3 ′ (SEQ ID NO: 153)] and prepared in the same manner (hereinafter referred to as “Control-siRNA”).
  • DOTAP manufactured by Avanti Polar Lipids
  • PEG-DSPE manufactured by NOF Corporation, the same shall apply hereinafter
  • distilled water was mixed to 40 mg / 16 mg / 1 mL, and the mixture was shaken and stirred with a vortex mixer.
  • the resulting dispersion was passed through a 0.4 ⁇ m polycarbonate membrane filter (manufactured by Whatman) at room temperature 4 times, through a 0.1 ⁇ m polycarbonate membrane filter 10 times, and further through a 0.05 ⁇ m polycarbonate membrane filter 24 times to prepare lead particles.
  • 0.625 mL of distilled water was gradually added to adjust the ethanol concentration to about 40 v / v%.
  • Liposomes were prepared. The resulting liposome dispersion is ultracentrifugated (1 hour, 110,000 ⁇ g, 25 ° C.), the supernatant is removed, and physiological saline is added to the precipitate for redispersion, and physiological saline is further added. The concentration was adjusted to obtain a composition.
  • Test example 1 Disease model causing pulmonary hypertension (Sugita T, Hyers TM, Dauber IM, Wagner WW, McMurtry IF, Reeves JT. Lung vessel leak precedes right ventricular hypertrophy in monocrotaline-treated rats.J Appl Physiol. Feb 1983; 54 (2) : 371-374.), And when the composition obtained in Example 1 was administered by the following method, it was confirmed that RNA specifically reached the lung in which pulmonary hypertension occurred. did. Monocrotaline (MCT) was subcutaneously administered to Wistar (5-week-old) male rats (100 to 120 g) (60 mg / kg) to obtain monocrotaline-induced pulmonary hypertension rats (MCT rats).
  • MCT Monocrotaline
  • physiological saline was similarly administered instead of MCT, and used as a control group. 10 days after administration of MCT or physiological saline, the composition obtained in Example 1 (0.2 mg / kg as Cy5-labeled MEKsiRNA) was administered from the tail vein of the rat, and 1, 2, 4, 7 days after administration of the composition Lungs were collected. The obtained lung was washed and perfused with physiological saline to remove blood, and 1 ⁇ L of 1% TritonX-100-containing PBS per 1.0 mg of lung was added to homogenize the lung. This was centrifuged (10000 ⁇ g, 4 ° C.), and the fluorescence intensity of Cy5 in the obtained supernatant was measured.
  • the concentration of Cy5-labeled MEK siRNA in each lung should be corrected by reducing the autofluorescence intensity in the lungs of MCT rats and control groups, which were similarly administered intravenously with physiological saline instead of the composition obtained in Example 1.
  • FIG. 1 shows changes over time in the accumulation of Cy5-labeled MEK siRNA in the lung 1, 2, 4, and 7 days after administration of the composition obtained in Example 1. From FIG. 1, in the lungs of MCT rats, the concentration of Cy5-labeled MEK siRNA was maximized 2 days after the administration of the composition obtained in Example 1, while the concentration of Cy5-labeled MEK siRNA in the lung of the control group was the composition administered. It can be seen that one day later, it showed a gentle peak. The concentration of Cy5-labeled MEK siRNA in the lung in MCT rats was significantly higher than the control group 2 days after administration of the composition.
  • Test example 2 When the composition obtained in Example 1 was administered, it was confirmed that RNA selectively reaches the lung, which is a disease site of pulmonary hypertension. Similar to Test Example 1, MCT rats and a control group were prepared. 10 days after administration of MCT or physiological saline, the composition obtained in Example 1 from rat tail vein (0.2 mg / kg as Cy5-labeled MEK siRNA) was administered, and the lung, liver, kidney, 48 hours after administration of the composition, Heart and brain were collected. The concentration of Cy5-labeled MEK siRNA in each obtained organ was measured and calculated in the same manner as in Test Example 1. FIG. 2 shows the concentration of Cy5-labeled MEK siRNA in each organ of MCT rats and control group 48 hours after administration of the composition obtained in Example 1. FIG.
  • Test example 3 When the composition obtained in Example 1 was administered, it was confirmed that RNA selectively reached the enlarged pulmonary vascular wall tissue and its vicinity.
  • MCT rats and a control group were prepared in the same manner as in Test Example 1, and the composition obtained in Example 1 was administered. The lung 48 hours after administration of the composition was removed, and a frozen section of the lower right lung was prepared. Vascular endothelial cells were immunostained (green), nuclei were stained by DAPI staining (blue), and the accumulation site of Cy5-labeled MEKsiRNA (red) was observed with a fluorescence microscope.
  • FIG. 3 shows a section of each rat immunostained pulmonary artery and its vicinity (left: control group, right: MCT rat).
  • Fig. 3 shows that in MCT rats with enlarged pulmonary vascular wall tissue (Fig. 3 right), the accumulation of Cy5-labeled MEK siRNA was higher than that in the control group without enlarged pulmonary vascular wall tissue (Fig. 3 left). It is localized at a high rate in the vicinity of the endothelial cells, and it can be seen that when the composition obtained in Example 1 is administered, RNA accumulates in the enlarged pulmonary vascular wall tissue and its vicinity.
  • Example 1 The Cy5-labeled MEKsiRNA in Example 1 was replaced with MEKsiRNA to obtain a composition in the same manner.
  • Test example 4 When the composition obtained in Example 2 was administered, it was confirmed that gene expression was suppressed in a dose-dependent manner in the lung in which pulmonary hypertension occurred.
  • the composition obtained in Example 2 was intravenously administered as MEK siRNA at doses of 0.2 mg / kg and 2 mg / kg 10 days after MCT administration. Further, the composition obtained in Comparative Example 1 was similarly administered as a control. Total RNA was extracted from each extracted lung using TRIzol Reagent (Invitrogen) and RNeasy Mini Kit (Qiagen).
  • GAPDH mRNA as an internal standard was reverse-transcribed in the same manner using the cDNA shown below. [GAPDH sense (5′-GAACATCATCCCCTGCATCCA-3 ′ (SEQ ID NO: 158)), GAPDH antisense (5′-CCAGTGAGCTTCCCGTTCA-3 ′ (SEQ ID NO: 159))].
  • GAPDH sense (5′-GAACATCATCCCCTGCATCCA-3 ′ (SEQ ID NO: 158)
  • GAPDH antisense 5′-CCAGTGAGCTTCCCGTTCA-3 ′ (SEQ ID NO: 159)
  • PCR amplification 1 ⁇ L of cDNA and 25 ⁇ L of Power SYBR Green PCR Master Mix (manufactured by Biosystems) were added, and an RNA solution with a total volume of 50 ⁇ L was used. Heat denaturation was performed at 95 ° C. for 15 seconds, and annealing and extension reaction were repeated at 60 ° C.
  • FIG. 4 shows the dose-dependent effects of MEK1 (left) and MEK2 (right) mRNA expression suppression in the lung 48 hours after administration of the composition. From FIG. 4, the group administered with the composition obtained in Example 2 was compared with the group administered with the composition obtained in Comparative Example 1 in a dose-dependent manner in the lungs of MEK1 and MEK2 mRNA. It can be seen that both expressions are suppressed.
  • Test Example 5 When the composition obtained in Example 2 was administered, changes over time in the gene expression suppression effect in the lungs in which pulmonary hypertension occurred were confirmed. Using the same disease state model rats as in Test Example 1, the composition obtained in Example 2 was intravenously administered (0.2 mg / kg) 10 days after MCT administration, and the lungs were removed 24, 48, and 96 hours after composition administration. Extracted. In addition, the composition obtained in Comparative Example 1 was similarly administered as a control, and the lungs were removed 48 hours after administration of the composition. The expression intensity of MEK1 and MEK2 mRNA in the lung was measured from the extracted lung in the same manner as in Test Example 4. FIG.
  • FIG. 5 shows temporal changes in the mRNA expression inhibitory effect of MEK1 (left) and MEK2 (right) in the lung 24, 48, and 96 hours after administration of the composition. From FIG. 5, when the composition obtained in Example 2 was administered, the expression of MEK1 and MEK2 mRNA in the lung was compared with the case where the composition (Control) obtained in Comparative Example 1 was administered. It is confirmed that the inhibitory effect is confirmed 24 hours after administration of the composition and lasts for 96 hours or more.
  • Test Example 6 It was confirmed that pulmonary hypertension was suppressed when the composition obtained in Example 2 was administered using the same disease state model rats as in Test Example 1.
  • the composition obtained in Example 2 was intravenously administered (0.2 mg / kg) to rats 3, 10, and 17 days after MCT administration, and hemodynamics were measured 21 days after MCT administration.
  • Rats anesthetized with isoflurane (1 to 1.5%) were inserted into the right ventricle via the right jugular vein using a polyethylene catheter (PE-50) (BD Bioscience), and systolic right ventricular pressure was measured. Systolic right ventricular pressure was calculated from each measured value during 20 consecutive heartbeats.
  • FIG. 6 is a bar graph showing systolic right ventricular pressure in each rat.
  • FIG. 6 shows that MCT rats of the pulmonary hypertension model showed significantly higher blood pressure than Sham, and that the group administered with the composition obtained in Comparative Example 1 did not suppress the hypertension. .
  • the group administered with the composition obtained in Example 2 significantly suppresses the MCT-induced increase in blood pressure compared to the group administered with the composition obtained in Comparative Example 1.
  • Test Example 7 It was confirmed that when the composition obtained in Example 2 was administered using the same disease state model rat as in Test Example 1, the right ventricular hypertrophy was suppressed. As in Test Example 6, the composition obtained in Example 2 was administered. After euthanizing the rat 21 days after MCT administration, the ventricle was removed, the right ventricle, left ventricle, and ventricular septum were weighed, and the right ventricular weight relative to the total weight of the left ventricle and ventricular septum The ratio was calculated (Kimura H, Kasahara Y, Kurosu K, Sugito K, Takiguchi Y, Terai M, Mikata A, Natsume M, Mukaida N, Matsushima K, Kuriyama T.
  • FIG. 7 is a bar graph showing the relative ratio of the right ventricular weight to the total weight of the left ventricle and the ventricular septum. From FIG. 7, in the pulmonary hypertension model MCT rat, the right ventricular weight increased with respect to Sham (MCT), and the composition obtained in Comparative Example 1 suppressed the increase in the right ventricular weight. I understand that there is no. On the other hand, it can be seen that the composition obtained in Example 2 significantly suppresses MCT-induced increase in the right ventricular weight relative to the group administered with the composition obtained in Comparative Example 1.
  • Test Example 8 Using the same disease state model rats as in Test Example 1, it was confirmed that when the composition obtained in Example 2 was administered, the pulmonary artery wall hypertrophy was suppressed.
  • the composition obtained in Example 2 was administered in the same manner as in Test Example 6, and the lungs were removed after euthanizing the rats. Among the excised lungs, 4 ⁇ m thick paraffin sections were prepared from the lower right lung. This was stained with Elastica-Wangieson staining (EV staining), and the hypertrophy of the pulmonary vascular wall was identified by staining the elastic fibers of the connective tissue.
  • EV staining Elastica-Wangieson staining
  • a control group was prepared and tested in the same manner as in Test Example 6.
  • FIG. 8 shows a microscopic image in which enlargement of the pulmonary artery in each rat is visualized by EV staining.
  • FIG. 8 shows a microscopic image in which enlargement of the pulmonary artery in each rat is visualized by EV staining.

Abstract

Disclosed are: a therapeutic agent for pulmonary hypertension, which comprises (i) RNA that comprises a sequence composed of contiguous 15 to 30 nucleotides contained in mRNA for a gene associated with the hypertrophy of a pulmonary blood vessel tissue and a nucleotide sequence complementary to the aforementioned sequence and (ii) a liposome in which the RNA is enclosed; and others. An example of the liposome in which the RNA is to be enclosed is a liposome that comprises a composite particle composed of a lead particle comprising a cationic substance and the RNA and a lipid bilayer membrane that covers the composite particle, wherein the lipid bilayer membrane comprises, for example, a lipid derivative composed of a neutral lipid and a water-soluble substance, a fatty acid derivative, or an aliphatic hydrocarbon derivative.

Description

核酸を含有する肺高血圧症治療剤Treatment for pulmonary hypertension containing nucleic acid
　本発明は、肺高血圧症治療剤および肺高血圧症治療用組成物等に関する。 The present invention relates to a therapeutic agent for pulmonary hypertension, a composition for treating pulmonary hypertension, and the like.
　収縮期圧で 30mmHg以上、平均圧で 20mmHg 以上の肺動脈圧が存在する場合に、これを肺高血圧と定義することが一般的であり、肺高血圧症とは、肺血管系の血管抵抗が増大し、肺動脈圧の持続的な亢進をきたす病態をいう。
　肺高血圧症には、原発性肺高血圧症と、心房中隔欠損、肺塞栓症、動脈管開存症、僧帽弁狭窄症、肺線維症、肺気腫、膠原病または肝硬変等を元疾患とする二次性肺高血圧症とがある。肺高血圧症では、何らかの原因で肺小動脈の血液の流れが細くなり、その結果、肺動脈内の圧力が高くなっている。肺動脈に血液が流れにくくなると、そこに血液を送り込んでいる右心室は、心筋を肥大させて血液を送り出すように変化する。しかし、このような負荷のかかった状態が続くと、心筋の収縮力は低下し、右心房を含む右心系の機能が障害された状態(右心不全という)になり、全身の血液の循環に障害がでてくると言われている。
　肺高血圧症においては、肺小動脈の肺血管壁組織の肥大が観察され、これが本疾患の発生または増悪の原因であると考えられている。 It is common to define pulmonary hypertension when there is a pulmonary arterial pressure of 30 mmHg or higher in terms of systolic pressure and 20 mmHg or higher in average pressure. Pulmonary hypertension is an increase in vascular resistance in the pulmonary vasculature. It refers to a pathological condition that causes a continuous increase in pulmonary artery pressure.
Pulmonary hypertension originates from primary pulmonary hypertension and atrial septal defect, pulmonary embolism, patent ductus arteriosus, mitral stenosis, pulmonary fibrosis, emphysema, collagen disease or cirrhosis There is secondary pulmonary hypertension. In pulmonary hypertension, the blood flow in the small pulmonary arteries is narrowed for some reason, and as a result, the pressure in the pulmonary artery is high. When blood becomes difficult to flow into the pulmonary artery, the right ventricle that is feeding blood changes to enlarge the myocardium and pump blood. However, if such a stressed state continues, the contraction force of the myocardium decreases, and the function of the right heart system including the right atrium is impaired (called right heart failure), and the circulation of blood throughout the body is lost. It is said that obstacles come out.
In pulmonary hypertension, enlargement of the pulmonary vascular wall tissue of small pulmonary arteries is observed, which is considered to be the cause of the occurrence or exacerbation of this disease.
　肥大した肺血管壁組織においては、細胞を増殖または成長させる因子が過剰に存在していることが知られている(非特許文献1、2参照)。それら細胞を増殖または成長させる因子をコードする遺伝子の発現を抑制することで、肺血管壁組織の肥大または硬化を抑制して、肺高血圧症を治療することが期待されている。
　しかしながら、細胞を増殖または成長させる因子をコードする遺伝子の発現を抑制するためには、肥大した肺血管壁組織に薬物を送達させる必要があり、例えば薬物として核酸を用いた場合には、生体内での安定性が極めて低いことから、肥大した肺血管壁組織に高い選択性をもって送達させなければならないが、これまでそのような送達手段の報告はなかった。 In the enlarged pulmonary vascular wall tissue, it is known that an excessive factor for cell proliferation or growth exists (see Non-Patent Documents 1 and 2). It is expected to suppress pulmonary vascular wall tissue hypertrophy or sclerosis and suppress pulmonary hypertension by suppressing the expression of a gene encoding a factor that proliferates or grows these cells.
However, in order to suppress the expression of a gene encoding a factor that proliferates or grows a cell, it is necessary to deliver a drug to an enlarged pulmonary vascular wall tissue. For example, when a nucleic acid is used as a drug, However, there is no report of such delivery means until now, although it has to be delivered with high selectivity to enlarged pulmonary vessel wall tissue.
　一方、核酸の細胞内への送達手段として、核酸封入リポソーム(リポソーム内に核酸を封入したリポソーム)が報告されている(特許文献1～3および非特許文献3参照)。特許文献1では、核酸等を封入するリポソームの製造方法として、例えば、カチオン性脂質をクロロホルムに予め溶解し、次いでオリゴデオキシヌクレオチド(ODN)の水溶液とメタノールを加えて混合後、遠心分離することでクロロホルム層にカチオン性脂質/ODNの複合体を移行させ、さらにクロロホルム層を取り出し、これにポリエチレングリコール化リン脂質と中性の脂質と水を加えて油中水型(W/O)エマルジョンを形成し、逆相蒸発法で処理してODN内包リポソームを製造する方法が報告され、特許文献2および非特許文献3では、ODNをpH3.8のクエン酸水溶液に溶解し、脂質(エタノール中)を加え、エタノール濃度を20v/v%まで下げてODN内包リポソームを調製し、サイジングろ過し、透析によって、過剰のエタノールを除去した後、試料をさらにpH7.5にて透析してリポソーム表面に付着したODNを除去してODN内包リポソームを製造する方法が報告され、それぞれ核酸等の有効成分を封入したリポソームが製造されている。 Meanwhile, nucleic acid-encapsulated liposomes (liposomes in which nucleic acids are encapsulated in liposomes) have been reported as means for delivering nucleic acids into cells (see Patent Documents 1 to 3 and Non-patent Document 3). In Patent Document 1, as a method for producing a liposome encapsulating nucleic acid or the like, for example, a cationic lipid is dissolved in chloroform in advance, and then an oligodeoxynucleotide (ODN) aqueous solution and methanol are added and mixed, followed by centrifugation. Transfer the cationic lipid / ODN complex to the chloroform layer, then remove the chloroform layer, and add polyethyleneglycolized phospholipid, neutral lipid and water to form a water-in-oil (W / O) emulsion. However, a method for producing ODN-encapsulated liposomes by the reverse phase evaporation method has been reported.In Patent Document 2 and Non-Patent Document 3, ODN is dissolved in an aqueous citric acid solution at pH 3.8, and lipid (in ethanol) is dissolved. In addition, the ODN-encapsulated liposomes were prepared by reducing the ethanol concentration to 20 v / v%, sizing filtered, excess ethanol was removed by dialysis, and the sample was further adjusted to pH 7.5. Dialysed to remove ODN adhering to the liposome surface is reported a method of producing the ODN encapsulated liposomes, liposomes were each encapsulating active ingredients such as nucleic acid has been produced.
　これらに対して特許文献3では、液体中で微粒子を脂質二重膜で被覆する方法で核酸等の有効成分を封入したリポソームを製造することが報告されている。該方法においては、微粒子が分散し、かつ脂質が溶解した極性有機溶媒含有水溶液中の極性有機溶媒の濃度を減少させることによって、微粒子が脂質二重膜で被覆されており、液体中において被覆が行われ、例えば静脈注射用微粒子等に好適な大きさの脂質二重膜で被覆された微粒子(被覆微粒子)が、すぐれた効率で製造されている。また、特許文献3では微粒子の例として例えば水溶性薬物とカチオン性脂質からなる静電的相互作用により形成される複合体が例示されている。複合粒子を被覆した被覆微粒子の粒子径は、被覆される複合粒子に応じて異なるが、ODN-脂質複合体を被覆して得られた被覆微粒子は、粒子径が小さく、注射剤として使用可能であること、該被覆微粒子は、静脈内に投与した場合、高い血中滞留性を示し、腫瘍組織に多く集積したことが報告されている。
　しかしながら、特許文献1～3および非特許文献3のいずれにも、肥大した肺血管壁組織に対して、選択的に核酸を送達することに関しての報告はない。 On the other hand, Patent Document 3 reports that a liposome in which an active ingredient such as a nucleic acid is encapsulated is produced by a method of coating fine particles with a lipid bilayer in a liquid. In this method, by reducing the concentration of the polar organic solvent in the aqueous solution containing the polar organic solvent in which the microparticles are dispersed and the lipid is dissolved, the microparticles are coated with the lipid bilayer membrane, and the coating is not performed in the liquid. For example, fine particles (coated fine particles) coated with a lipid bilayer having a size suitable for fine particles for intravenous injection or the like are produced with excellent efficiency. Patent Document 3 exemplifies a complex formed by electrostatic interaction composed of, for example, a water-soluble drug and a cationic lipid as an example of fine particles. The particle size of the coated fine particles coated with the composite particles varies depending on the coated composite particles, but the coated fine particles obtained by coating the ODN-lipid complex have a small particle size and can be used as an injection. In fact, it has been reported that the coated microparticles show a high blood retention when administered intravenously and accumulate in tumor tissues in large amounts.
However, none of Patent Documents 1 to 3 and Non-Patent Document 3 have reports on selective delivery of nucleic acids to enlarged pulmonary vascular wall tissues.
特表2002-508765号公報Special Table 2002-508765 特表2002-501511号公報Special table 2002-501511 gazette 国際公開第02/28367号パンフレットInternational Publication No. 02/28367 Pamphlet
　本発明の目的は、核酸を含有する肺高血圧症治療剤等を提供することにある。 An object of the present invention is to provide a therapeutic agent for pulmonary hypertension and the like containing a nucleic acid.
　本発明は以下の(1)～(60)に関する。
(1)　(i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、組成物。
(2)　リポソームが、静脈内投与可能な大きさのリポソームである、(1)記載の組成物。
(3)　RNAが、RNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAである、(1)または(2)記載の組成物。
(4)　肺血管壁組織の肥大に関連した遺伝子が、細胞増殖関連因子、分裂促進因子活性化タンパク質キナーゼ（MAPキナーゼ;MAPK）シグナル伝達関連因子およびアポトーシス関連因子のいずれかについての遺伝子である、(1)～(3)のいずれかに記載の組成物。
(5)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPキナーゼシグナル伝達関連因子についてのmRNAである、(1)～(3)のいずれかに記載の組成物。
(6)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPK/ERKキナーゼ(MEK)についてのmRNAである、(1)～(3)のいずれかに記載の組成物。
(7)　mRNAがヒトのmRNAである、(1)～(6)のいずれかに記載の組成物。
(8)　RNAを封入したリポソームが、リード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能である、(1)～(7)のいずれかに記載の組成物。
(9)　極性有機溶媒がアルコールである、(8)記載の組成物。
(10)　極性有機溶媒がエタノールである、(8)記載の組成物。
(11)　リード粒子が、カチオン性物質を含むリード粒子であり、脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(8)～(10)のいずれかに記載の組成物。
(12)　RNAを封入したリポソームが、カチオン性物質を含むリード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(1)～(7)のいずれかに記載の組成物。
(13)　カチオン性物質がN-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムおよび3β-［N-(N’,N’-ジメチルアミノエチル)カルバモイル］コレステロールから選ばれる一つ以上である、(11)または(12)記載の組成物。
(14)　水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体が、ポリエチレングリコール-ホスファチジルエタノールアミンである、(11)～(13)のいずれかに記載の組成物。
(15)　中性脂質が卵黄ホスファチジルコリンである、(11)～(14)のいずれかに記載の組成物。
(16)　(i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、肺高血圧症治療剤。
(17)　リポソームが、静脈内投与可能な大きさのリポソームである、(16)記載の肺高血圧症治療剤。
(18)　RNAが、RNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAである、(16)または(17)記載の肺高血圧症治療剤。
(19)　肺血管壁組織の肥大に関連した遺伝子が、細胞増殖関連因子、MAPキナーゼシグナル伝達関連因子およびアポトーシス関連因子のいずれかについての遺伝子である、(16)～(18)のいずれかに記載の肺高血圧症治療剤。
(20)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPキナーゼシグナル伝達関連因子についてのmRNAである、(16)～(18)のいずれかに記載の肺高血圧症治療剤。
(21)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMEKのmRNAである、(16)～(18)のいずれかに記載の肺高血圧症治療剤。
(22)　mRNAがヒトのmRNAである、(16)～(21)のいずれかに記載の肺高血圧症治療剤。
(23)　RNAを封入したリポソームが、リード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能である、(16)～(22)のいずれかに記載の肺高血圧症治療剤。
(24)　極性有機溶媒がアルコールである、(23)記載の肺高血圧症治療剤。
(25)　極性有機溶媒がエタノールである、(23)記載の肺高血圧症治療剤。
(26)　リード粒子が、カチオン性物質を含むリード粒子であり、脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(23)～(25)のいずれかに記載の肺高血圧症治療剤。
(27)　RNAを封入したリポソームが、カチオン性物質を含むリード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(16)～(22)のいずれかに記載の肺高血圧症治療剤。
(28)　カチオン性物質がN-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムおよび3β-［N-(N’,N’-ジメチルアミノエチル)カルバモイル］コレステロールから選ばれる一つ以上である、(26)または(27)記載の肺高血圧症治療剤。
(29)　水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体が、ポリエチレングリコール-ホスファチジルエタノールアミンである、(26)～(28)のいずれかに記載の肺高血圧症治療剤。
(30)　中性脂質が卵黄ホスファチジルコリンである、(26)～(29)のいずれかに記載の肺高血圧症治療剤。
(31)　(i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、組成物を哺乳動物に投与する肺高血圧症の治療方法。
(32)　リポソームが、静脈内投与可能な大きさのリポソームである、(31)記載の肺高血圧症の治療方法。
(33)　RNAが、RNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAである、(31)または(32)記載の肺高血圧症の治療方法。
(34)　肺血管壁組織の肥大に関連した遺伝子が、細胞増殖関連因子、MAPキナーゼシグナル伝達関連因子およびアポトーシス関連因子のいずれかについての遺伝子である、(31)～(33)のいずれかに記載の肺高血圧症の治療方法。
(35)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPキナーゼシグナル伝達関連因子についてのmRNAである、(31)～(33)のいずれかに記載の肺高血圧症の治療方法。
(36)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMEKのmRNAである、(31)～(33)のいずれかに記載の肺高血圧症の治療方法。
(37)　mRNAがヒトのmRNAである、(31)～(36)のいずれかに記載の肺高血圧症の治療方法。
(38)　RNAを封入したリポソームが、リード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能である、(31)～(37)のいずれかに記載の肺高血圧症の治療方法。
(39)　極性有機溶媒がアルコールである、(38)記載の肺高血圧症の治療方法。
(40)　極性有機溶媒がエタノールである、(38)記載の肺高血圧症の治療方法。
(41)　リード粒子が、カチオン性物質を含むリード粒子であり、脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(38)～(40)のいずれかに記載の肺高血圧症の治療方法。
(42)　RNAを封入したリポソームが、カチオン性物質を含むリード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(31)～(37)のいずれかに記載の肺高血圧症の治療方法。
(43)　カチオン性物質がN-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムおよび3β-［N-(N’,N’-ジメチルアミノエチル)カルバモイル］コレステロールから選ばれる一つ以上である、(41)または(42)記載の肺高血圧症の治療方法。
(44)　水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体が、ポリエチレングリコール-ホスファチジルエタノールアミンである、(41)～(43)のいずれかに記載の肺高血圧症の治療方法。
(45)　中性脂質が卵黄ホスファチジルコリンである、(41)～(44)のいずれかに記載の肺高血圧症の治療方法。
(46)　(i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、組成物の肺高血圧症治療剤の製造のための使用。
(47)　リポソームが、静脈内投与可能な大きさのリポソームである、(46)記載の使用。
(48)　RNAが、RNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAである、(46)または(47)記載の使用。
(49)　肺血管壁組織の肥大に関連した遺伝子が、細胞増殖関連因子、MAPキナーゼシグナル伝達関連因子およびアポトーシス関連因子のいずれかについての遺伝子である、(46)～(48)のいずれかに記載の使用。
(50)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPキナーゼシグナル伝達関連因子についてのmRNAである、(46)～(48)のいずれかに記載の使用。
(51)　肺血管壁組織の肥大に関連した遺伝子のmRNAがMEKのmRNAである、(46)～(48)のいずれかに記載の使用。
(52)　mRNAがヒトのmRNAである、(46)～(51)のいずれかに記載の使用。
(53)　RNAを封入したリポソームが、リード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能である、(46)～(52)のいずれかに記載の使用。
(54)　極性有機溶媒がアルコールである、(53)記載の使用。
(55)　極性有機溶媒がエタノールである、(53)記載の使用。
(56)　リード粒子が、カチオン性物質を含むリード粒子であり、脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(53)～(55)のいずれかに記載の使用。
(57)　RNAを封入したリポソームが、カチオン性物質を含むリード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、(46)～(52)のいずれかに記載の使用。
(58)　カチオン性物質がN-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムおよび3β-［N-(N’,N’-ジメチルアミノエチル)カルバモイル］コレステロールから選ばれる一つ以上である、(56)または(57)記載の使用。
(59)　水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体が、ポリエチレングリコール-ホスファチジルエタノールアミンである、(56)～(58)のいずれかに記載の使用。
(60)　中性脂質が卵黄ホスファチジルコリンである、(56)～(59)のいずれかに記載の使用。 The present invention relates to the following (1) to (60).
(1) (i) RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside A composition containing the prepared liposomes.
(2) The composition according to (1), wherein the liposome is a liposome having a size that can be administered intravenously.
(3) The composition according to (1) or (2), wherein the RNA is RNA having an action of suppressing expression of the gene using RNA interference (RNAi).
(4) The gene related to hypertrophy of pulmonary vascular wall tissue is a gene for any of cell growth-related factor, mitogen-activated protein kinase (MAP kinase; MAPK) signaling-related factor and apoptosis-related factor. The composition according to any one of (1) to (3).
(5) The composition according to any one of (1) to (3), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signaling-related factor.
(6) The composition according to any one of (1) to (3), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for MAPK / ERK kinase (MEK).
(7) The composition according to any one of (1) to (6), wherein the mRNA is human mRNA.
(8) The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a constituent, 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. , (1) to (7).
(9) The composition according to (8), wherein the polar organic solvent is an alcohol.
(10) The composition according to (8), wherein the polar organic solvent is ethanol.
(11) The lead particles are lead particles containing a cationic substance, and 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 as a constituent component. The composition according to any one of (8) to (10), which is a film.
(12) The liposome encapsulating RNA is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent component, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (1) to (7), wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative. Composition.
(13) The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- (11) one or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, Or the composition according to (12).
(14) The composition according to any one of (11) to (13), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
(15) The composition according to any one of (11) to (14), wherein the neutral lipid is egg yolk phosphatidylcholine.
(16) (i) an RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and a base sequence complementary to the sequence; and (ii) enclosing the RNA inside A therapeutic agent for pulmonary hypertension, comprising the prepared liposome.
(17) The therapeutic agent for pulmonary hypertension according to (16), wherein the liposome is a liposome having a size that can be administered intravenously.
(18) The therapeutic agent for pulmonary hypertension according to (16) or (17), wherein the RNA is RNA having an action of suppressing expression of the gene using RNA interference (RNAi).
(19) The gene associated with hypertrophy of the pulmonary vascular wall tissue is a gene for any one of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor, according to any one of (16) to (18) The therapeutic agent for pulmonary hypertension as described.
(20) The therapeutic agent for pulmonary hypertension according to any of (16) to (18), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signaling-related factor.
(21) The therapeutic agent for pulmonary hypertension according to any of (16) to (18), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is MEK mRNA.
(22) The therapeutic agent for pulmonary hypertension according to any of (16) to (21), wherein the mRNA is human mRNA.
(23) A liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a 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 therapeutic agent for pulmonary hypertension according to any one of (16) to (22).
(24) The therapeutic agent for pulmonary hypertension according to (23), wherein the polar organic solvent is alcohol.
(25) The therapeutic agent for pulmonary hypertension according to (23), wherein the polar organic solvent is ethanol.
(26) A lipid particle comprising lead particles containing a cationic substance and a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance. The therapeutic agent for pulmonary hypertension according to any one of (23) to (25), which is a membrane.
(27) The RNA encapsulated liposome is a liposome composed of a lead particle containing a cationic substance, a composite particle containing the RNA as a constituent component, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (16) to (22), 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. For treating pulmonary hypertension.
(28) The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- One or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, (26) Or the therapeutic agent for pulmonary hypertension according to (27).
(29) The therapeutic agent for pulmonary hypertension according to any one of (26) to (28), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
(30) The therapeutic agent for pulmonary hypertension according to any of (26) to (29), wherein the neutral lipid is egg yolk phosphatidylcholine.
(31) (i) RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside A method for treating pulmonary hypertension, comprising administering a composition containing a prepared liposome to a mammal.
(32) The method for treating pulmonary hypertension according to (31), wherein the liposome is a liposome having a size that can be administered intravenously.
(33) The method for treating pulmonary hypertension according to (31) or (32), wherein the RNA is RNA having an action of suppressing expression of the gene using RNA interference (RNAi).
(34) The gene associated with hypertrophy of pulmonary vascular wall tissue is a gene for any of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor, and any of (31) to (33) The method for treating pulmonary hypertension as described.
(35) The method for treating pulmonary hypertension according to any of (31) to (33), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signaling-related factor.
(36) The method for treating pulmonary hypertension according to any of (31) to (33), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is MEK mRNA.
(37) The method for treating pulmonary hypertension according to any of (31) to (36), wherein the mRNA is human mRNA.
(38) RNA encapsulated liposome is a liposome composed of a lead particle, a composite particle comprising the RNA as a constituent, 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 method for treating pulmonary hypertension according to any of (31) to (37).
(39) The method for treating pulmonary hypertension according to (38), wherein the polar organic solvent is alcohol.
(40) The method for treating pulmonary hypertension according to (38), wherein the polar organic solvent is ethanol.
(41) The lipid bilayer in which the lead particle is a lead particle containing a cationic substance, and the lipid bilayer membrane is a lipid, fatty acid derivative or aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance. The method for treating pulmonary hypertension according to any one of (38) to (40), wherein the method is a membrane.
(42) The liposome encapsulating RNA is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent component, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (31) to (37), 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. For treating pulmonary hypertension in children.
(43) Cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- (41) one or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, Or the method for treating pulmonary hypertension according to (42).
(44) The method for treating pulmonary hypertension according to any of (41) to (43), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
(45) The method for treating pulmonary hypertension according to any of (41) to (44), wherein the neutral lipid is egg yolk phosphatidylcholine.
(46) (i) RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside Use of the composition for the manufacture of a therapeutic agent for pulmonary hypertension, comprising the prepared liposomes.
(47) The use according to (46), wherein the liposome is a liposome of a size that can be administered intravenously.
(48) The use according to (46) or (47), wherein the RNA is RNA having an action of suppressing the expression of the gene using RNA interference (RNAi).
(49) The gene related to hypertrophy of the pulmonary vascular wall tissue is a gene for any of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor, according to any of (46) to (48) Use of description.
(50) The use according to any one of (46) to (48), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signaling-related factor.
(51) The use according to any one of (46) to (48), wherein the mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is MEK mRNA.
(52) The use according to any one of (46) to (51), wherein the mRNA is human mRNA.
(53) The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising the RNA as a constituent, 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. , Use according to any of (46) to (52).
(54) The use according to (53), wherein the polar organic solvent is an alcohol.
(55) The use according to (53), wherein the polar organic solvent is ethanol.
(56) A lipid particle comprising lead particles containing a cationic substance and a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance. The use according to any one of (53) to (55), which is a membrane.
(57) RNA encapsulated liposome is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (46) to (52), 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. Use of.
(58) Cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- One or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, (56) Or use according to (57).
(59) The use according to any one of (56) to (58), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
(60) The use according to any one of (56) to (59), wherein the neutral lipid is egg yolk phosphatidylcholine.
　本発明の肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAを封入したリポソームを含有する組成物を、ほ乳類等に投与することにより、気管支または肺の肥大した肺血管壁組織において、肺血管壁組織の肥大に関連した遺伝子の発現を抑制することができる。また、気管支または肺において、肺血管壁組織の肥大に関連した前記遺伝子の発現を抑制することで、肺高血圧症を治療することができる。 A composition containing liposomes encapsulating RNA containing a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue of the present invention and a base sequence complementary to the sequence, such as mammals, etc. Administration to the pulmonary vascular wall tissue with enlarged bronchi or lung can suppress the expression of genes related to pulmonary vascular wall tissue hypertrophy. Moreover, pulmonary hypertension can be treated by suppressing the expression of the gene associated with hypertrophy of pulmonary vascular wall tissue in bronchi or lung.
実施例1で得られた組成物を投与した場合に、肺高血圧症を生じるモデルであるMCTラット特異的に、RNAが高濃度で集積することを示している。横軸は投与開始後の日数、縦軸は肺のCy5標識MEKsiRNAの濃度(nM)を表わしている。○は対照群、●はモノクロタリン誘発性肺高血圧症ラット（MCTラット）群の平均値と標準偏差を示す。また、＊は統計解析(student t テスト)の結果、ベースラインに対して有意差p値＜0.05の認められた点を示す。†は、統計解析(student t テスト)の結果、群間で有意差p値＜0.05 の認められた点を示す。When the composition obtained in Example 1 is administered, RNA is accumulated at a high concentration specifically in the MCT rat, which is a model causing pulmonary hypertension. The horizontal axis represents the number of days after the start of administration, and the vertical axis represents the concentration (nM) of Cy5-labeled MEK siRNA in the lung. ○ indicates the mean and standard deviation of the control group, and ● indicates the monocrotaline-induced pulmonary hypertension rat (MCT rat) group. Moreover, * shows the point by which significant difference p value <0.05 was recognized with respect to the base line as a result of statistical analysis (student t test). † indicates a point where a significant difference between groups was found as a result of statistical analysis (student t test) <0.05. 実施例1で得られた組成物を投与した場合に、肺高血圧症の疾患部位である肺に、RNAが選択的に集積することを示した。横軸は臓器または組織の名称、縦軸は各臓器または組織のCy5標識MEKsiRNAの濃度(nM)を表わしている。白抜きカラムは対照群、黒ぬりカラムはMCTラット群の平均値と標準偏差を示す。また、＊は統計解析(student t テスト)の結果、群間で有意差p値＜0.05の認められた点を示す。It was shown that when the composition obtained in Example 1 was administered, RNA was selectively accumulated in the lung, which is a disease site of pulmonary hypertension. The horizontal axis represents the organ or tissue name, and the vertical axis represents the concentration (nM) of Cy5-labeled MEK siRNA in each organ or tissue. The white column represents the mean and standard deviation of the control group, and the black column represents the MCT rat group. Moreover, * shows the point by which significant difference p value <0.05 was recognized between groups as a result of the statistical analysis (student | t test). 対照群(左図)、MCTラット群(右図)の肺動脈およびその近傍を免疫染色した切片を示した。図中の白色部分が緑色に染色された血管内皮細胞を示し、矢印が赤色の蛍光が強く認められたCy5標識MEKsiRNAの集積を示す。Shown are immunostained sections of the pulmonary artery and its vicinity in the control group (left figure) and MCT rat group (right figure). In the figure, the white part indicates vascular endothelial cells stained in green, and the arrow indicates the accumulation of Cy5-labeled MEK siRNA in which strong red fluorescence was observed. 実施例2で得られた組成物を投与した場合に、肺高血圧症を生じた肺において、投与量依存的に遺伝子の発現を抑制することを示した。縦軸は肺におけるMEK1(左図)およびMEK2(右図)のmRNAの発現強度の、GAPDHのmRNA発現強度に対する比を示した。0.2および2は、実施例2で得られた組成物を0.2mg/kgおよび2mg/kgの各投与量で静脈内投与した群を表し、Controlは、比較例1で得られた組成物を投与した群を表す。It was shown that when the composition obtained in Example 2 was administered, gene expression was suppressed in a dose-dependent manner in the lungs in which pulmonary hypertension occurred. The vertical axis represents the ratio of the expression intensity of MEK1 (left figure) and MEK2 (right figure) mRNA in the lung to that of GAPDH. 0.2 and 2 represent groups in which the composition obtained in Example 2 was intravenously administered at doses of 0.2 mg / kg and 2 mg / kg, and Control administered the composition obtained in Comparative Example 1. Represents a group. 実施例2で得られた組成物を投与した場合において、肺高血圧症を生じた肺での遺伝子の発現抑制効果の経時的変化を示した。縦軸は肺におけるMEK1(左図)およびMEK2(右図)のmRNAの発現強度の、GAPDHのmRNA発現強度に対する比を示した。24、48および96は、それぞれ実施例2で得られた組成物の投与後24、48および96時間後を表し、Controlは、比較例1で得られた組成物を投与した群を表す。When the composition obtained in Example 2 was administered, changes over time in the gene expression suppression effect in the lungs in which pulmonary hypertension occurred were shown. The vertical axis represents the ratio of the expression intensity of MEK1 (left figure) and MEK2 (right figure) mRNA in the lung to that of GAPDH. 24, 48, and 96 represent 24, 48, and 96 hours after administration of the composition obtained in Example 2, respectively, and Control represents a group that was administered with the composition obtained in Comparative Example 1. 実施例2で得られた組成物を投与した場合に、肺高血圧を抑制することを示した。縦軸は収縮期右心室圧(mmHg)を示した。MCT、比較例1および実施例2は、それぞれ生理食塩水、比較例1で得られた組成物および実施例2で得られた組成物を投与したMCTラットの群、Shamは、MCTの代わりに生理食塩水を皮下投与し、生理食塩水を静脈内投与したラットの群を表す。＊は、統計解析(student t テスト)の結果、実施例2で得られた組成物を投与した群と比較例1で得られた組成物を投与した群との間で有意差p値＜0.05 の認められたことを示す。It was shown that pulmonary hypertension is suppressed when the composition obtained in Example 2 is administered. The vertical axis represents systolic right ventricular pressure (mmHg). MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. * Indicates a significant difference between the group administered with the composition obtained in Example 2 and the group administered with the composition obtained in Comparative Example 1 as a result of statistical analysis (student t test). Indicates that 実施例2で得られた組成物を投与した場合に、右心室の肥大化を抑制することを示した。縦軸は左心室と心室中隔の総重量に対する右心室の重量の相対比を示した。MCT、比較例1および実施例2は、それぞれ生理食塩水、比較例1で得られた組成物および実施例2で得られた組成物を投与したMCTラットの群、Shamは、MCTの代わりに生理食塩水を皮下投与し、生理食塩水を静脈内投与したラットの群を表す。＊は、統計解析(student t テスト)の結果、実施例2で得られた組成物を投与した群と比較例1で得られた組成物を投与した群との間で有意差p値＜0.05 の認められたことを示す。It was shown that when the composition obtained in Example 2 was administered, hypertrophy of the right ventricle was suppressed. The vertical axis represents the relative ratio of the right ventricular weight to the total weight of the left ventricle and the ventricular septum. MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. * Indicates a significant difference between the group administered with the composition obtained in Example 2 and the group administered with the composition obtained in Comparative Example 1 as a result of statistical analysis (student t test). Indicates that 各ラットにおける肺動脈の肥大化をEV染色によって可視化した顕微鏡画像を示した。MCT、比較例1および実施例2は、それぞれ生理食塩水、比較例1で得られた組成物および実施例2で得られた組成物を投与したMCTラットの群、Shamは、MCTの代わりに生理食塩水を皮下投与し、生理食塩水を静脈内投与したラットの群を表す。The microscopic image which visualized the enlargement of the pulmonary artery in each rat by EV staining was shown. MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. 実施例2で得られた組成物を投与した場合に、肺動脈壁の肥大化を抑制することを示した。縦軸は肺動脈壁の肥大化の度合いを示した。MCT、比較例1および実施例2は、それぞれ生理食塩水、比較例1で得られた組成物および実施例2で得られた組成物を投与したMCTラットの群、Shamは、MCTの代わりに生理食塩水を皮下投与し、生理食塩水を静脈内投与したラットの群を表す。＊は、統計解析(student t テスト)の結果、実施例2で得られた組成物を投与した群と比較例1で得られた組成物を投与した群との間で有意差p値＜0.05 の認められたことを示す。It was shown that when the composition obtained in Example 2 was administered, enlargement of the pulmonary artery wall was suppressed. The vertical axis shows the degree of enlargement of the pulmonary artery wall. MCT, Comparative Example 1 and Example 2 are groups of MCT rats administered with saline, the composition obtained in Comparative Example 1 and the composition obtained in Example 2, respectively, and Sham instead of MCT 1 represents a group of rats administered with physiological saline subcutaneously and physiological saline intravenously. * Indicates a significant difference between the group administered with the composition obtained in Example 2 and the group administered with the composition obtained in Comparative Example 1 as a result of statistical analysis (student t test). Indicates that
　本発明で用いられる肺血管壁組織の肥大に関連した遺伝子としては、ほ乳類の気管支または肺においてmRNAを産生して発現する肺血管壁組織の肥大に関連した遺伝子であれば特に限定されないが、例えば肺血管の増殖または成長に関連する遺伝子があげられ、好ましくは細胞増殖関連因子、MAPキナーゼシグナル伝達関連因子、アポトーシス関連因子等についての遺伝子があげられる。細胞増殖関連因子としては、例えば、線維芽細胞増殖因子、線維芽細胞増殖因子受容体、上皮成長因子、上皮成長因子受容体、血管内皮増殖因子(VGEF)、血管内皮増殖因子受容体、血小板由来増殖因子、血小板由来増殖因子受容体、肝細胞増殖因子、肝細胞増殖因子受容体、クルッペル様因子、Ets転写因子、低酸素誘導因子、核因子等があげられ、MAPキナーゼシグナル伝達関連因子としては、例えば、分裂促進因子活性化タンパク質キナーゼ(MAPK)、MAPKキナーゼ(MAPKK)、MAPKKキナーゼ(MAPKKK)、c-Jun N末端キナーゼ(JNK)、p38MAPK等があげられ、アポトーシス関連因子としては、例えば、サバイビン、FLICE抑制タンパク質(FLIP)、アポトーシス抑制系関連タンパク質Bcl-2、Bcl-xL、Mcl-1および微小管結合タンパク質等があげられ、好ましくは線維芽細胞増殖因子、線維芽細胞増殖因子受容体、MAPキナーゼシグナル伝達関連因子、クルッペル様因子、Bcl-2およびBcl-xLがあげられ、さらに好ましくはMAPキナーゼシグナル伝達関連因子、Bcl-2があげられる。 The gene associated with hypertrophy of pulmonary vascular wall tissue used in the present invention is not particularly limited as long as it is a gene associated with hypertrophy of pulmonary vascular wall tissue that is produced and expressed in mammalian bronchi or lung, for example, Examples include genes related to proliferation or growth of pulmonary blood vessels, preferably genes related to cell proliferation-related factors, MAP kinase signal transduction-related factors, apoptosis-related factors and the like. Cell growth-related factors include, for example, fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor receptor, vascular endothelial growth factor (VGEF), vascular endothelial growth factor receptor, platelet-derived Growth factor, platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor, Ets transcription factor, hypoxia inducer, nuclear factor, etc. For example, mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), MAPKK kinase (MAPKKK), c-Jun N-terminal kinase (JNK), p38MAPK and the like, as apoptosis-related factors, for example, Survivin, FLICE inhibitory protein (FLIP), apoptosis inhibitor related proteins Bcl-2, Bcl-xL, Mcl-1, and microtubule-binding proteins are preferred, preferably fibroblast growth factor Fibroblast growth factor receptor, MAP kinase signaling-related factor, Kruppel-like factor, Bcl-2 and Bcl-xL, and the like, more preferably MAP kinase signaling associated factors, Bcl-2 and the like.
　分裂促進因子活性化タンパク質キナーゼ(Mitogen-activated Protein Kinase、MAPK)とはセリン/スレオニンキナーゼの一つであり、何らかの刺激(酸化ストレス、サイトカインなど)を受けて活性化される。全身の細胞に広く発現しており、様々な細胞の機能発現において重要な働きをしている。単にMAP(マップ)キナーゼと略して呼ばれることが多い。
細胞外からの刺激が入ると低分子量Gタンパク質であるRasが活性化され、さらにその下流に続くシグナルカスケードの活性化が引き起こされる。また、MAPKホスファターゼ(MAPK Phosphatase:MKP)による脱リン酸化がMAPKを不活性化し、この機構に対して抑制的に働いている。狭義にはMAPKは細胞外シグナル調節キナーゼ(Extracellular Signal-regulated Kinase、ERK)1/2のみを指すが、広義にはこれに加えてc-Jun N末端キナーゼ(c-jun N-terminal kinase、JNK)、p38 MAPK、ERK5及びERK7等の分子をも含み、MAPKファミリーとも呼ばれる。 Mitogen-activated protein kinase (MAPK) is one of serine / threonine kinases and is activated by receiving some kind of stimulation (oxidative stress, cytokine, etc.). It is widely expressed in cells throughout the body and plays an important role in the functional expression of various cells. Often referred to simply as MAP kinase.
When a stimulus from outside the cell enters, Ras, which is a low molecular weight G protein, is activated, and further activation of the signal cascade downstream thereof is caused. In addition, dephosphorylation by MAPK phosphatase (MAPK Phosphatase: MKP) inactivates MAPK and acts on this mechanism in a suppressive manner. In the narrow sense, MAPK refers only to extracellular signal-regulated kinase (ERK) 1/2, but in a broad sense, c-Jun N-terminal kinase (JNK) ), Including molecules such as p38 MAPK, ERK5 and ERK7, and is also referred to as the MAPK family.
　ERK1/2はMAPKファミリーの中でも最初に同定されたものであり、古典的MAPKとも称される。ERK1/2は分子量が44kDaのERK1と42kDaのERK2から成り、これらのタンパク質のアミノ酸配列は互いに85%の相同性がある。
　ERK1/2の活性化は以下のような機序で生じる。上皮増殖因子受容体(Epidermal Growth Factor Receptor、EGFR)等のチロシンキナーゼ関連型受容体にリガンドが結合すると受容体細胞内ドメインのリン酸化が生じる。Grb2等のSH2ドメインを含むアダプタータンパク質が受容体のリン酸化チロシンに結合すると、Grb2はSH3ドメインを介してSosと結合し、Sosを活性化させる。活性化したSosはRasのGDP-GTP交換反応によりRasを活性化させる。以下、RasはMAPキナーゼカスケードへとシグナルを伝えていくが、RasはMAPキナーゼキナーゼキナーゼ(MAPKKK)であるRafを、RafはMAPキナーゼキナーゼであるMAPK/ERK kinase(MEK)を活性化し、このMEKによりERKの活性化が引き起こされる。なお、ERK1に対するMEKがMEK1、ERK2に対するMEKがMEK2である。ERKの活性化にはThr183及びTyr185が重要な働きをしている。通常ERK1/2は細胞質に優位に存在するが、活性化されることにより核内へ移行して転写因子と相互作用することにより転写の制御を行っている。 ERK1 / 2 was first identified in the MAPK family and is also referred to as classical MAPK. ERK1 / 2 consists of ERK1 with a molecular weight of 44 kDa and ERK2 with a 42 kDa molecular weight, and the amino acid sequences of these proteins are 85% homologous to each other.
The activation of ERK1 / 2 occurs by the following mechanism. When a ligand binds to a tyrosine kinase-related receptor such as epidermal growth factor receptor (EGFR), phosphorylation of the receptor intracellular domain occurs. When an adapter protein containing an SH2 domain such as Grb2 binds to phosphorylated tyrosine of the receptor, Grb2 binds to Sos through the SH3 domain and activates Sos. Activated Sos activates Ras by the GDP-GTP exchange reaction of Ras. Ras then transmits signals to the MAP kinase cascade. Causes activation of ERK. The MEK for ERK1 is MEK1, and the MEK for ERK2 is MEK2. Thr183 and Tyr185 play an important role in the activation of ERK. Normally, ERK1 / 2 exists predominantly in the cytoplasm, but when activated, it translocates into the nucleus and interacts with transcription factors to control transcription.
　JNKはc-JunのSer63とSer73をリン酸化する活性を持つキナーゼとして同定された。JNKは放射線やリポ多糖、IL-1、浸透圧及び熱ショックなどのストレスにより活性化し、ストレス応答性MAPK(Stress-activated Protein Kinase、SAPK)とも呼ばれる。
　JNK遺伝子にはJNK1～JNK3が存在する。JNK1はアポトーシスや神経変性、細胞の分化・増殖、炎症性サイトカインの産生などの過程に関与している。JNKは様々な細胞内タンパク質のリン酸化を行い、それらの機能修飾をしている。 JNK was identified as a kinase with the activity to phosphorylate Ser63 and Ser73 of c-Jun. JNK is activated by stress such as radiation, lipopolysaccharide, IL-1, osmotic pressure and heat shock, and is also called stress-responsive MAPK (Stress-activated Protein Kinase, SAPK).
There are JNK1 to JNK3 in the JNK gene. JNK1 is involved in processes such as apoptosis and neurodegeneration, cell differentiation and proliferation, and production of inflammatory cytokines. JNK phosphorylates various intracellular proteins and modifies their functions.
　本発明で用いられるRNAとしては、前記遺伝子のmRNAの連続する15～30塩基、好ましくは17～25塩基、より好ましくは19～23塩基の配列および該配列と相補的な塩基の配列を含んでいるRNAがあげられる。
　本発明で用いられるRNAは、リボースの一部または全部が、デオキシリボースに置換されているもの、すなわちDNAも包含する。さらに、本発明で用いられるRNA中のリボヌクレオチド、デオキシリボヌクレオチドに修飾を施していてもよく、例えば、糖部修飾ヌクレオチド類似体やリン酸ジエステル結合修飾ヌクレオチド類似体等になっていてもよい。また、本発明で用いられるRNAとしては、該RNA中のリン酸部、エステル部等に含まれる酸素原子等が、例えば硫黄原子等の他の原子に置換された誘導体も包含される。
　本発明において、本発明で用いられるRNA中のリボヌクレオチドをデオキシリボヌクレオチドとすること、本発明で用いられるRNA中のリボヌクレオチドおよびデオキシリボヌクレオチドに修飾を施すこと、本発明で用いられるRNA中のリン酸部、エステル部等に含まれる酸素原子等を、例えば硫黄原子等の他の原子に置換することは、RNAまたはDNAに比べ、ヌクレアーゼ耐性を向上させるため、安定化させるため、相補鎖核酸とのアフィニティーを上げるため、細胞透過性を上げるため、あるいは可視化させるため等のいかなる目的で成されたものであってもよい。 The RNA used in the present invention includes a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of the mRNA of the gene, and a base sequence complementary to the sequence. RNA.
The RNA used in the present invention also includes DNA in which part or all of ribose is substituted with deoxyribose, that is, DNA. Furthermore, ribonucleotides and deoxyribonucleotides in RNA used in the present invention may be modified, for example, sugar-modified nucleotide analogs, phosphodiester bond-modified nucleotide analogs, and the like. The RNA used in the present invention also includes derivatives in which an oxygen atom or the like contained in a phosphate part, an ester part, or the like in the RNA is substituted with another atom such as a sulfur atom.
In the present invention, the ribonucleotide in the RNA used in the present invention is deoxyribonucleotide, the ribonucleotide and deoxyribonucleotide in the RNA used in the present invention are modified, the phosphate in the RNA used in the present invention Substituting oxygen atoms, etc., contained in the ester part, ester part, etc., with other atoms, such as sulfur atoms, improves the nuclease resistance compared to RNA or DNA, and stabilizes it. It may be formed for any purpose such as increasing affinity, increasing cell permeability, or visualizing.
　糖部修飾ヌクレオチド類似体とは、ヌクレオチドの糖の化学構造の一部あるいは全てに対し、任意の化学構造物質を付加あるいは置換したものであればいかなるものでもよく、例えば、2’-O-メチルリボースで置換されたヌクレオチド類似体、2’-O-プロピルリボースで置換されたヌクレオチド類似体、2’-メトキシエトキシリボースで置換されたヌクレオチド類似体、2’-O-メトキシエチルリボースで置換されたヌクレオチド類似体、2’-O-［2-(グアニジウム)エチル］リボースで置換されたヌクレオチド類似体、2’-O-フルオロリボースで置換されたヌクレオチド類似体、糖部に架橋構造を導入することにより2つの環状構造を有する架橋構造型人工核酸(Bridged Nucleic Acid)(BNA)、より具体的には、2’位の酸素原子と4’位の炭素原子がメチレンを介して架橋したロックト人工核酸(Locked Nucleic Acid)(LNA)、エチレン架橋構造型人工核酸(Ethylene bridged nucleic acid)(ENA)［Nucleic Acid Research, 32, e175(2004)］等があげられ、さらにペプチド核酸(PNA)[Acc. Chem. Res., 32, 624 (1999)]、オキシペプチド核酸(OPNA)[J. Am. Chem. Soc., 123, 4653 (2001)]、ペプチドリボ核酸(PRNA)[J. Am. Chem. Soc., 122, 6900 (2000)]等をあげることができる。 The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to part or all of the chemical structure of the sugar of the nucleotide. For example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged Nucleic Acid (BNA), more specifically, 2′-position oxygen atom and 4′-position carbon atom via methylene Crosslinked locked artificial nucleic acid (Locked Nucleic Acid) (LNA), ethylene bridged structure type artificial nucleic acid (Ethylene bridged nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)], etc. Nucleic acid (PNA) [Acc. Chem. Res., 32, 624) (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].
　リン酸ジエステル結合修飾ヌクレオチド類似体とは、ヌクレオチドのリン酸ジエステル結合の化学構造の一部あるいは全てに対し、任意の化学物質を付加あるいは置換したものであればいかなるものでもよく、例えば、ホスフォロチオエート結合に置換されたヌクレオチド類似体、N3'-P5'ホスフォアミデート結合に置換されたヌクレオチド類似体等をあげることができる［細胞工学, 16, 1463-1473 (1997)］［RNAi法とアンチセンス法、講談社(2005)］。 The phosphodiester bond-modified nucleotide analogue may be any one in which any chemical substance is added or substituted to a part or all of the chemical structure of the phosphodiester bond of a nucleotide. Examples include nucleotide analogues substituted with thioate linkages, nucleotide analogues substituted with N3'-P5 'phosphoramidate linkages [Cell engineering, 16, 1463-1473 (1997)] [RNAi method And Antisense, Kodansha (2005)].
　また、本発明で用いられるRNAとしては、好ましくはRNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAがあげられる。ここではMEK遺伝子の発現を抑制するRNAを例にとって、RNA干渉(RNAi)を利用した遺伝子の発現を抑制するRNAについて説明する。他の遺伝子の場合も同様の構造を有し、また同様の操作で得ることができる。 The RNA used in the present invention is preferably an RNA having an action of suppressing the expression of the gene using RNA interference (RNAi). Here, RNA that suppresses gene expression using RNA interference (RNAi) will be described using RNA that suppresses MEK gene expression as an example. Other genes have similar structures and can be obtained by similar operations.
　MEK遺伝子の発現を抑制するRNAは、MEK mRNAの連続する15～30塩基、好ましくは17～25塩基、より好ましくは19～23塩基の配列(以下配列Xとする)および該配列と相補的な塩基の配列(以下相補配列X’とする)を含んでいる。RNAとしては、(A)配列Xの鎖(センス鎖)および相補配列X’の鎖(アンチセンス鎖)からなる二本鎖RNA、(B)配列Xの鎖(センス鎖)および相補配列X’の鎖(アンチセンス鎖)からなる二本鎖RNAの配列Xの鎖もしくは相補配列X’の鎖またはそれぞれの鎖の3’端に1～6個、好ましくは2～4個のヌクレオチドが同一または異なって付加された二本鎖RNAであってMEK遺伝子の発現を抑制するRNA(以下、(A)および(B)のような構造のRNAをMEKsiRNAとよぶ)、(C)配列XからなるRNAおよび相補配列X’からなるRNAが、スペーサーオリゴヌクレオチドでつながれ、ヘアピン構造を有するRNAであってMEK遺伝子の発現を抑制するRNA、(D)配列XからなるRNAおよび相補配列X’からなるRNAが、スペーサーオリゴヌクレオチドでつながれ、3’端に1～6個、好ましくは2～4個のヌクレオチドが付加された、ヘアピン構造を有するRNAであってMEK遺伝子の発現を抑制するRNA(以下、(C)および(D)のようなRNAをMEKshRNAとよぶ)等があげられる。これらのRNAに付加されるヌクレオチドの塩基は、グアニン、アデニン、シトシン、チミンおよびウラシルのいずれか1種または複数種でもよく、またRNAでもDNAでもよいが、ウリジル酸(U)およびデオキシチミジル酸(dT)のいずれか1種または2種が好ましい。またスペーサーオリゴヌクレオチドとしては6～12塩基のRNAが好ましく、その5’端の配列は2個のUであるのが好ましい。スペーサーオリゴヌクレオチドの例として、UUCAAGAGAの配列からなるRNAがあげられる。スペーサーオリゴヌクレオチドによってつながれる2つのRNAの順番はどちらが5’側になってもよい。また、いずれの場合も、相補配列X’の3’端側に隣接して付加されるヌクレオチドの塩基の配列を、mRNA内で配列Xと隣接する塩基の配列と相補的な塩基の配列としてもよく、この構造がより好ましい。配列Xは、MEK mRNAの連続する15～30塩基の配列、好ましくは17～25塩基、より好ましくは19～23塩基の配列であれば、いずれの配列でもよいが、遺伝子の発現を抑制したい動物のMEK cDNAの塩基配列から、AAではじまる21塩基の部分配列を取り出す。取り出した配列のGC含量を計算し、GC含量が20～80%、好ましくは30%～70%、より好ましくは40～60%の配列を複数個選択して設計した配列がより好ましい。 The RNA that suppresses the expression of the MEK gene is a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of MEK 配 列 mRNA (hereinafter referred to as sequence X) and complementary to the sequence. It contains a base sequence (hereinafter referred to as complementary sequence X ′). RNA includes: (A) double-stranded RNA consisting of the strand of sequence X (sense strand) and complementary strand X ′ (antisense strand); (B) the strand of sequence X (sense strand) and complementary sequence X ′ 1 to 6, preferably 2 to 4 nucleotides are the same at the 3 ′ end of the strand of the sequence X or the complementary sequence X ′ of the double-stranded RNA comprising the strands (antisense strands) RNA that consists of differently added double-stranded RNAs that suppress the expression of the MEK gene (hereinafter referred to as RNA with a structure like (A) and (B) is called MEKsiRNA), and (C) RNA consisting of sequence X And RNA consisting of the complementary sequence X ′ is an RNA having a hairpin structure that is connected by a spacer oligonucleotide and suppresses the expression of the MEK gene, (D) RNA consisting of the sequence X and RNA consisting of the complementary sequence X ′ , Linked by spacer oligonucleotides, with 1 to 6, preferably 2 to 4 nucleotides added to the 3 ′ end, It inhibits the expression of MEK genes a RNA having a hairpin structure RNA (hereinafter, (C) and RNA is referred to as a MEKshRNA such as (D)) and the like. The nucleotide base added to these RNAs may be one or more of guanine, adenine, cytosine, thymine and uracil, and may be RNA or DNA, but uridylic acid (U) and deoxythymidylic acid ( Any one or two of dT) are preferred. The spacer oligonucleotide is preferably RNA of 6 to 12 bases, and the sequence at the 5 'end is preferably 2 U. An example of the spacer oligonucleotide is RNA having the sequence UUCAAGAGA. Either of the two RNAs connected by the spacer oligonucleotide may be on the 5 'side. In either case, the nucleotide sequence of the nucleotide added adjacent to the 3 ′ end side of the complementary sequence X ′ may be the base sequence complementary to the sequence of the nucleotide adjacent to the sequence X in the mRNA. Well, this structure is more preferred. The sequence X may be any sequence as long as it is a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of MEK mRNA. The partial sequence of 21 bases beginning with AA is extracted from the base sequence of MEK cDNA. More preferable is a sequence designed by calculating the GC content of the extracted sequence and selecting a plurality of sequences having a GC content of 20 to 80%, preferably 30% to 70%, more preferably 40 to 60%.
　MEK遺伝子の発現を抑制するRNAは、配列XによってMEK遺伝子の発現の抑制の強さが異なり、抑制が弱い場合もあるので、配列Xとして複数の配列を設計して、それぞれの配列XをもとにしたRNAを調製し、RNAをMEK遺伝子が発現している細胞に導入してMEK遺伝子の発現を測定し、MEK遺伝子の発現をより強く抑制するRNAを選択することより、本発明のRNAを取得できる。
　MEK1遺伝子の発現を抑制するRNAとして、例えば表１に示すNo.1～No.40のRNAがあげられる。また、MEK2遺伝子の発現を抑制するRNAとして、例えば表２に示すNo.41～No.74のRNAがあげられる。 RNA that suppresses MEK gene expression has different strength of suppression of MEK gene expression depending on the sequence X, and there are cases where the suppression is weak. The RNA of the present invention is prepared by introducing RNA into a cell in which the MEK gene is expressed, measuring the expression of the MEK gene, and selecting an RNA that strongly suppresses the expression of the MEK gene. Can be obtained.
Examples of RNA that suppresses the expression of the MEK1 gene include No. 1 to No. 40 RNA shown in Table 1. Examples of RNA that suppresses the expression of the MEK2 gene include RNA No. 41 to No. 74 shown in Table 2.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　本発明で用いられるRNAを合成する方法としては、特に限定されず、公知の化学合成を用いる方法、あるいは、酵素的転写法等にて合成することができる。公知の化学合成を用いる方法として、ホスホロアミダイト法、ホスフォロチオエート法、ホスホトリエステル法等をあげることができ、例えば、ABI3900ハイスループット核酸合成機(アプライドバイオシステムズ社製)により合成することができる。酵素的転写法としては、目的の塩基配列を有したプラスミドまたはDNAを鋳型として典型的なファージRNAポリメラーゼ、例えば、T7ポリメラーゼ、T3ポリメラーゼ、SP6RNAポリメラーゼ等を用いて転写し、合成することができる。 The method for synthesizing the RNA used in the present invention is not particularly limited, and it can be synthesized by a method using a known chemical synthesis, an enzymatic transcription method or the like. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, etc., for example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do. As an enzymatic transcription method, transcription or synthesis can be performed using a plasmid or DNA having a target base sequence as a template and using a typical phage RNA polymerase, for example, T7 polymerase, T3 polymerase, SP6 RNA polymerase, or the like.
　なお、表1のMEKsiRNA　No.1は、例えば株式会社日本バイオサービス等に依頼して化学合成し、アニーリングさせることにより調製できる。表1および表２のMEKsiRNA　No.2～74はサイレンサーsiRNA作製キット(Silencer(登録商標)siRNA Construction Kit、アンビオン社製)を利用したインビトロ転写により調製できる。インビトロ転写の鋳型作製に用いるDNAは、例えば北海道システムサイエンス株式会社等に化学合成を依頼して得ることができる。 In addition, MEKsiRNA No. 1 in Table 1 can be prepared by, for example, requesting Nippon Bio Service Co., Ltd., chemical synthesis, and annealing. MEK siRNA Nos. 2 to 74 in Tables 1 and 2 can be prepared by in vitro transcription using a silencer siRNA preparation kit (Silencer (registered trademark) siRNA-Construction-Kit, manufactured by Ambion). The DNA used for template production for in vitro transcription can be obtained, for example, by requesting chemical synthesis from Hokkaido System Science Co., Ltd.
　本発明の組成物におけるリポソーム(以下リポソームA)としては、本発明で用いられるRNAを内部に封入したリポソームであれば特に限定されないが、例えばカチオン性脂質/RNAの複合体を疎水性の有機溶媒層に分散させ、ポリエチレングリコール化脂質と中性の脂質と水を加えて油中水型(W/O)エマルジョンを形成し、逆相蒸発法で処理して製造されたリポソーム(特許文献1参照)、RNAを、酸性の電解質水溶液に溶解し、脂質(エタノール中)を加え、エタノール濃度を下げてRNA内包リポソームを調製した後、試料のpHを上げて透析してリポソーム表面に付着した前記RNAを除去して製造されたリポソーム(特許文献2および非特許文献3参照)、リード粒子と前記RNAから構成される複合粒子および該複合粒子を封入した脂質二重膜から構成されたリポソーム(特許文献3および国際公開第2006/080118号パンフレット参照)等があげられ、リード粒子と前記RNAから構成される複合粒子および該複合粒子を封入した脂質二重膜から構成されたリポソームが好ましく、該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定濃度で該極性有機溶媒を含む液に分散可能であることがより好ましい。また、リポソームAとしては、好ましくはカチオン性物質を含むリード粒子と前記RNAを構成成分とする複合粒子、および該複合粒子を被覆する脂質二重膜から構成され、該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とするリポソームもあげられ、該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定濃度で該極性有機溶媒を含む液に分散可能であることがより好ましい。
　なお、本発明において、分散とは、溶解せずに分散することを意味する。 The liposome in the composition of the present invention (hereinafter referred to as liposome A) is not particularly limited as long as it is a liposome encapsulating RNA used in the present invention. For example, a cationic lipid / RNA complex is a hydrophobic organic solvent. Liposomes produced by dispersing in layers, adding polyethylene glycolated lipids, neutral lipids and water to form water-in-oil (W / O) emulsions and processing by reverse phase evaporation (see Patent Document 1) ), RNA is dissolved in an acidic electrolyte aqueous solution, lipid (in ethanol) is added, and RNA-encapsulated liposomes are prepared by lowering the ethanol concentration, and then the RNA attached to the liposome surface by dialysis by raising the pH of the sample (See Patent Document 2 and Non-Patent Document 3), composite particles composed of lead particles and RNA, and liposomes composed of lipid bilayers encapsulating the composite particles (special Reference 3 and International Publication No. 2006/080118 pamphlet), etc. are preferred, and a liposome composed of a composite particle composed of a lead particle and the RNA and a lipid bilayer membrane encapsulating the composite particle is preferred. More preferably, the components of the 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. preferable. The liposome A is preferably composed of a lead particle containing a cationic substance, a composite particle containing the RNA as a constituent component, and a lipid bilayer covering the composite particle. And liposomes comprising lipid derivatives, fatty acid derivatives or aliphatic hydrocarbon derivatives of water-soluble substances as a constituent, and the lipid bilayer constituent is soluble in a specific polar organic solvent. 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.
In the present invention, the term “dispersing” means dispersing without dissolving.
　本発明におけるリード粒子としては、例えば、脂質集合体、リポソーム(以下リポソームB)、エマルジョン粒子、高分子ミセル、金属コロイド等を構成成分とする微粒子があげられ、好ましくはリポソームBを構成成分とする微粒子があげられる。本発明におけるリード粒子は、脂質集合体、リポソームB、エマルジョン粒子、高分子ミセル、金属コロイド等を2つ以上組み合わせた複合体を構成成分としていてもよく、脂質集合体、リポソームB、エマルジョン粒子、高分子ミセル、金属コロイド等と他の化合物(例えば糖、脂質、無機化合物等)とを組み合わせた複合体を構成成分としていてもよい。 The lead particles in the present invention include, for example, fine particles comprising lipid aggregates, liposomes (hereinafter referred to as liposome B), emulsion particles, polymer micelles, metal colloids, etc., preferably liposome B as a constituent component. Fine particles. The lead particles in the present invention may be composed of a complex comprising a combination of two or more lipid aggregates, liposome B, emulsion particles, polymer micelles, metal colloids, etc., and lipid aggregates, liposome B, emulsion particles, A complex formed by combining polymer micelles, metal colloids, and the like with other compounds (for example, sugars, lipids, inorganic compounds, etc.) may be used as a constituent component.
　リード粒子の構成成分としての脂質集合体またはリポソームBは、例えば親水性と疎水性の両方の性質を兼ね備えた両親媒性を持つ、水中において脂質二重層構造をとる極性脂質等によって構成されるのが好ましい。該脂質としては、単純脂質、複合脂質または誘導脂質のいかなるものであってもよく、例えばリン脂質、グリセロ糖脂質、スフィンゴ糖脂質、スフィンゴイド、ステロール、カチオン性脂質等があげられるがこれらに限定されない。好ましくはリン脂質またはカチオン性脂質があげられる。 Lipid aggregates or liposomes B as constituents of lead particles are composed of, for example, polar lipids that have a lipid bilayer structure in water with amphiphilic properties that combine both hydrophilic and hydrophobic properties. Is preferred. The lipid may be any of simple lipids, complex lipids or derived lipids, such as phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids, sterols, and cationic lipids, but are not limited thereto. Not. Preferable examples include phospholipids and cationic lipids.
　上記リード粒子を構成する脂質におけるリン脂質としては、例えばホスファチジルコリン(具体的には大豆ホスファチジルコリン、卵黄ホスファチジルコリン(EPC)、ジステアロイルホスファチジルコリン、ジパルミトイルホスファチジルコリン、パルミトイルオレオイルホスファチジルコリン(POPC)、ジミリストイルホスファチジルコリン、ジオレオイルホスファチジルコリン等)、ホスファチジルエタノールアミン(具体的にはジステアロイルホスファチジルエタノールアミン(DSPE)、ジパルミトイルホスファチジルエタノールアミン(DPPE)、ジオレオイルホスファチジルエタノールアミン(DORE)、ジミリストイルホスホエタノールアミン(DMPE)、パルミトイルオレオイル-ホスファチジルエタノールアミン(POPE)、1 -ステアロイル- 2 -オレオイル-ホスファチジルエタノールアミン(SOPE)等)、グリセロリン脂質(具体的にはホスファチジルセリン、ホスファチジン酸、ホスファチジルグリセロール、ホスファチジルイノシトール、パルミトイルオレヨールホスファチジルグリセロール(POPG)、リゾホスファチジルコリン等)、スフィンゴリン脂質(具体的にはスフィンゴミエリン、セラミドホスホエタノールアミン、セラミドホスホグリセロール、セラミドホスホグリセロリン酸等)、グリセロホスホノ脂質、スフィンゴホスホノ脂質、天然レシチン(具体的には卵黄レシチン、大豆レシチン等)または水素添加リン脂質(具体的には水素添加大豆ホスファチジルコリン等)等の天然または合成のリン脂質があげられる。 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)) , Palmitoyl oleoyl-phosphatidylethanolamine (POPE), 1 -stearoyl- 2 -oleoyl-phosphine Glycidylphosphoamine (specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoylphosphatidylglycerol (POPG), lysophosphatidylcholine, etc.), sphingophospholipid (specifically Is sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate, etc.), glycerophosphonolipid, sphingophosphonolipid, natural lecithin (specifically egg yolk lecithin, soybean lecithin, etc.) or hydrogenated phospholipid ( Specific examples include natural or synthetic phospholipids such as hydrogenated soybean phosphatidylcholine.
　上記リード粒子を構成する脂質におけるグリセロ糖脂質としては、例えばスルホキシリボシルグリセリド、ジグリコシルジグリセリド、ジガラクトシルジグリセリド、ガラクトシルジグリセリドまたはグリコシルジグリセリド等があげられる。 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.
　上記リード粒子を構成する脂質におけるスフィンゴ糖脂質としては、例えばガラクトシルセレブロシド、ラクトシルセレブロシドまたはガングリオシド等があげられる。 Examples of the glycosphingolipid in the lipid constituting the lead particle include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.
　上記リード粒子を構成する脂質におけるスフィンゴイドとしては、例えばスフィンガン、イコサスフィンガン、スフィンゴシン等、またはそれらの誘導体等があげられる。誘導体としては、例えばスフィンガン、イコサスフィンガンまたはスフィンゴシン等の-NH2を-NHCO(CH2)xCH3(式中、xは0～18の整数を表し、中でも6、12または18が好ましい)に変換したもの等があげられる。 Examples of the sphingoid in the lipid constituting the lead particles include sphingan, icosasphingan, sphingosine, and derivatives thereof. As the derivative, for example, —NH 2 such as sphingan, icosasphingan or sphingosine —NHCO (CH 2 ) x CH 3 (wherein x represents an integer of 0 to 18, among which 6, 12 or 18 is preferable. ) And the like.
　上記リード粒子を構成する脂質におけるステロールとしては、例えばコレステロール、ジヒドロコレステロール、ラノステロール、β-シトステロール、カンペステロール、スチグマステロール、ブラシカステロール、エルゴカステロール、フコステロール等があげられる。 Examples of the sterol in the lipid constituting the lead particle include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brush casterol, ergocasterol, fucostosterol and the like.
　上記リード粒子を構成する脂質におけるカチオン性脂質としては、親水性と疎水性の両方の性質を兼ね備えた両親媒性を持つ、水中において脂質二重層構造をとる極性脂質のうち、親水性部に第一級アミン、第二級アミン、第三級アミン、第四級アンモニウム、窒素原子を含む複素環等を有する構造を持つものであり、例えば、N-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム(DOTAP)、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン(DODAP)、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム(DOTMA)、2,3-ジオレイルオキシ-N-［2-(スペルミンカルボキシアミド)エチル］-N,N-ジメチル-1-プロパナミニウムトリフルオロ酢酸(DOSPA)、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム(DMRIE)、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム(DORIE)、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムまたは3β-［N-(N',N'-ジメチルアミノエチル)カルバモイル]コレステロール(DC-Chol)等があげられる。 As 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-dimethyl -1-propanaminium trifluoroacetic acid (DOSPA), N- [1- (2,3-ditetradecyloxypropyl)]-N, N-dimethyl-N-hydroxy ester Ammonium bromide (DMRIE), N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl-N-hydroxyethylammonium bromide (DORIE), 1,2-dilinoleyloxy -N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride or 3β- [N- ( N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like.
　リポソームBにおいては、これら脂質は、1種単独でまたは2種以上を組み合わせて用いられ、好ましくは2種以上組み合わせて用いられる。2種以上組み合わせて用いる場合の組み合わせとしては、例えば水素添加大豆ホスファチジルコリン、ポリエチレングリコール化脂質(後記のポリエチレングリコール化脂質と同義)およびコレステロールから選ばれる2成分以上の組み合わせ、ジステアロイルホスファチジルコリン、ポリエチレングリコール化脂質およびコレステロールから選ばれる2成分以上の組み合わせ、EPCおよびDOTAPの組み合わせ、DOTAPおよびポリエチレングリコール化脂質の組み合わせ、EPC、DOTAPおよびポリエチレングリコール化脂質の組み合わせ、EPC、DOTAP、コレステロールおよびポリエチレングリコール化脂質の組み合わせ等があげられる。 In liposome B, these lipids are used singly or in combination of two or more, preferably in combination of two or more. As a combination when used in combination of two or more, for example, hydrogenated soybean phosphatidylcholine, polyethyleneglycolized lipid (synonymous with polyethyleneglycolized lipid described later) and cholesterol, a combination of two or more components, distearoylphosphatidylcholine, polyethyleneglycolated Combination of two or more components selected from lipid and cholesterol, combination of EPC and DOTAP, combination of DOTAP and polyethylene glycolated lipid, combination of EPC, DOTAP and polyethylene glycolated lipid, combination of EPC, DOTAP, cholesterol and polyethylene glycolated lipid Etc.
　また、リポソームBは、必要に応じて、例えばコレステロール等のステロール等の膜安定化剤、例えばトコフェロール等の抗酸化剤等の安定化剤を含有していてもよい。これら安定化剤は1種単独でまたは2種以上組み合わせて使用し得る。 Liposomes B may contain a film stabilizer such as sterol such as cholesterol, for example, and a stabilizer such as antioxidant such as tocopherol, if necessary. These stabilizers may be used alone or in combination of two or more.
　脂質集合体としては、例えば球状ミセル、球状逆ミセル、ソーセージ状ミセル、ソーセージ状逆ミセル、板状ミセル、板状逆ミセル、ヘキサゴナルI、ヘキサゴナルIIまたは脂質2分子以上からなる会合体等があげられる。 Examples of 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 or aggregates composed of two or more lipid molecules. .
　エマルジョン粒子としては、例えば脂肪乳剤、非イオン性界面活性剤と大豆油等の油からなるエマルジョン、リピッドエマルジョン、リピッドナノスフェアー等の水中油型(O/W)エマルジョンや水中油中水型(W/O/W)エマルジョン粒子等があげられる。 Examples of emulsion particles include fat emulsions, emulsions composed of nonionic surfactants and oils such as soybean oil, oil-in-water (O / W) emulsions such as lipid emulsions and lipid nanospheres, and water-in-oil-in-water (W / O / W) emulsion particles and the like.
　上記リード粒子を構成するエマルジョン粒子における非イオン性界面活性剤としては、例えばモノオレイン酸ポリオキシエチレンソルビタン(具体的にはポリソルベート80等)、ポリオキシエチレンポリオキシプロピレングリコール(具体的にはプルロニックF68等)、ソルビタン脂肪酸エステル(具体的にはソルビタンモノラウレート、ソルビタンモノオレエート等)、ポリオキシエチレン誘導体(具体的にはポリオキシエチレン硬化ヒマシ油60、ポリオキシエチレンラウリルアルコール等)またはグリセリン脂肪酸エステル等があげられる。 Examples of the nonionic surfactant in the emulsion particles constituting the lead particles include polyoxyethylene sorbitan monooleate (specifically polysorbate 80), polyoxyethylene polyoxypropylene glycol (specifically Pluronic F68). ), Sorbitan fatty acid esters (specifically sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.) or glycerin fatty acid Examples include esters.
　高分子ミセルとしては、例えばアルブミン、デキストラン、ポリフェクト(polyfect)、キトサン、デキストラン硫酸またはDNA等の天然高分子、例えばポリ-L-リジン、ポリエチレンイミン、ポリアスパラギン酸、スチレンマレイン酸共重合体、イソプロピルアクリルアミド-アクリルピロリドン共重合体、ポリエチレングリコール修飾デンドリマー、ポリ乳酸、ポリ乳酸ポリグリコール酸またはポリエチレングリコール化ポリ乳酸等の高分子あるいはそれらの塩の１以上からなるミセルがあげられる。 Examples of the polymer micelle include natural polymers such as albumin, dextran, polyfect, chitosan, dextran sulfate or DNA, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropyl Examples include micelles composed of one or more polymers such as acrylamide-acrylpyrrolidone copolymer, polyethylene glycol-modified dendrimer, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or salts thereof.
　ここで、高分子における塩は、例えば金属塩、アンモニウム塩、酸付加塩、有機アミン付加塩、アミノ酸付加塩等を包含する。金属塩としては、例えばリチウム塩、ナトリウム塩、カリウム塩等のアルカリ金属塩、マグネシウム塩、カルシウム塩等のアルカリ土類金属塩、アルミニウム塩または亜鉛塩等があげられる。アンモニウム塩としては、例えばアンモニウムまたはテトラメチルアンモニウム等の塩があげられる。酸付加塩としては、例えば塩酸塩、硫酸塩、硝酸塩またはリン酸塩等の無機酸塩、および酢酸塩、マレイン酸塩、フマル酸塩またはクエン酸塩等の有機酸塩があげられる。有機アミン付加塩としては、例えばモルホリンまたはピペリジン等の付加塩があげられる。アミノ酸付加塩としては、例えばグリシン、フェニルアラニン、アスパラギン酸、グルタミン酸またはリジン等の付加塩があげられる。 Here, 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. Examples of 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. Examples of the ammonium salt include salts such as ammonium and tetramethylammonium. Examples of 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. Examples of organic amine addition salts include addition salts such as morpholine and piperidine. Examples of amino acid addition salts include addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid or lysine.
　金属コロイドとしては、例えば金、銀、白金、銅、ロジウム、シリカ、カルシウム、アルミニウム、鉄、インジウム、カドミウム、バリウムまたは鉛等を含む金属コロイドがあげられる。 Examples of the metal colloid include metal colloids containing gold, silver, platinum, copper, rhodium, silica, calcium, aluminum, iron, indium, cadmium, barium or lead.
　また、本発明におけるリード粒子は、糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂質誘導体もしくは脂肪酸誘導体または界面活性剤等を含有することが好ましく、糖脂質、または水溶性高分子の脂質誘導体もしくは脂肪酸誘導体を含有することがより好ましく、水溶性高分子の脂質誘導体または脂肪酸誘導体を含有することがさらに好ましい。糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂質誘導体もしくは脂肪酸誘導体または界面活性剤は、分子の一部がリード粒子の他の構成成分と例えば疎水性親和力、静電的相互作用等で結合する性質を有し、他の部分がリード粒子の製造時の溶媒と、例えば親水性親和力、静電的相互作用等で結合する性質を有する、2面性をもつ物質であるのが好ましい。糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂質誘導体もしくは脂肪酸誘導体または界面活性剤は、リード粒子の構成成分として含有されてもよく、リード粒子の構成成分に加えて用いてもよい。 In addition, the lead particles in the present invention preferably contain a lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers. It is more preferable to contain a lipid derivative or fatty acid derivative of a water-soluble polymer, and it is further preferable to contain a lipid derivative or fatty acid derivative of a water-soluble polymer. 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 with the solvent at the time of lead particle production, for example, hydrophilic affinity, electrostatic interaction, etc. Preferably there is. The lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugar, peptide, nucleic acid and water-soluble polymer may be contained as a component of the lead particle, and in addition to the component of the lead particle It may be used.
　糖、ペプチドおよび核酸から選ばれる１つ以上の物質の脂質誘導体または脂肪酸誘導体としては、例えばショ糖、ソルビトール、乳糖等の糖、例えばカゼイン由来ペプチド、卵白由来ペプチド、大豆由来ペプチド、グルタチオン等のペプチド、例えばDNA、RNA、プラスミド、siRNA、ODN等の核酸と、例えば前記リード粒子の定義の中であげた脂質、または例えばステアリン酸、パルミチン酸、ミリスチン酸、ラウリン酸等の脂肪酸とが結合してなるもの等があげられる。 Examples of lipid derivatives or fatty acid derivatives of one or more substances 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, and glutathione peptides. For example, a nucleic acid such as DNA, RNA, plasmid, siRNA, or ODN and a lipid listed in the definition of the lead particle or a fatty acid such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. And the like.
　また、糖の脂質誘導体または脂肪酸誘導体には、例えば前記リード粒子の定義の中であげたグリセロ糖脂質またはスフィンゴ糖脂質等も含まれる。 Further, the sugar lipid derivatives or fatty acid derivatives include, for example, glyceroglycolipids or sphingoglycolipids mentioned in the definition of the lead particles.
　水溶性高分子の脂質誘導体または脂肪酸誘導体としては、例えばポリエチレングリコール、ポリグリセリン、ポリエチレンイミン、ポリビニルアルコール、ポリアクリル酸、ポリアクリルアミド、オリゴ糖、デキストリン、水溶性セルロース、デキストラン、コンドロイチン硫酸、ポリグリセリン、キトサン、ポリビニルピロリドン、ポリアスパラギン酸アミド、ポリ-L-リジン、マンナン、プルラン、オリゴグリセロール等またはそれらの誘導体と、例えば前記リード粒子の定義の中であげた脂質、または例えばステアリン酸、パルミチン酸、ミリスチン酸もしくはラウリン酸等の脂肪酸とが結合してなるもの等があげられ、より好ましくは、ポリエチレングリコール誘導体、ポリグリセリン誘導体等の脂質誘導体または脂肪酸誘導体があげられ、さらに好ましくは、ポリエチレングリコール誘導体の脂質誘導体または脂肪酸誘導体があげられる。 Examples of the water-soluble polymer lipid derivative or fatty acid derivative include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like or their derivatives and the lipids mentioned in the definition of lead particles, for example, stearic acid, palmitic acid, Examples include those formed by bonding with fatty acids such as myristic acid or lauric acid, and more preferred are lipid derivatives such as polyethylene glycol derivatives and polyglycerin derivatives, or fatty acid derivatives. Is, more preferably, a lipid derivative or a fatty acid derivative of a polyethylene glycol derivative.
　ポリエチレングリコール誘導体の脂質誘導体または脂肪酸誘導体としては、例えばポリエチレングリコール化脂質(具体的にはポリエチレングリコール-ホスファチジルエタノールアミン(より具体的には1,2-ジステアロイル-sn-グリセロ-3-ホスホエタノールアミン-N-［メトキシ(ポリエチレングリコール)-2000］(PEG-DSPE)等)、ポリオキシエチレン硬化ヒマシ油60、クレモフォアイーエル(CREMOPHOR　EL)等)、ポリエチレングリコールソルビタン脂肪酸エステル類(具体的にはモノオレイン酸ポリオキシエチレンソルビタン等)またはポリエチレングリコール脂肪酸エステル類等があげられ、より好ましくは、ポリエチレングリコール化脂質があげられる。 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.
　ポリグリセリン誘導体の脂質誘導体または脂肪酸誘導体としては、例えばポリグリセリン化脂質(具体的にはポリグリセリン-ホスファチジルエタノールアミン等)またはポリグリセリン脂肪酸エステル類等があげられ、より好ましくは、ポリグリセリン化脂質があげられる。 Examples of 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.
　界面活性剤としては、例えばモノオレイン酸ポリオキシエチレンソルビタン(具体的にはポリソルベート80等)、ポリオキシエチレンポリオキシプロピレングリコール(具体的にはプルロニックF68等)、ソルビタン脂肪酸エステル(具体的にはソルビタンモノラウレート、ソルビタンモノオレエート等)、ポリオキシエチレン誘導体(具体的にはポリオキシエチレン硬化ヒマシ油60、ポリオキシエチレンラウリルアルコール等)、グリセリン脂肪酸エステルまたはポリエチレングリコールアルキルエーテル等があげられ、好ましくは、ポリオキシエチレンポリオキシプロピレングリコール、グリセリン脂肪酸エステルまたはポリエチレングリコールアルキルエーテル等があげられる。 Examples of the surfactant 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.
　上記したリード粒子は、正電荷をもつものが好ましい。ここで述べる、正電荷とは、本発明で用いられるRNA内の電荷、分子内分極等に対して静電的引力を生じる電荷、表面分極等を包含する。リード粒子が正電荷をもつには、リード粒子は、カチオン性物質を含有するのが好ましく、リード粒子は、カチオン性脂質を含有するのがより好ましい。 The above-described lead particles preferably have a positive charge. The positive charge described here includes a charge in RNA used in the present invention, a charge that generates an electrostatic attraction with respect to intramolecular polarization, a surface polarization, and the like. In order for the lead particles to have a positive charge, the lead particles preferably contain a cationic substance, and the lead particles more preferably contain a cationic lipid.
　リード粒子に含有されるカチオン性物質は、カチオン性を呈する物質であるが、カチオン性の基とアニオン性の基の両方をもつ両性の物質であっても、pHや、他の物質との結合等により相対的な陰性度が変化するので、その時々に応じてカチオン性物質に分類され得るものも含まれる。これらカチオン性物質は、リード粒子の構成成分として含有されてもよく、リード粒子の構成成分に加えて用いてもよい。 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 a constituent component of lead particles, or may be used in addition to the constituent components of lead particles.
　カチオン性物質としては、例えば前記のリード粒子の定義で例示したもののうちのカチオン性物質［具体的には、カチオン性脂質(前記と同義)、カチオン性高分子等］、等電点以下の値のpHで複合体の形成を行える蛋白質またはペプチド等があげられ、好ましくはカチオン性脂質があげられる。 As a cationic substance, for example, a cationic substance [specifically, a cationic lipid (as defined above), a cationic polymer, etc.] among those exemplified in the definition of the lead particle, a value below the isoelectric point Examples thereof include proteins or peptides capable of forming a complex at a pH of, preferably cationic lipids.
　カチオン性高分子としては、例えばポリ-L-リジン、ポリエチレンイミン、ポリフェクト(polyfect)またはキトサン等があげられる。 Examples of the cationic polymer include poly-L-lysine, polyethyleneimine, polyfect, and chitosan.
　等電点以下の値のpHで複合体の形成を行える蛋白質またはペプチドとしては、その物質の等電点以下の値のpHで、複合体の形成を行える蛋白質またはペプチドであれば特に限定されない。該蛋白質またはペプチドとしては、例えば、アルブミン、オロソムコイド、グロブリン、フィブリノーゲン、ペプシンまたはリボヌクレアーゼT1等があげられる。 The protein or peptide capable of forming a complex at a pH below the isoelectric point is not particularly limited as long as it is a protein or peptide capable of forming a complex 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.
　本発明におけるリード粒子は、公知の製造方法またはそれに準じる方法で製造することができ、いかなる製造方法で製造されたものであってよい。例えば、リード粒子の1つであるリポソームBを構成成分とするリード粒子の製造には、公知のリポソームの調製方法が適用できる。公知のリポソームの調製方法としては、例えばバンガム(Bangham)らのリポソーム調製法［“ジャーナル・オブ・モレキュラー・バイオロジー(J.Mol.Biol.)”,1965年,第13巻,p.238-252参照］、エタノール注入法［“ジャーナル・オブ・セル・バイオロジー(J.Cell Biol.)”,1975年,第66巻,p.621-634参照］、フレンチプレス法［“エフイービーエス・レターズ(FEBS Lett.)”,1979年,第99巻,p.210-214参照］、凍結融解法［“アーカイブス・オブ・バイオケミストリー・アンド・バイオフィジックス(Arch. Biochem. Biophys.)”,1981年,第212巻,p.186-194参照］、逆相蒸発法［“プロシーディングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンス・ユナイテッド・ステイツ・オブ・アメリカ(Proc. Natl. Acad. Sci. USA)”,1978年,第75巻, p.4194-4198参照］またはpH勾配法(例えば特許第2572554号公報、特許第2659136号公報等参照)等があげられる。リポソームBの製造の際にリポソームBを分散させる溶液としては、例えば水、酸、アルカリ、種々の緩衝液、生理的食塩液またはアミノ酸輸液等を用いることができる。また、リポソームBの製造の際には、例えばクエン酸、アスコルビン酸、システインまたはエチレンジアミン四酢酸(EDTA)等の抗酸化剤、例えばグリセリン、ブドウ糖または塩化ナトリウム等の等張化剤等の添加も可能である。また、脂質等を例えばエタノール等の有機溶媒に溶解し、溶媒を留去した後、生理食塩水等を添加、振とう撹拌し、リポソームを形成させることによってもリポソームBを製造することができる。 The lead particles in the present invention can be produced by a known production method or a method according thereto, and may be produced by any production method. For example, a known liposome preparation method can be applied to the production of lead particles containing liposome B, which is one of the lead particles, as a constituent component. 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. Biophys. ””, 1981 Year 212, p. 186-194], reversed-phase evaporation [“Proceedings of the National Academy of Sciences United States of America (Proc. Natl. Acad. Sci. USA) ”, 1978, Vol. 75, p.4194-4198] or pH gradient method (for example, Patent No. 2572). No. 554, Japanese Patent No. 2659136, etc.). As a solution for dispersing liposome B in the production of liposome B, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, or the like can be used. In the production of 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.
　また、例えばカチオン性物質、高分子、ポリオキシエチレン誘導体等によるリポソームB等のリード粒子の表面改質も任意に行うことができる［ラジック(D.D.Lasic)、マーティン(F.Martin)編,“ステルス・リポソームズ(Stealth Liposomes)”(米国),シーアールシー・プレス・インク(CRC Press Inc),1995年,p.93-102参照］。表面改質に使用し得る高分子としては、例えばデキストラン、プルラン、マンナン、アミロペクチンまたはヒドロキシエチルデンプン等があげられる。ポリオキシエチレン誘導体としては、例えばポリソルベート80、プルロニックF68、ポリオキシエチレン硬化ヒマシ油60、ポリオキシエチレンラウリルアルコールまたはPEG-DSPE等があげられる。リポソームB等のリード粒子の表面改質は、リード粒子に糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂質誘導体もしくは脂肪酸誘導体または界面活性剤を含有させる方法の1つである。 Also, for example, 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]. Examples of the polymer that can be used for the surface modification include dextran, pullulan, mannan, amylopectin, and hydroxyethyl starch. Examples of the polyoxyethylene derivative include polysorbate 80, Pluronic F68, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, and PEG-DSPE. Surface modification of lead particles such as liposome B is 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.
　リポソームBの平均粒子径は、所望により自由に選択できるが、下記の粒子径とするのが好ましい。リポソームBの平均粒子径を調節する方法としては、例えばエクストルージョン法、大きな多重膜リポソーム(MLV)を機械的に粉砕(具体的にはマントンゴウリン、マイクロフルイダイザー等を使用)する方法［ミュラー(R.H.Muller)、ベニタ(S.Benita)、ボーム(B.Bohm)編著,“エマルジョン・アンド・ナノサスペンジョンズ・フォー・ザ・フォーミュレーション・オブ・ポアリー・ソラブル・ドラッグズ(Emulsion and Nanosuspensions for the Formulation of Poorly Soluble Drugs)”,ドイツ,サイエンティフィック・パブリッシャーズ・スチュットガルト(Scientific Publishers Stuttgart),1998年,p.267-294参照］等があげられる。 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].
　また、リード粒子を構成する例えば脂質集合体、リポソームB、エマルジョン粒子、高分子ミセル、金属コロイド等から選ばれる2つ以上を組み合わせた複合体の製造方法は、例えば水中で例えば脂質、高分子等を混合するだけでもよく、所望によりさらに整粒工程や無菌化工程等を加えることもできる。また、前記複合体の製造は例えばアセトンまたはエーテル等種々の溶媒中で行うことも可能である。 In addition, a method for producing a composite comprising a combination of two or more selected from lipid aggregates, liposome B, emulsion particles, polymer micelles, metal colloids and the like constituting the lead particles, for example, lipids, polymers, etc. in water May be mixed, and a granulation step, a sterilization step, and the like may be added if desired. In addition, the complex can be produced in various solvents such as acetone or ether.
　本発明におけるリード粒子の大きさは、平均粒子径が約10nm～1000nmであるのが好ましく、約30nm～300nmであるのがより好ましく、約50nm～200nmであるのがさらに好ましい。 The average size of the lead particles in the present invention is preferably about 10 nm to 1000 nm, more preferably about 30 nm to 300 nm, and further preferably about 50 nm to 200 nm.
　本発明におけるリード粒子とRNAを含む複合粒子を被覆する脂質二重膜の構成成分としては、例えば前記リード粒子の定義の中であげた脂質または界面活性剤等があげられ、好ましくは、脂質のうちの中性脂質があげられる。ここで、中性脂質とは、脂質のうちの、前記リード粒子が正電荷をもつ場合におけるカチオン性物質の中であげたカチオン性脂質および後記の付着競合剤の中であげたアニオン性脂質を除いたもののことであり、中性脂質としてより好ましくは、リン脂質、グリセロ糖脂質またはスフィンゴ糖脂質等があげられる。より好ましくはリン脂質があげられ、さらに好ましくはEPCがあげられる。これら脂質は1種単独でまたは2種以上を組み合わせて用いることができる。 Examples of the component of the lipid bilayer membrane covering the composite particles containing lead particles and RNA in the present invention include the lipids and surfactants mentioned in the definition of the lead particles. Our neutral lipids. Here, the neutral lipid refers to the cationic lipids mentioned in the cationic substance and the anionic lipids mentioned in the adhesion competitor described later when the lead particles have a positive charge. More preferably, neutral lipids include phospholipids, glyceroglycolipids or sphingoglycolipids. More preferred are phospholipids, and more preferred is EPC. These lipids can be used alone or in combination of two or more.
　複合粒子を被覆する脂質二重膜の構成成分は、特定の極性有機溶媒に可溶であることが好ましく、特定の濃度で該極性有機溶媒を含む液中には、分散可能であることが好ましい。特定の濃度で該極性溶媒を含む液中の該極性溶媒の濃度は、該脂質二重膜の構成成分が分散可能で、該複合粒子も分散可能な濃度が好ましい。該極性有機溶媒としては、例えばメタノール、エタノール、n-プロパノール、2-プロパノール、n-ブタノール、2-ブタノール、tert-ブタノール等のアルコール、グリセリン、エチレングリコール、プロピレングリコール等のグリコールまたはポリエチレングリコール等のポリアルキレングリコール等があげられ、中でも、アルコールが好ましく、エタノールがより好ましい。
　本発明における極性有機溶媒を含む液中の、極性有機溶媒以外の溶媒としては、例えば、水、液体二酸化炭素、液体炭化水素、ハロゲン化炭素またはハロゲン化炭化水素等があげられ、好ましくは水があげられる。また、イオンまたは緩衝成分等を含んでいてもよい。溶媒は1種または2種以上を用いることができるが、2種以上用いる場合は、相溶する組み合わせが好ましい。 The components of the lipid bilayer membrane covering the composite particles are preferably soluble in a specific 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. Examples of 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 give. Moreover, an ion or a buffer component etc. may be included. One or more solvents can be used, but when two or more solvents are used, a compatible combination is preferred.
　複合粒子を被覆する脂質二重膜は、水溶性物質の脂質誘導体、脂肪酸誘導体もしくは脂肪族炭化水素誘導体、ポリオキシエチレンポリオキシプロピレングリコール、グリセリン脂肪酸エステルまたはポリエチレングリコールアルキルエーテルを含有することが好ましく、水溶性物質の脂質誘導体、脂肪酸誘導体もしくは脂肪族炭化水素誘導体を含有することがより好ましい。水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体としては、例えば前記の糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂質誘導体もしくは脂肪酸誘導体、または糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂肪族炭化水素誘導体があげられ、好ましくは前記水溶性高分子の脂質誘導体または脂肪酸誘導体があげられ、より好ましくは前記ポリエチレングリコール化脂質があげられ、さらに好ましくはポリエチレングリコール-ホスファチジルエタノールアミンがあげられる。なお、本発明における水溶性物質の脂肪族炭化水素誘導体としては、水溶性物質と、例えば長鎖脂肪族アルコール、ポリオキシプロピレンアルキルまたはグリセリン脂肪酸エステルのアルコール性残基等とが結合してなるものもあげられる。 The lipid bilayer coating the composite particles preferably contains a lipid derivative of a water-soluble substance, a fatty acid derivative or an aliphatic hydrocarbon derivative, polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether, More preferably, it contains a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance. Examples of 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. An aliphatic hydrocarbon derivative of one or more substances selected from nucleic acids and water-soluble polymers, preferably lipid derivatives or fatty acid derivatives of the water-soluble polymers, more preferably the polyethylene glycolated lipids. More preferred is polyethylene glycol-phosphatidylethanolamine. In addition, as the 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 also be raised.
　糖、ペプチドまたは核酸の脂肪族炭化水素誘導体としては、例えばショ糖、ソルビトールまたは乳糖等の糖、例えばカゼイン由来ペプチド、卵白由来ペプチド、大豆由来ペプチドまたはグルタチオン等のペプチド、あるいは例えばDNA、RNA、プラスミド、siRNAまたはODN等の核酸の脂肪族炭化水素誘導体があげられる。 Examples of the 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.
　水溶性高分子の脂肪族炭化水素誘導体としては、例えばポリエチレングリコール、ポリグリセリン、ポリエチレンイミン、ポリビニルアルコール、ポリアクリル酸、ポリアクリルアミド、オリゴ糖、デキストリン、水溶性セルロース、デキストラン、コンドロイチン硫酸、キトサン、ポリビニルピロリドン、ポリアスパラギン酸アミド、ポリ-L-リジン、マンナン、プルラン、オリゴグリセロール等またはそれらの誘導体の脂肪族炭化水素誘導体があげられ、より好ましくは、ポリエチレングリコール誘導体またはポリグリセリン誘導体等の脂肪族炭化水素誘導体があげられ、さらに好ましくは、ポリエチレングリコール誘導体の脂肪族炭化水素誘導体があげられる。 Examples of the aliphatic hydrocarbon derivatives of water-soluble polymers include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, chitosan, polyvinyl Examples thereof include aliphatic hydrocarbon derivatives of pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like or derivatives thereof, more preferably aliphatic carbonization such as polyethylene glycol derivatives or polyglycerin derivatives. Examples thereof include hydrogen derivatives, and more preferable examples include aliphatic hydrocarbon derivatives of polyethylene glycol derivatives.
　リード粒子がリポソームBを構成成分とする微粒子である場合、リポソームBと本発明で用いられるRNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるものがリポソームAとなり、その構成から狭義のリポソームと分類され、リード粒子がリポソームBを構成成分とする微粒子以外である場合でも、脂質二重膜で被覆されているので、広義のリポソームと分類される。本発明において、リード粒子がリポソームBを構成成分とする微粒子であることがより好ましい。 When the lead particle is a fine particle comprising liposome B as a constituent, the liposome A is composed of a composite particle comprising liposome B and RNA used in the present invention and a lipid bilayer coating the composite particle. Thus, it is classified as a liposome in a narrow sense based on its configuration, and even when the lead particle is other than a fine particle containing liposome B as a constituent component, it is classified as a liposome in a broad sense because it is covered with a lipid bilayer membrane. In the present invention, the lead particles are more preferably fine particles containing liposome B as a constituent component.
　本発明におけるリード粒子と本発明で用いられるRNAを構成成分とする複合粒子は、該リード粒子を製造後または該リード粒子の製造と同時に、本発明で用いられるRNAをリード粒子に付着または封入して複合粒子を製造でき、さらに該複合粒子の製造後または複合粒子の製造と同時に、脂質二重膜で該複合粒子を被覆することによりリポソームAを製造することができる。リポソームAは、例えば、特許文献1、2、3、非特許文献3等に記載の公知の製造方法またはそれに準じる方法で製造するか、例えばリード粒子に本発明で用いられるRNAを付着または封入して複合粒子を製造後、該複合粒子および被覆層成分を、該被覆層成分が可溶な極性有機溶媒を含み、該複合粒子が溶解せず、該被覆層成分が分散状態で存在することが可能な濃度の液中に分散させる工程および該複合粒子を該被覆層成分で被覆する工程を含む製造方法で製造することができる。 The composite particles comprising the lead particles in the present invention and the RNA used in the present invention are prepared by attaching or enclosing the RNA used in the present invention to the lead particles after the lead particles are produced or simultaneously with the production of the lead particles. Further, 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 produced by, for example, a known production method described in Patent Documents 1, 2, 3 and Non-Patent Document 3 or a method similar thereto, or, for example, RNA used in the present invention is attached to or encapsulated in lead particles. After the composite particles are manufactured, the composite particles and the coating layer component contain a polar organic solvent in which the coating layer component is soluble, the composite particles do not dissolve, and the coating layer component exists in a dispersed state. It can be produced by a production method including a step of dispersing in a liquid having a possible concentration and a step of coating the composite particles with the coating layer component.
　本発明の組成物におけるリポソームAの好ましい製造方法としては、以下のリード粒子と本発明で用いられるRNAを構成成分とする複合粒子を製造する工程(工程1)および該複合粒子を脂質二重膜で被覆する工程(工程2または工程3)を含む製造方法があげられる。 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 RNA used in the present invention (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).
　工程1)　リード粒子と本発明で用いられるRNAを構成成分とする複合粒子を製造する工程
　リード粒子を、例えば水等の溶媒中に分散させ、リード粒子が分散した液中に、本発明で用いられるRNAを分散または溶解して含有させて混合し、リード粒子に本発明で用いられるRNAを付着させることが好ましい。工程1において、リード粒子の凝集を抑制するために、リード粒子は凝集抑制物質を含有するリード粒子であることが好ましい。凝集抑制物質としては、前記糖、ペプチド、核酸および水溶性高分子から選ばれる1つ以上の物質の脂質誘導体もしくは脂肪酸誘導体または界面活性剤が好ましくあげられる。また、リード粒子が、正電荷をもつものである場合、リード粒子が分散した液中で、本発明で用いられるRNAと付着競合剤を共存させ、付着競合剤を該RNAとともにリード粒子に付着させてもよく、さらにリード粒子が凝集抑制物質を含有するリード粒子である場合にも、リード粒子の凝集をより抑制させるために付着競合剤を用いてもよい。リード粒子と本発明で用いられるRNAのいずれかの組み合わせとしては、複合粒子が極性有機溶媒を含有する液に分散可能となる組み合わせを選択することが好ましく、極性有機溶媒に対しての複合粒子の溶解度が、工程2または3で用いる脂質二重膜の構成成分のそれよりも低いことがより好ましく、また、該極性有機溶媒を含む液中に、該脂質二重膜の構成成分が分散可能で、該複合粒子も分散可能な濃度で該極性有機溶媒を含む液が存在する組み合わせを選択することがより好ましい。 Step 1) Step of producing composite particles comprising lead particles and RNA used in the present invention as constituent components Lead particles are dispersed in a solvent such as water, and used in the present invention in a liquid in which the lead particles are dispersed. It is preferable to disperse or dissolve and mix the RNA to be used, and to attach the RNA used in the present invention to the lead particles. In step 1, in order to suppress the aggregation of 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. In addition, when the lead particle has a positive charge, the RNA used in the present invention and the adhesion competitor are coexisted in the liquid in which the lead particle is dispersed, and the adhesion competitor is attached to the lead particle together with the RNA. In addition, when the lead particles are lead particles containing an aggregation inhibitor, an adhesion competitor may be used to further suppress the aggregation of the lead particles. As a combination of any of the lead particles and RNA used in the present invention, it is preferable to select a combination in which the composite particles can be dispersed in a liquid containing a polar organic solvent. More preferably, the solubility 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 a concentration capable of dispersing the composite particles.
　付着競合剤としては、例えばアニオン性物質等があげられる。該アニオン性物質は、分子内の電荷、分子内分極等による静電的引力により、リード粒子の構成成分に静電的に付着する物質を包含する。付着競合剤としてのアニオン性物質は、アニオン性を呈する物質であるが、アニオン性の基とカチオン性の基の両方をもつ両性の物質であっても、pHや他の物質との結合等により相対的な陰性度が変化するので、その時々に応じてアニオン性物質に分類され得る。 Examples of adhesion competitors include anionic substances. The anionic substance includes a substance that adheres electrostatically to the constituent components of the lead particles by electrostatic attraction due to intramolecular charge, intramolecular polarization, and the like. An anionic substance as an adhesion competing agent is an anionic substance, but even an amphoteric substance having both an anionic group and a cationic group is affected by pH, binding to other substances, etc. Since the relative negative degree changes, it can be classified into anionic substances depending on the occasion.
　アニオン性物質としてはアニオン性脂質、アニオン性界面活性剤、アニオン性高分子等または等電点以上の値のpHで複合体の形成を行える蛋白質、ペプチドもしくは核酸等があげられ、好ましくはデキストラン硫酸、デキストラン硫酸ナトリウム、コンドロイチン硫酸、コンドロイチン硫酸ナトリウム、ヒアルロン酸、コンドロイチン、デルマタン硫酸、ヘパラン硫酸、ヘパリン、ケラタン硫酸またはデキストランフルオレセインアニオニック等があげられる。これらアニオン性物質は1種単独でまたは2種以上を組み合わせて用いることができる。 Examples of the anionic substance include anionic lipids, anionic surfactants, anionic polymers, etc., and proteins, peptides, or nucleic acids that can form a complex at a pH higher than the isoelectric point, and preferably dextran sulfate. Dextran sodium sulfate, chondroitin sulfate, chondroitin sulfate sodium, hyaluronic acid, chondroitin, dermatan sulfate, heparan sulfate, heparin, keratan sulfate or dextran fluorescein anionic. These anionic substances can be used alone or in combination of two or more.
　アニオン性脂質としては、例えばホスファチジルセリン、ホスファチジルグリセロール、ホスファチジルイノシトールまたはホスファチジン酸等があげられる。
　アニオン性界面活性剤としては、例えばアシルサルコシン、アルキル硫酸ナトリウム、アルキルベンゼンスルホン酸塩、炭素数7～22の脂肪酸ナトリウム等があげられる。具体的にはドデシル硫酸ナトリウム、ラウリル硫酸ナトリウム、コール酸ナトリウム、デオキシコール酸ナトリウムまたはタウロデオキシコール酸ナトリウム等があげられる。 Examples of the anionic lipid include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, and the like.
Examples of the anionic surfactant 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.
　アニオン性高分子としては、例えばポリアスパラギン酸、スチレンマレイン酸共重合体、イソプロピルアクリルアミド-アクリルピロリドン共重合体、ポリエチレングリコール修飾デンドリマー、ポリ乳酸、ポリ乳酸ポリグリコール酸、ポリエチレングリコール化ポリ乳酸、デキストラン硫酸、デキストラン硫酸ナトリウム、コンドロイチン硫酸、コンドロイチン硫酸ナトリウム、ヒアルロン酸、コンドロイチン、デルマタン硫酸、ヘパラン硫酸、ヘパリン、ケラタン硫酸またはデキストランフルオレセインアニオニック等があげられる。
　等電点以上の値のpHで複合体の形成を行える蛋白質またはペプチドとしては、その物質の等電点以上の値のpHで、複合体の形成を行える蛋白質またはペプチドであれば特に限定されない。例えば、アルブミン、オロソムコイド、グロブリン、フィブリノーゲン、ヒストン、プロタミン、リボヌクレアーゼまたはリゾチーム等があげられる。 Examples of the anionic polymer include polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acrylpyrrolidone 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 capable of forming a complex 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 capable of forming a complex 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.
　アニオン性物質としての核酸としては、例えばDNA、RNA、プラスミド、siRNAまたはODN等があげられ、生理活性を示さないものであれば、どのような長さ、配列のものであってもよい。 Examples of the nucleic acid as the anionic substance 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.
　付着競合剤は、リード粒子の構成成分に静電的に付着することが好ましく、リード粒子の構成成分に付着してもリード粒子の構成成分を凝集させるような架橋を形成しない大きさの物質であるか、分子内に付着する部分と、付着に反発してリード粒子の凝集を抑制する部分をもつ物質であることが好ましい。 The adhesion competing agent preferably adheres electrostatically to the constituents of the lead particles, and is a substance having a size that does not form a crosslink that causes the constituents of the lead particles to aggregate even if attached to the constituents of the lead particles. It is preferable that the substance has a part that adheres in the molecule and a part that repels the adhesion and suppresses the aggregation of the lead particles.
　工程1は、より具体的には、例えば凝集抑制物質を含有するリード粒子が分散した液を製造する操作および該リード粒子が分散した液中に、本発明で用いられるRNAを分散または溶解して含有させる操作(例えば該リード粒子が分散した液中に、本発明で用いられるRNAを加えて分散または溶解させる操作、該リード粒子が分散した液中に、本発明で用いられるRNAが分散または溶解した液を加える操作等)を含む製造方法において実施することができる。ここで、リード粒子が分散した液中に、本発明で用いられるRNAを分散または溶解して含有させる工程により得られる複合粒子としては、具体的には、例えばカチオン性脂質を含有するリポソームBを構成成分とする微粒子に本発明で用いられるRNAが付着して形成される複合粒子、カチオン性脂質を含有する脂質集合体を構成成分とする微粒子に本発明で用いられるRNAが付着して形成される複合粒子、ポリ-L-リジン等のカチオン性高分子を含有する高分子ミセルを構成成分とする微粒子に本発明で用いられるRNAが付着して形成される複合粒子、カチオン性高分子を含有するリポソームBを構成成分とする微粒子に本発明で用いられるRNAが付着して形成される複合粒子、カチオン性脂質を含有する高分子ミセルを構成成分とする微粒子に本発明で用いられるRNAが付着して形成される複合粒子等があげられる。また、リード粒子が分散した液中に、本発明で用いられるRNAを分散または溶解して含有させる操作が、本発明で用いられるRNAが分散または溶解した液に、さらに付着競合剤を含有させて、これを該リード粒子が分散した液中に加える操作であることが好ましく、この場合、該リード粒子に、本発明で用いられるRNAと該付着競合剤が共に付着して複合粒子が製造され、該複合粒子の製造中におけるリード粒子の凝集も、製造後における複合粒子の凝集もより抑制されて製造できる。 More specifically, step 1 includes, for example, an operation for producing a liquid in which lead particles containing an aggregation-inhibiting substance are dispersed, and RNA used in the present invention is dispersed or dissolved in the liquid in which the lead particles are dispersed. Operation of containing (for example, an operation of adding and dispersing or dissolving RNA used in the present invention in a liquid in which the lead particles are dispersed, and RNA used in the present invention being dispersed or dissolved in a liquid in which the lead particles are dispersed) For example, an operation of adding the prepared liquid). Here, as the composite particles obtained by the step of dispersing or dissolving the RNA used in the present invention in the liquid in which the lead particles are dispersed, specifically, for example, liposome B containing a cationic lipid. Composite particles formed by adhering RNA used in the present invention to fine particles as constituent components, formed by adhering RNA used in the present invention to fine particles containing lipid aggregates containing cationic lipids Composite particles formed by adhering RNA used in the present invention to fine particles containing polymer micelles containing cationic polymers such as poly-L-lysine as constituents, and containing cationic polymers Used in the present invention for composite particles formed by attaching the RNA used in the present invention to fine particles comprising liposome B as a constituent, and polymer micelles containing cationic lipids as constituents Composite particles and the like that RNA to be is formed by deposition. Further, the operation of dispersing or dissolving the RNA used in the present invention in the liquid in which the lead particles are dispersed includes adding an adhesion competitor to the liquid in which the RNA used in the present invention is dispersed or dissolved. The lead particles are preferably added to a liquid in which the lead particles are dispersed. In this case, the RNA used in the present invention and the adhesion competitor are both 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.
　リード粒子のリード粒子が分散する液に対する割合は、リード粒子に本発明で用いられるRNAが付着できれば特に限定されるものではないが、約1μg/mL～1g/mLであるのが好ましく、約0.1～500mg/mLであるのがより好ましい。 The ratio of the lead particles to the liquid in which the lead particles are dispersed is not particularly limited as long as the RNA used in the present invention can adhere to the lead particles, but it is preferably about 1 μg / mL to 1 g / mL, and about 0.1 More preferably, it is ˜500 mg / mL.
　工程2)　複合粒子を脂質二重膜で被覆する工程(その1)
　工程1で得られた複合粒子が分散し、かつ脂質二重膜の構成成分の全部または一部が溶解した極性有機溶媒を含む液(液A)を調製する操作、次いで、液A中の極性有機溶媒の濃度を減少させることによって、複合粒子を脂質二重膜で被覆する操作を含む製造方法によってリポソームAが製造できる。この場合、リポソームAは分散液(液B)の形態で得られる。液Aにおける溶媒は、該脂質二重膜の構成成分が可溶で、該複合粒子が分散可能な極性有機溶媒の濃度の該極性有機溶媒を含む溶媒であり、液A中の極性有機溶媒の濃度を減少させた液Bでは、該脂質二重膜の構成成分が分散可能で、該複合粒子も分散可能であることが好ましい。液A中の溶媒が、極性有機溶媒と極性有機溶媒以外の溶媒との混合液である場合、例えば該極性有機溶媒と混合可能な極性有機溶媒以外の溶媒を含む溶媒(液C)を加えること、および/または、蒸発留去、半透膜分離、分留等によって、選択的に極性有機溶媒を取り除くことで、極性有機溶媒の濃度を減少させることができる。ここで、液Cは、極性有機溶媒以外の溶媒を含む液が好ましいが、極性有機溶媒も液Aにおける極性有機溶媒の濃度より低ければ含んでいてよい。 Step 2) Step of coating composite particles with lipid bilayer (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 all or part of the components of the lipid bilayer are dissolved, and then the polarity in the liquid A By reducing the concentration of the organic solvent, liposome A can be produced by a production method including an operation of coating the composite particles with a lipid bilayer membrane. In this case, 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. In the liquid B having a reduced concentration, it is preferable that the constituent components of the lipid bilayer membrane can be dispersed and the composite particles can also be dispersed. When 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. Here, 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.
　工程2における極性有機溶媒以外の溶媒としては、例えば、水、液体二酸化炭素、液体炭化水素、ハロゲン化炭素またはハロゲン化炭化水素等があげられ、好ましくは水があげられる。また、液Aおよび液Cは、イオンまたは緩衝成分等を含んでいてもよい。これら溶媒は1種単独でまたは2種以上を組み合わせて用いることができる。 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. Moreover, 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.
　極性有機溶媒と極性有機溶媒以外の溶媒の組み合わせは、相互に混合可能である組み合わせであるのが好ましく、液Aおよび液B中の溶媒ならびに液Cに対する複合粒子および脂質二重膜の構成成分の溶解度等を考慮して選択できる。一方、複合粒子については、液Aおよび液B中の溶媒ならびに液Cのいずれに対しての溶解度も低いことが好ましく、また極性有機溶媒および極性有機溶媒以外の溶媒のいずれに対しての溶解度も低いことが好ましく、脂質二重膜の構成成分は、液B中の溶媒および液Cに対しての溶解度が低いことが好ましく、液A中の溶媒に対しての溶解度が高いことが好ましく、また極性有機溶媒に対しての溶解度が高いことが好ましく、極性有機溶媒以外の溶媒に対する溶解度が低いことが好ましい。ここで、「複合粒子の溶解度が低い」とは、複合粒子に含有されるリード粒子、本発明で用いられるRNAおよび付着競合剤等の各成分の、溶媒中における溶出性が小さいことであり、各成分の個々の溶解度が高くても各成分間の結合等によって各成分の溶出性が小さくなっていればよい。例えば、リード粒子に含まれる成分のいずれかの液A中の溶媒に対する溶解度が高い場合でも、リード粒子が正電荷をもつ場合、本発明で用いられるRNA内の電荷、分子内分極等と静電的に結合することで、複合粒子中の成分の溶出が抑制され、複合粒子の液A中の溶媒に対する溶解度を低くすることが可能である。すなわち、リード粒子が正電荷をもつことは、リポソームAの製造において、複合粒子の成分の溶出を抑制し、製造性と歩留まりを向上させる効果も備えている。 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. The solvent in the liquid A and the liquid B and the components of the composite particles and the lipid bilayer membrane for the liquid C It can be selected in consideration of solubility. On the other hand, it is preferable that the composite particles have low solubility in both the solvent in liquid A and liquid B and liquid C, and the solubility in both polar organic solvents and solvents other than polar organic solvents is also low. Preferably, the lipid bilayer component is preferably low in solubility in the solvent in solution B and in solution C, preferably high in solubility in the solvent in solution A, and The solubility in a polar organic solvent is preferably high, and the solubility in a solvent other than the polar organic solvent is preferably low. Here, “the solubility of the composite particles is low” means that each component such as the lead particles contained in the composite particles, the RNA used in the present invention, and the adhesion competing agent has low elution in a solvent, Even if the individual solubility of each component is high, it is sufficient that the elution property of each component is reduced by the binding between the components. For example, even if 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 RNA used in the present invention, the intramolecular polarization, etc. Thus, the 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.
　液Aにおける極性有機溶媒の濃度は、脂質二重膜の構成成分が可溶で、複合粒子が分散可能であれば特に限定されるものではなく、用いる溶媒や複合粒子、脂質二重膜の構成成分の種類等により異なるが、好ましくは約30v/v%以上、より好ましくは約60～90v/v%である。また、液Bにおける極性有機溶媒の濃度は、液Aよりも低い濃度で該極性有機溶媒を含み、脂質二重膜の構成成分が分散可能で、複合粒子も分散可能であれば特に限定されるものではないが、好ましくは約50v/v%以下である。 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%. Further, 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.
　液Aを調製する工程としては、極性有機溶媒、複合粒子および脂質二重膜の構成成分、必要により極性有機溶媒以外の溶媒を混合して液Aを調製する工程があげられる。極性有機溶媒、複合粒子および脂質二重膜の構成成分、必要により極性有機溶媒以外の溶媒は、複合粒子が溶解しなければ、それらを加える順序に特に制限はないが、好ましくは、例えば該複合粒子が分散した極性有機溶媒を含む液(液D)を調製し、液D中の極性有機溶媒と同一または異なった極性有機溶媒を含む溶媒に該脂質二重膜の構成成分を溶解させた液(液E)を調製し、液Dと液Eを混合して調製する工程があげられる。液Dと液Eを混合して液Aを調製する際には、徐々に混合することが好ましい。 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. When preparing liquid A by mixing liquid D and liquid E, it is preferable to mix gradually.
　工程3)　複合粒子を脂質二重膜で被覆する工程(その2)
　工程1で得られた複合粒子および脂質二重膜の構成成分を、該脂質二重膜の構成成分が可溶な極性有機溶媒を含む、該複合粒子が溶解せず、該脂質二重膜の構成成分が分散状態で存在することが可能な濃度の液中に分散させる操作(得られる液を液Fとする)を含む製造方法でリポソームAが製造でき、この場合、リポソームAは分散液の状態で得られる。液Fにおける溶媒は、該脂質二重膜の構成成分が可溶な極性有機溶媒を含む溶媒であり、該脂質二重膜の構成成分および該複合粒子がともに分散可能な特定の濃度で液Fに含まれている。 Step 3) Step of coating composite particles with lipid bilayer (Part 2)
A 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, the composite particle does not dissolve, and the lipid bilayer membrane Liposome A can be produced by a production method including an operation of dispersing in a liquid having a concentration that allows the constituent components to exist in a dispersed state (the liquid obtained is liquid F). Obtained in the state. The solvent in the liquid F is a solvent containing a polar organic solvent in which the components of the lipid bilayer membrane are soluble, and the liquid F at a specific concentration at which both the components of the lipid bilayer membrane and the composite particles can be dispersed. Included.
　液Fの調製方法はいかなる形態をも取ることができる。例えば複合粒子の分散液と、脂質二重膜の構成成分の溶解液または分散液を調製した後、両液を混合して液Fを調製してもよく、複合粒子または脂質二重膜の構成成分のどちらか一方の分散液を調製し、その分散液に、固体状態の複合粒子または脂質二重膜の構成成分の残る一方を加えて分散させて液Fを調製してもよい。複合粒子の分散液と、脂質二重膜の構成成分の溶解液または分散液を混合する場合には、複合粒子の分散媒は、あらかじめ極性有機溶媒を含んでいてもよく、脂質二重膜の構成成分の溶媒または分散媒は極性有機溶媒を含む液または極性有機溶媒のみで構成される液であってもよい。一方、複合粒子または脂質二重膜の構成成分のどちらか一方の分散液を調製し、該分散液に、固体状態の複合粒子または脂質二重膜の構成成分の残る一方を加える場合には、該分散液は、極性有機溶媒を含む液であることが好ましい。なお、液Fを調製した後に複合粒子が溶解せず、脂質二重膜の構成成分が分散している場合には、複合粒子が溶解せず、脂質二重膜の構成成分が分散する極性有機溶媒濃度の範囲であれば極性有機溶媒を加えてもよく、極性有機溶媒を除去してもよく、または濃度を減少させてもよい。一方、液Fを調製した後に複合粒子は溶解していないが、脂質二重膜の構成成分が溶解している場合には、複合粒子が溶解せず、脂質二重膜の構成成分が分散する極性有機溶媒濃度の範囲で極性有機溶媒を除去するかまたは濃度を減少させればよい。また、複合粒子と脂質二重膜の構成成分をあらかじめ極性有機溶媒以外の溶媒中で混合し、そこに複合粒子が溶解せず、脂質二重膜の構成成分が分散する極性有機溶媒濃度の範囲で極性有機溶媒を加えてもよい。その場合には、複合粒子および脂質二重膜の構成成分のそれぞれを極性有機溶媒以外の溶媒中に分散させ、両分散液を混合した後で、極性有機溶媒を加えてもよく、複合粒子または脂質二重膜の構成成分のどちらか一方を極性有機溶媒以外の溶媒中に分散させ、その分散液に、固体状態の複合粒子または脂質二重膜の構成成分の残る一方を加えて分散させた後で、極性有機溶媒を加えてもよい。また、複合粒子および脂質二重膜の構成成分が分散し、極性有機溶媒を含有する液を、複合粒子が脂質二重膜で被覆されるに充分な時間、静置または混合する操作を含むことが好ましい。複合粒子と脂質二重膜の構成成分を、極性有機溶媒を含有する液中に分散させた後、静置または混合する時間は、複合粒子および脂質二重膜の構成成分を、極性有機溶媒を含有する液中に分散させた後に瞬時に終了させるのでなければ制限はないが、脂質二重膜の構成成分や、極性有機溶媒を含有する液の種類に応じて任意に設定することができ、得られたリポソームAの収率が定常量となる時間を設定することが好ましく、例えば約3秒～30分である。なお、複合粒子および脂質二重膜の構成成分を、極性有機溶媒を含有する液中に分散させると、複合粒子への脂質二重膜の被覆が開始され、速やかに複合粒子への脂質二重膜の被覆が完了することもあり、例えば、脂質二重膜の構成成分の溶解液を調製した後、複合粒子の分散液と、脂質二重膜の構成成分の溶解液とを混合して液Fを調製する場合において、脂質二重膜の構成成分の液Fへの溶解性が低いと、脂質二重膜の構成成分が極性有機溶媒を含有する液中に分散するのとほぼ同時に、複合粒子への脂質二重膜の被覆が完了することもある。 The method for preparing liquid F can take any form. For example, after preparing a dispersion of composite particles and a solution or dispersion of the components of the lipid bilayer membrane, 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. When mixing the composite particle dispersion and the lipid bilayer component solution or 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. On the other hand, when preparing a dispersion of either the composite particles or the components of the lipid bilayer membrane, and adding the remaining one of the solid-state composite particles or the components of the lipid bilayer to the dispersion, The dispersion is preferably a liquid containing a polar organic solvent. In addition, when the composite particles are not dissolved after the preparation of the liquid F and the components of the lipid bilayer are dispersed, the polar organic particles are not dissolved and the components of the lipid bilayer are dispersed. A polar organic solvent may be added within the solvent concentration range, the polar organic solvent may be removed, or the concentration may be decreased. On the other hand, the composite particles are not dissolved after preparing the liquid F. However, when the components of the lipid bilayer membrane are dissolved, 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. In addition, 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. In that case, 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. In addition, 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. After dispersing the components of the composite particles and the lipid bilayer membrane in a liquid containing a polar organic solvent, 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. When the components of the composite particle and the lipid bilayer are dispersed in a liquid containing a polar organic solvent, 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. When preparing F, if the solubility of the lipid bilayer components in liquid F is low, the lipid bilayer components are complexed almost simultaneously with the dispersion in the liquid containing the polar organic solvent. Sometimes the coating of the lipid bilayer on the particles is complete.
　液Fにおける極性有機溶媒以外の溶媒としては、例えば工程2における極性有機溶媒以外の溶媒で例示した物があげられ、好ましくは水があげられる。 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.
　液Fにおける極性有機溶媒の濃度は、複合粒子と、脂質二重膜の構成成分がともに分散されている条件さえ満たしていれば特に限定されるものではなく、用いる溶媒や複合粒子、脂質二重膜の構成成分の種類等により異なるが、好ましくは約1～80v/v%、より好ましくは約10～60v/v%、さらに好ましくは約20～50v/v%、最も好ましくは約30～40v/v%である。 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%.
　本発明において、「脂質二重膜の構成成分が極性有機溶媒に対して可溶」とは、脂質二重膜の構成成分が極性有機溶媒に溶解する性質をもつ場合、可溶化剤等を用いることにより脂質二重膜の構成成分が極性有機溶媒に溶解する性質をもつ場合、脂質二重膜の構成成分が極性有機溶媒中で凝集体またはミセル等を形成して乳濁もしくはエマルジョン化し得る性質をもつ場合等を包含する。
　また、「脂質二重膜の構成成分が分散する」とは、脂質二重膜の構成成分の全部が凝集体またはミセル等を形成して乳濁もしくはエマルジョン化している状態、脂質二重膜の構成成分の一部が凝集体またはミセル等を形成して乳濁もしくはエマルジョン化し、残る部分が溶解している状態、脂質二重膜の構成成分の一部が凝集体またはミセル等を形成して乳濁もしくはエマルジョン化し、残る部分が沈殿している状態等を包含し、脂質二重膜の構成成分の全部が溶解している状態を包含しない。 In the present invention, “the component of the lipid bilayer membrane is soluble in the polar organic solvent” means that when the component of the lipid bilayer membrane has the property of being dissolved in the polar organic solvent, a solubilizer or the like is used. When the components of the lipid bilayer membrane have the property of being dissolved in a polar organic solvent, the components of the lipid bilayer membrane can form emulsions or micelles in the polar organic solvent and become emulsion or emulsion The case where it has is included.
In addition, “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 where the emulsion is emulsified or emulsified, and the remaining part is precipitated, and does not include a state where all the components of the lipid bilayer are dissolved.
　本発明において、「複合粒子が分散する」とは、複合粒子が懸濁または乳濁もしくはエマルジョン化している状態のことであり、複合粒子の一部が懸濁または乳濁もしくはエマルジョン化し、残る部分が溶解している状態、複合粒子の一部が乳濁もしくはエマルジョン化し、残る部分が沈殿している状態等を包含し、複合粒子の全部が溶解している状態を包含しない。「複合粒子が溶解しない」とは、前記の「複合粒子が分散する」と同義である。 In the present invention, “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. Including a state in which a part of the composite particles is emulsified or emulsified and a remaining part is precipitated, and does not include a state in which all of the composite particles are dissolved. “Composite particles do not dissolve” has the same meaning as “composite particles are dispersed”.
　本発明におけるリポソームAの製造方法において用いられる、極性有機溶媒含有水溶液中の複合粒子の濃度は、複合粒子を脂質二重膜で被覆できれば特に限定されるものではないが、約1μg/mL～1g/mLであるのが好ましく、約0.1～500mg/mLであるのがより好ましい。また、用いられる脂質二重膜の構成成分の濃度は、複合粒子を被覆できれば特に限定されるものではないが、約1μg/mL～1g/mLであるのが好ましく、約0.1～400mg/mLであるのがより好ましい。 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 μg / 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 μg / mL to 1 g / mL, preferably about 0.1 to 400 mg / mL. More preferably.
　本発明のリポソームAに対する脂質二重膜の割合は、重量比で約1:0.1～1:1000が好ましく、約1:1～1:10がより好ましい。 The ratio of the lipid bilayer membrane to the liposome A of the present invention is preferably about 1: 0.1 to 1: 1000, more preferably about 1: 1 to 1:10 by weight.
　また、本発明におけるリポソームAの大きさは、例えば注射可能な大きさであることが好ましい。具体的には、平均粒子径が約10nm～1000nmであるのが好ましく、約50nm～300nmであるのがより好ましく、約70nm～200nmであるのがさらに好ましい。 In addition, the size of the liposome A in the present invention is preferably an injectable size, for example. Specifically, the average particle size is preferably about 10 nm to 1000 nm, more preferably about 50 nm to 300 nm, and further preferably about 70 nm to 200 nm.
　さらに、上記で得られるリポソームAに抗体等の蛋白質、糖類、糖脂質、アミノ酸、核酸、種々の低分子化合物または高分子化合物等の物質による修飾を行うこともでき、これらで得られる被覆複合粒子もリポソームAに包含される。例えば、ターゲッティングに応用するため、上記で得られるリポソームAに対して、さらに抗体等の蛋白質、ペプチドまたは脂肪酸類等による脂質二重膜の表面修飾を行うこともできる［ラジック(D. D. Lasic)、マーティン(F. Martin)編,“ステルス・リポソームズ(Stealth Liposomes)”(米国),シーアールシー・プレス・インク(CRC Press Inc),1995年,p.93-102参照］。また、リポソームAに例えば水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体による表面改質も任意に行うことができ、これら表面改質に用いられる水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体は、前記脂質二重膜の構成成分としての水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体と同義である。 Furthermore, 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. For example, for application to targeting, 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]. In addition, 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.
　本発明の組成物を、肺高血圧症または気管支もしくは肺における肥大した肺血管壁組織を有する哺乳動物(人を含む)に投与することで、本発明で用いられるRNAを肺血管壁組織の肥大に関連した遺伝子の発現部位である気管支または肺における肥大した肺血管壁組織へ送達することができ、該遺伝子の発現が抑制される。肺血管壁組織の肥大に関連した遺伝子の発現が抑制されることで、肺血管壁組織の肥大が抑制され、肺高血圧症を治療または予防する。また、気管支または肺における肺血管壁組織の肥大が抑制されることで、肺動脈内の圧力を下げ、肺動脈の血液が流れやすなり、心筋の肥大や右心房を含む右心系の機能障害が改善され、肺高血圧症に起因する右心不全または全身の血液の循環障害が治療または予防される。
　即ち、本発明は、上記説明した本発明の組成物を哺乳動物に投与する肺高血圧症の治療方法も提供する。投与対象は、肺高血圧症に罹患している人または気管支もしくは肺における肥大した肺血管壁組織を有する人であることが好ましく、肺高血圧症に罹患している人がより好ましい。
　また、本発明の組成物におけるRNAを、補酵素や抗体等のペプチドおよび蛋白質、オリゴヌクレオチドやプラスミド等の核酸等に換えた組成物も、同様に気管支または肺における肥大した肺血管壁組織へ送達することができ、気管支または肺における疾患の治療剤または予防剤として使用することができる。 By administering the composition of the present invention to mammals (including humans) having pulmonary hypertension or enlarged pulmonary vascular wall tissue in the bronchus or lung, the RNA used in the present invention can be used to enlarge the pulmonary vascular wall tissue. The gene can be delivered to the enlarged pulmonary vascular wall tissue in the bronchus or lung, which is the site of expression of the related gene, and the expression of the gene is suppressed. By suppressing the expression of genes related to hypertrophy of pulmonary vascular wall tissue, hypertrophy of pulmonary vascular wall tissue is suppressed, thereby treating or preventing pulmonary hypertension. In addition, suppression of pulmonary vascular wall tissue enlargement in the bronchus or lungs reduces the pressure in the pulmonary artery and facilitates blood flow in the pulmonary artery, improving myocardial hypertrophy and dysfunction of the right heart system including the right atrium Right heart failure or systemic blood circulation disorders due to pulmonary hypertension are treated or prevented.
That is, the present invention also provides a method for treating pulmonary hypertension, wherein the above-described composition of the present invention is administered to a mammal. The administration subject is preferably a person suffering from pulmonary hypertension or a person having an enlarged pulmonary vascular wall tissue in the bronchi or lung, more preferably a person suffering from pulmonary hypertension.
Similarly, a composition in which RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, and nucleic acids such as oligonucleotides and plasmids is also delivered to the enlarged pulmonary vascular wall tissue in the bronchi or lung. And can be used as a therapeutic or prophylactic agent for diseases in the bronchi or lungs.
　また、本発明の組成物、および本発明の組成物におけるRNAを補酵素や抗体等のペプチドおよび蛋白質、オリゴヌクレオチドやプラスミド等の核酸等に換えた組成物は、送達の量を直接または間接的に測定して、肺高血圧症に罹患しているかどうかまたは気管支もしくは肺における肥大した肺血管壁組織を有するかどうかを診断するための診断薬として使用することもできる。
　また、本発明の組成物、および本発明の組成物におけるRNAを補酵素や抗体等のペプチドおよび蛋白質、オリゴヌクレオチドやプラスミド等の核酸等に換えた組成物は、例えば血液成分等の生体成分(例えば血液、消化管等)中での該RNA、ペプチド、蛋白質または核酸の安定化、副作用の低減または気管支または肺への薬剤集積性の増大等を目的とする製剤としても使用できる。 In addition, the composition of the present invention, and the composition in which the RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, etc., are delivered directly or indirectly. It can also be used as a diagnostic to measure whether it suffers from pulmonary hypertension or has enlarged pulmonary vascular wall tissue in the bronchi or lung.
In addition, the composition of the present invention, and the composition in which the RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, and the like, for example, biological components such as blood components ( For example, it can also be used as a preparation for the purpose of stabilizing the RNA, peptide, protein or nucleic acid in blood, gastrointestinal tract, etc., reducing side effects or increasing drug accumulation in bronchi or lung.
　本発明の組成物、および本発明の組成物におけるRNAを補酵素や抗体等のペプチドおよび蛋白質、オリゴヌクレオチドやプラスミド等の核酸等に換えた組成物を、気管支または肺における肺高血圧症等の疾患の治療剤または予防剤として使用する場合、投与経路としては、治療に際し最も効果的な投与経路を使用するのが望ましく、口腔内、気道内、直腸内、皮下、筋肉内または静脈内等の非経口投与または経口投与をあげることができ、好ましくは静脈内投与または筋肉内投与をあげることができ、より好ましくは静脈内投与があげられる。
　投与量は、投与対象の病状や年齢、投与経路などによって異なるが、例えばRNAに換算した1日投与量が約0.1μg～1000mgとなるように投与すればよい。 Diseases such as pulmonary hypertension in the bronchus or lungs in the composition of the present invention, and compositions in which RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, etc. When using as a therapeutic or prophylactic agent, it is desirable to use the most effective route for treatment, such as oral, respiratory tract, rectal, subcutaneous, intramuscular or intravenous. Oral administration or oral administration can be exemplified, preferably intravenous administration or intramuscular administration can be exemplified, and more preferably intravenous administration is exemplified.
The dose varies depending on the disease state, age, route of administration, etc. of the administration subject, but for example, it may be administered so that the daily dose converted to RNA is about 0.1 μg to 1000 mg.
　静脈内投与または筋肉内投与に適当な剤形としては、例えば注射剤があげられ、上述の方法により調製したリポソームAの分散液をそのまま例えば注射剤等の形態として用いることも可能であるが、該分散液から例えば濾過、遠心分離等によって溶媒を除去して使用することも、該分散液を凍結乾燥して使用すること、または例えばマンニトール、ラクトース、トレハロース、マルトースまたはグリシン等の賦形剤を加えた分散液を凍結乾燥して使用することもできる。
　注射剤の場合、前記のリポソームAの分散液または前記の溶媒を除去または凍結乾燥したリポソームAに、例えば水、酸、アルカリ、種々の緩衝液、生理的食塩液またはアミノ酸輸液等を混合して調製された本発明の組成物を使用することが好ましい。また、例えばクエン酸、アスコルビン酸、システインもしくはEDTA等の抗酸化剤またはグリセリン、ブドウ糖もしくは塩化ナトリウム等の等張化剤等を添加することも可能である。また、例えばグリセリン等の凍結保存剤を加えて凍結保存することもできる。 Examples of suitable dosage forms 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, for example, in the form of injections. The dispersion can be used after removing the solvent by, for example, filtration, centrifugation, etc., or the dispersion can be used by lyophilization, or an excipient such as mannitol, lactose, trehalose, maltose or glycine can be used. The added dispersion can be lyophilized for use.
In the case of injections, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, etc. are mixed into the liposome A dispersion or the liposome A from which the solvent is removed or lyophilized. It is preferred to use the prepared composition of the invention. Further, for example, an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA, or an isotonic agent such as glycerin, glucose or sodium chloride can be added. Moreover, it can also be cryopreserved by adding a cryopreservation agent such as glycerin.
　本発明の肺高血圧症治療剤としては、肺高血圧症の治療または予防に用いることを意図した本発明の組成物があげられるが、例えば、本発明の組成物のうち、リポソームAが、リード粒子と前記RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成され、該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能であるリポソーム、またはカチオン性物質を含むリード粒子と前記RNAを構成成分とする複合粒子、および該複合粒子を被覆する脂質二重膜から構成され、該脂質二重膜の構成成分が極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能であるリポソームを含有することが好ましい。
　また、本発明は、上記説明した本発明の組成物の、肺高血圧症の治療剤の製造のための使用も提供する。 Examples of the therapeutic agent for pulmonary hypertension of the present invention include the composition of the present invention intended to be used for the treatment or prevention of pulmonary hypertension. For example, among the compositions of the present invention, liposome A is a lead particle. And a lipid bilayer coating the composite particle, the component of the lipid bilayer is soluble in a specific polar organic solvent, and the lipid bilayer And a composite particle comprising a lead particle containing a cationic substance and the RNA as constituents, and the composite particle, wherein the component and the composite particle are dispersible in a liquid containing the polar organic solvent at a specific concentration A component of the lipid bilayer membrane is soluble in a polar organic solvent, and the component of the lipid bilayer membrane and the composite particle are mixed with the polar organic membrane at a specific concentration. Dispersible in liquid containing solvent It is preferable to contain a certain liposome.
The present invention also provides use of the composition of the present invention described above for the manufacture of a therapeutic agent for pulmonary hypertension.
　次に、実施例および試験例により、本発明を具体的に説明する。ただし、本発明はこれら実施例および試験例に限定されるものではない。なお、実施例1において用いたRNAは、ラットMEK1/2遺伝子に相同性を有する19塩基の配列および該配列と相補的な塩基の配列を含むRNAであり、それぞれの3’端にdTdTを付加したRNA［5’-GCAUCUGCAUGGAGCACAUdTdT-3’(配列a、配列番号149)、5’-AUGUGCUCCAUGCAGAUGCdTdT-3’(配列b、配列番号150)］であり、配列aの1本鎖RNAの5’端をCyanine5(Cy5)で修飾したRNA［5’-Cy5-GCAUCUGCAUGGAGCACAUdTdT-3’(配列c、配列番号151)］を、Eurogenetec社から入手し、配列cと配列bをアニーリングさせることにより調製した(以下、「Cy5標識MEKsiRNA」という)。また、実施例2において用いたRNAは、配列aおよび配列bのRNAを用いて同様に調製した(以下、「MEKsiRNA」という)。比較例1において用いたRNAは、ラットのMEK1/2遺伝子に相同性のない19塩基の配列および該配列と相補的な塩基の配列を含むRNA［5’-UACACGAGGUACGUCUACGdTdT-3’(配列番号152)、5’-CGUAGACGUACCUCGUGUAdTdT-3’(配列番号153)］であり、同様に調製した(以下、「Control-siRNA」という)。 Next, the present invention will be specifically described with reference to examples and test examples. However, the present invention is not limited to these examples and test examples. The RNA used in Example 1 is an RNA containing a 19-base sequence homologous to the rat MEK1 / 2 gene and a base sequence complementary to the sequence, and dTdT is added to each 3 ′ end. RNA [5'-GCAUCUGCAUGGAGCACAUdTdT-3 '(sequence a, SEQ ID NO: 149), 5'-AUGUGCUCCAUGCAGAUGCdTdT-3' (sequence b, SEQ ID NO: 150)], the 5 'end of the single-stranded RNA of sequence a RNA modified with Cyanine5 (Cy5) [5′-Cy5-GCAUCUGCAUGGAGCACAUdTdT-3 ′ (sequence c, SEQ ID NO: 151)] was obtained from Eurogenetec and prepared by annealing sequence c and sequence b (hereinafter, "Cy5-labeled MEKsiRNA"). The RNA used in Example 2 was similarly prepared using RNAs of sequence a and sequence b (hereinafter referred to as “MEKsiRNA”). The RNA used in Comparative Example 1 is an RNA [5′-UACACGAGGUACGUCUACGdTdT-3 ′ (SEQ ID NO: 152) containing a 19-base sequence not homologous to the rat MEK1 / 2 gene and a base sequence complementary to the sequence. 5′-CGUAGACGUACCUCGUGUAdTdT-3 ′ (SEQ ID NO: 153)] and prepared in the same manner (hereinafter referred to as “Control-siRNA”).
　DOTAP(アバンチポーラルリピッズ社製)/PEG-DSPE(日本油脂製、以下同様)/蒸留水を40mg/16mg/1mLになるように混合し、ボルテックスミキサーで振とう攪拌した。得られた分散液を室温で0.4μmのポリカーボネートメンブランフィルター(ワットマン製)に4回、0.1μmのポリカーボネートメンブランフィルターに10回、さらに0.05μmのポリカーボネートメンブランフィルターに24回通してリード粒子を調製した。得られたリード粒子の分散液0.375mLに、Cy5標識MEKsiRNAの24mg/mL水溶液0.125mLを添加し、複合粒子を調製した。得られた複合粒子の分散液0.5mLを、脂質二重膜の構成成分のEPC(日本油脂製)/PEG-DSPE/エタノール/蒸留水を15mg/3.125mg/0.625mL/0.375mLになるように混合して得られた溶液2mL（エタノール濃度はおよそ62.5v/v%）に添加した。次に蒸留水0.625mLを徐々に加え、エタノールの濃度をおよそ40v/v %とした。次に、EPC/PEG-DSPE/エタノール/蒸留水を62.5mg/62.5mg/0.4mL/0.6mLになるように混合して得られた溶液を0.2mL添加し、蒸留水23.275mLを添加してリポソームを調製した。得られたリポソームの分散液を超遠心分離(1時間、110,000×g、25℃)し、上清を除去し、沈殿に生理食塩水を添加して再分散させ、生理食塩水をさらに添加して濃度を調整し、組成物を得た。 DOTAP (manufactured by Avanti Polar Lipids) / PEG-DSPE (manufactured by NOF Corporation, the same shall apply hereinafter) / distilled water was mixed to 40 mg / 16 mg / 1 mL, and the mixture was shaken and stirred with a vortex mixer. The resulting dispersion was passed through a 0.4 μm polycarbonate membrane filter (manufactured by Whatman) at room temperature 4 times, through a 0.1 μm polycarbonate membrane filter 10 times, and further through a 0.05 μm polycarbonate membrane filter 24 times to prepare lead particles. To 0.375 mL of the resulting lead particle dispersion, 0.125 mL of a 24 mg / mL aqueous solution of Cy5-labeled MEK siRNA was added to prepare composite particles. 0.5 mL of the resulting composite particle dispersion is adjusted to 15 mg / 3.125 mg / 0.625 mL / 0.375 mL of EPC (manufactured by NOF Corporation) / PEG-DSPE / ethanol / distilled water as a component of the lipid bilayer membrane. It added to 2 mL (ethanol concentration is about 62.5 v / v%) of the solution obtained by mixing. Next, 0.625 mL of distilled water was gradually added to adjust the ethanol concentration to about 40 v / v%. Next, add 0.2 mL of the solution obtained by mixing EPC / PEG-DSPE / ethanol / distilled water to 62.5 mg / 62.5 mg / 0.4 mL / 0.6 mL, and add 23.275 mL of distilled water. Liposomes were prepared. The resulting liposome dispersion is ultracentrifugated (1 hour, 110,000 × g, 25 ° C.), the supernatant is removed, and physiological saline is added to the precipitate for redispersion, and physiological saline is further added. The concentration was adjusted to obtain a composition.
試験例1
　肺高血圧症を生じる疾患モデル(Sugita T, Hyers TM, Dauber IM, Wagner WW, McMurtry IF, Reeves JT. Lung vessel leak precedes right ventricular hypertrophy in monocrotaline-treated rats. J Appl Physiol. Feb 1983;54(2):371-374. 参照)を用い、次の方法により、実施例1で得られた組成物を投与した場合には、肺高血圧症を生じた肺に、RNAが特異的に到達することを確認した。
　Wistar(5週齢)雄性ラット(100～120g)の皮下にモノクロタリン(MCT)を投与(60mg/kg)し、モノクロタリン誘発性肺高血圧症ラット(MCTラット)とした。また、MCTの代わりに生理食塩水を同様に投与し、対照群とした。
　MCTまたは生理食塩水投与10日後、ラット尾静脈より実施例1で得られた組成物(Cy5標識MEKsiRNAとして0.2 mg/kg)を投与し、組成物投与1日、2日、4日、7日後の肺を採取した。得られた肺を生理食塩水で洗浄灌流して血液を除いた後、肺1.0mgあたり1μLの1%TritonX-100含有PBSを添加し、肺をホモジナイズした。これを遠心分離(10000×g、4℃)し、得られた上清中におけるCy5の蛍光強度を測定した。各肺におけるCy5標識MEKsiRNAの濃度は、実施例1で得られた組成物の代わりに生理食塩水を同様に静脈内投与したMCTラットおよび対照群それぞれの肺における自家蛍光強度を減じて補正することで算出した。
　図1に、実施例1で得られた組成物の投与1、2、4、および7日後の、肺におけるCy5標識MEKsiRNAの集積の時間変化を示した。
　図1から、MCTラットの肺において、実施例1で得られた組成物の投与2日後にCy5標識MEKsiRNAの濃度が最大となり、一方、対照群の肺におけるCy5標識MEKsiRNAの濃度は、組成物投与から1日後に緩やかなピークを示したことがわかる。MCTラットにおける肺でのCy5標識MEKsiRNAの濃度は、組成物投与2日後において、対照群に対して有意に高い濃度であった。 Test example 1
Disease model causing pulmonary hypertension (Sugita T, Hyers TM, Dauber IM, Wagner WW, McMurtry IF, Reeves JT. Lung vessel leak precedes right ventricular hypertrophy in monocrotaline-treated rats.J Appl Physiol. Feb 1983; 54 (2) : 371-374.), And when the composition obtained in Example 1 was administered by the following method, it was confirmed that RNA specifically reached the lung in which pulmonary hypertension occurred. did.
Monocrotaline (MCT) was subcutaneously administered to Wistar (5-week-old) male rats (100 to 120 g) (60 mg / kg) to obtain monocrotaline-induced pulmonary hypertension rats (MCT rats). In addition, physiological saline was similarly administered instead of MCT, and used as a control group.
10 days after administration of MCT or physiological saline, the composition obtained in Example 1 (0.2 mg / kg as Cy5-labeled MEKsiRNA) was administered from the tail vein of the rat, and 1, 2, 4, 7 days after administration of the composition Lungs were collected. The obtained lung was washed and perfused with physiological saline to remove blood, and 1 μL of 1% TritonX-100-containing PBS per 1.0 mg of lung was added to homogenize the lung. This was centrifuged (10000 × g, 4 ° C.), and the fluorescence intensity of Cy5 in the obtained supernatant was measured. The concentration of Cy5-labeled MEK siRNA in each lung should be corrected by reducing the autofluorescence intensity in the lungs of MCT rats and control groups, which were similarly administered intravenously with physiological saline instead of the composition obtained in Example 1. Calculated with
FIG. 1 shows changes over time in the accumulation of Cy5-labeled MEK siRNA in the lung 1, 2, 4, and 7 days after administration of the composition obtained in Example 1.
From FIG. 1, in the lungs of MCT rats, the concentration of Cy5-labeled MEK siRNA was maximized 2 days after the administration of the composition obtained in Example 1, while the concentration of Cy5-labeled MEK siRNA in the lung of the control group was the composition administered. It can be seen that one day later, it showed a gentle peak. The concentration of Cy5-labeled MEK siRNA in the lung in MCT rats was significantly higher than the control group 2 days after administration of the composition.
試験例2
　実施例1で得られた組成物を投与した場合に、肺高血圧症の疾患部位である肺に、RNAが選択的に到達することを確認した。
　試験例1と同様にMCTラットおよび対照群を用意した。MCTまたは生理食塩水の投与10日後、ラット尾静脈より実施例1で得られた組成物(Cy5標識MEKsiRNAとして0.2 mg/kg)を投与し、組成物投与48時間後の肺、肝臓、腎臓、心臓、脳を採取した。得られた各臓器中におけるCy5標識MEKsiRNAの濃度を、試験例1と同様に測定し算出した。
　図2に、実施例1で得られた組成物の投与48時間後のMCTラットおよび対照群の、各臓器中におけるCy5標識MEKsiRNA濃度を示した。
　図2から、48時間後のMCTラットの肺は対照群の肺と比較して、有意に高いCy5標識MEKsiRNA濃度を示すことが分かる。一方、肺以外の臓器ではそれぞれのラットにおいてCy5標識MEKsiRNAの濃度に有意な差がないことが分かる。 Test example 2
When the composition obtained in Example 1 was administered, it was confirmed that RNA selectively reaches the lung, which is a disease site of pulmonary hypertension.
Similar to Test Example 1, MCT rats and a control group were prepared. 10 days after administration of MCT or physiological saline, the composition obtained in Example 1 from rat tail vein (0.2 mg / kg as Cy5-labeled MEK siRNA) was administered, and the lung, liver, kidney, 48 hours after administration of the composition, Heart and brain were collected. The concentration of Cy5-labeled MEK siRNA in each obtained organ was measured and calculated in the same manner as in Test Example 1.
FIG. 2 shows the concentration of Cy5-labeled MEK siRNA in each organ of MCT rats and control group 48 hours after administration of the composition obtained in Example 1.
FIG. 2 shows that the lungs of MCT rats after 48 hours show significantly higher Cy5-labeled MEK siRNA concentrations compared to the lungs of the control group. On the other hand, in organs other than the lung, it can be seen that there is no significant difference in the concentration of Cy5-labeled MEK siRNA in each rat.
試験例3
　実施例1で得られた組成物を投与した場合において、肥大した肺血管壁組織およびその近傍に、RNAが選択的に到達することを確認した。
　試験例1と同様にMCTラットおよび対照群を用意して実施例1で得られた組成物を投与した。組成物投与48時間後の肺を摘出し、右肺下部の凍結切片を作成した。血管内皮細胞を免疫染色し(緑色)、DAPI染色によって核を染色し(青色)、Cy5標識MEKsiRNA(赤色)の集積部位を蛍光顕微鏡で観察した。
　図3に各ラットにおける肺動脈およびその近傍を免疫染色した切片を示した（左：対照群、右：MCTラット）。図中の白色部分が緑色に染色された血管内皮細胞を示し、矢印が赤色の蛍光が強く認められたCy5標識MEKsiRNAの集積を示す。
　図3より、肥大した肺血管壁組織を有するMCTラット（図3右）において、Cy5標識MEKsiRNAの集積が、肥大した肺血管壁組織を有さない対照群（図3左）と比較して血管内皮細胞の近傍に高い割合で局在しており、実施例1で得られた組成物を投与した場合に、肥大した肺血管壁組織およびその近傍にRNAが集積することが分かる。 Test example 3
When the composition obtained in Example 1 was administered, it was confirmed that RNA selectively reached the enlarged pulmonary vascular wall tissue and its vicinity.
MCT rats and a control group were prepared in the same manner as in Test Example 1, and the composition obtained in Example 1 was administered. The lung 48 hours after administration of the composition was removed, and a frozen section of the lower right lung was prepared. Vascular endothelial cells were immunostained (green), nuclei were stained by DAPI staining (blue), and the accumulation site of Cy5-labeled MEKsiRNA (red) was observed with a fluorescence microscope.
FIG. 3 shows a section of each rat immunostained pulmonary artery and its vicinity (left: control group, right: MCT rat). In the figure, the white part indicates vascular endothelial cells stained in green, and the arrow indicates the accumulation of Cy5-labeled MEK siRNA in which strong red fluorescence was observed.
Fig. 3 shows that in MCT rats with enlarged pulmonary vascular wall tissue (Fig. 3 right), the accumulation of Cy5-labeled MEK siRNA was higher than that in the control group without enlarged pulmonary vascular wall tissue (Fig. 3 left). It is localized at a high rate in the vicinity of the endothelial cells, and it can be seen that when the composition obtained in Example 1 is administered, RNA accumulates in the enlarged pulmonary vascular wall tissue and its vicinity.
　実施例1におけるCy5標識MEKsiRNAを、MEKsiRNAに換え、同様に組成物を得た。 The Cy5-labeled MEKsiRNA in Example 1 was replaced with MEKsiRNA to obtain a composition in the same manner.
比較例1
　実施例1におけるCy5標識MEKsiRNAを、Control-siRNAに換え、同様に組成物を得た。 Comparative Example 1
The Cy5-labeled MEK siRNA in Example 1 was replaced with Control-siRNA to obtain a composition in the same manner.
試験例4
　実施例2で得られた組成物を投与した場合において、肺高血圧症を生じた肺で、投与量依存的に遺伝子の発現を抑制することを確認した。
　試験例1と同じ病態モデルラットを用いて、MCT投与10日後に実施例2で得られた組成物をMEKsiRNAとして0.2mg/kgおよび2mg/kgの各投与量で静脈内投与した。また、比較例1で得られた組成物を対照として同様に投与した。摘出した各肺からTRIzol Reagent(インビトロジェン製)および、RNeasy Mini Kit(キアゲン製)を用いて全RNAを抽出した。全RNAはQuantiTect　Reverse Transcription　Kit(キアゲン製)と以下に示す鋳型DNAプライマー(cDNA)を用いてMEK1およびMEK2のmRNAを逆転写させた［MEK1 sense(5’-CAGGTGCTGCATGAGTGCAA-3’(配列番号154))、MEK1 antisense(5’-CTTGATCCAAGGACCCACCATC-3’(配列番号155))、MEK2 sense(5’-TGCAACTCACCGTACATCGT-3’(配列番号156))、MEK2 antisense(5’-CCTTCAGTACCTGGTCCAGT-3’(配列番号157))］。また、内部標準としてGAPDHのmRNAも以下に示すcDNAを用いて同様に逆転写させた。［GAPDH sense(5’-GAACATCATCCCTGCATCCA-3’(配列番号158))、GAPDH antisense(5’-CCAGTGAGCTTCCCGTTCA-3’(配列番号159))］。PCR増幅では1μLのcDNAと25μLのPower SYBR Green PCR Master Mix(バイオシステム製)を添加して、全量を50μLとしたRNA溶液を用いた。95℃、15秒間で熱変性し、60℃、1分間でアニーリングおよび伸張反応を繰り返して、これを40サイクル行った。肺におけるMEK1およびMEK2のmRNAの発現強度は、各摘出肺でのGAPDHのmRNA発現強度で補正して算出した。
　図4に、組成物投与48時間後の肺における、投与量依存的なMEK1（左）およびMEK2（右）のmRNAの発現抑制効果を示した。
　図4から、実施例2で得られた組成物を投与した群が、比較例1で得られた組成物を投与した群と比較して、肺において投与量依存的にMEK1およびMEK2のmRNAの発現をどちらも抑制されていることが分かる。 Test example 4
When the composition obtained in Example 2 was administered, it was confirmed that gene expression was suppressed in a dose-dependent manner in the lung in which pulmonary hypertension occurred.
Using the same disease state model rats as in Test Example 1, the composition obtained in Example 2 was intravenously administered as MEK siRNA at doses of 0.2 mg / kg and 2 mg / kg 10 days after MCT administration. Further, the composition obtained in Comparative Example 1 was similarly administered as a control. Total RNA was extracted from each extracted lung using TRIzol Reagent (Invitrogen) and RNeasy Mini Kit (Qiagen). Total RNA was reverse-transcribed from MEK1 and MEK2 mRNA using QuantiTect Reverse Transcription Kit (Qiagen) and template DNA primer (cDNA) shown below [MEK1 sense (5'-CAGGTGCTGCATGAGTGCAA-3 '(SEQ ID NO: 154) ), MEK1 antisense (5'-CTTGATCCAAGGACCCACCATC-3 '(SEQ ID NO: 155)), MEK2 sense (5'-TGCAACTCACCGTACATCGT-3' (SEQ ID NO: 156)), MEK2 antisense (5'-CCTTCAGTACCTGGTCCAGT-3 '(SEQ ID NO: 157) ))]. In addition, GAPDH mRNA as an internal standard was reverse-transcribed in the same manner using the cDNA shown below. [GAPDH sense (5′-GAACATCATCCCCTGCATCCA-3 ′ (SEQ ID NO: 158)), GAPDH antisense (5′-CCAGTGAGCTTCCCGTTCA-3 ′ (SEQ ID NO: 159))]. In PCR amplification, 1 μL of cDNA and 25 μL of Power SYBR Green PCR Master Mix (manufactured by Biosystems) were added, and an RNA solution with a total volume of 50 μL was used. Heat denaturation was performed at 95 ° C. for 15 seconds, and annealing and extension reaction were repeated at 60 ° C. for 1 minute, and this was performed for 40 cycles. The expression intensity of MEK1 and MEK2 mRNA in the lung was calculated by correcting the expression intensity of GAPDH mRNA in each isolated lung.
FIG. 4 shows the dose-dependent effects of MEK1 (left) and MEK2 (right) mRNA expression suppression in the lung 48 hours after administration of the composition.
From FIG. 4, the group administered with the composition obtained in Example 2 was compared with the group administered with the composition obtained in Comparative Example 1 in a dose-dependent manner in the lungs of MEK1 and MEK2 mRNA. It can be seen that both expressions are suppressed.
試験例5
　実施例2で得られた組成物を投与した場合において、肺高血圧症を生じた肺での遺伝子の発現抑制効果の経時的変化を確認した。
　試験例1と同じ病態モデルラットを用いて、MCT投与10日後に実施例2で得られた組成物を静脈内投与し(0.2 mg/kg)、組成物投与24、48、96時間後に肺を摘出した。また、比較例1で得られた組成物を対照として同様に投与し、組成物投与48時間後に肺を摘出した。摘出した肺から試験例4と同様に、肺におけるMEK1およびMEK2のmRNAの発現強度を測定した。
　図5に、組成物投与24、48、96時間後の肺におけるMEK1（左）およびMEK2（右）のmRNAの発現抑制効果の経時的変化を示した。
　図5から、実施例2で得られた組成物を投与した場合に、比較例1で得られた組成物(Control)を投与した場合と比較して、肺においてMEK1およびMEK2のmRNAの発現を抑制し、その抑制効果は組成物の投与24時間後には確認され、96時間以上持続することが分かる。 Test Example 5
When the composition obtained in Example 2 was administered, changes over time in the gene expression suppression effect in the lungs in which pulmonary hypertension occurred were confirmed.
Using the same disease state model rats as in Test Example 1, the composition obtained in Example 2 was intravenously administered (0.2 mg / kg) 10 days after MCT administration, and the lungs were removed 24, 48, and 96 hours after composition administration. Extracted. In addition, the composition obtained in Comparative Example 1 was similarly administered as a control, and the lungs were removed 48 hours after administration of the composition. The expression intensity of MEK1 and MEK2 mRNA in the lung was measured from the extracted lung in the same manner as in Test Example 4.
FIG. 5 shows temporal changes in the mRNA expression inhibitory effect of MEK1 (left) and MEK2 (right) in the lung 24, 48, and 96 hours after administration of the composition.
From FIG. 5, when the composition obtained in Example 2 was administered, the expression of MEK1 and MEK2 mRNA in the lung was compared with the case where the composition (Control) obtained in Comparative Example 1 was administered. It is confirmed that the inhibitory effect is confirmed 24 hours after administration of the composition and lasts for 96 hours or more.
試験例6
　試験例1と同じ病態モデルラットを用いて、実施例2で得られた組成物を投与した場合に、肺高血圧を抑制することを確認した。
　MCT投与3、10、17日後のラットに実施例2で得られた組成物を静脈内投与(0.2 mg/kg)し、MCT投与21日後に血行動態を測定した。イソフルラン(1～1.5%)で麻酔したラットにポリエチレンカテーテル(PE-50)(BDバイオサイエンス製)を用いて右頚静脈を介して右心室に挿入し、収縮期右心室圧を測定した。収縮期右心室圧は連続20回の心拍動中における各測定値から算出した。
　対照群として、比較例1で得られた組成物(0.2 mg/kg)を静脈内投与したMCTラット（比較例1）、生理食塩水を同様に静脈内投与したMCTラット(MCT)、およびMCTの代わりに生理食塩水を皮下投与し、生理食塩水を静脈内投与したラット(Sham)においても同様に試験した。
　図6に、各ラットにおける収縮期右心室圧を棒グラフで示した。
　図6から、肺高血圧症モデルのMCTラットではShamに対して有意に高い血圧を示しており、比較例1で得られた組成物を投与した群は、その高血圧を抑制していないことが分かる。一方、実施例2で得られた組成物を投与した群は、MCT誘発性の血圧上昇を、比較例1で得られた組成物を投与した群に対して有意に抑えることが分かる。 Test Example 6
It was confirmed that pulmonary hypertension was suppressed when the composition obtained in Example 2 was administered using the same disease state model rats as in Test Example 1.
The composition obtained in Example 2 was intravenously administered (0.2 mg / kg) to rats 3, 10, and 17 days after MCT administration, and hemodynamics were measured 21 days after MCT administration. Rats anesthetized with isoflurane (1 to 1.5%) were inserted into the right ventricle via the right jugular vein using a polyethylene catheter (PE-50) (BD Bioscience), and systolic right ventricular pressure was measured. Systolic right ventricular pressure was calculated from each measured value during 20 consecutive heartbeats.
As control groups, MCT rats (Comparative Example 1) intravenously administered with the composition (0.2 mg / kg) obtained in Comparative Example 1, MCT rats (MCT) intravenously administered with physiological saline, and MCT In the same manner, rats (Sham) were administered subcutaneously with physiological saline instead of and intravenously administered with physiological saline.
FIG. 6 is a bar graph showing systolic right ventricular pressure in each rat.
FIG. 6 shows that MCT rats of the pulmonary hypertension model showed significantly higher blood pressure than Sham, and that the group administered with the composition obtained in Comparative Example 1 did not suppress the hypertension. . On the other hand, it can be seen that the group administered with the composition obtained in Example 2 significantly suppresses the MCT-induced increase in blood pressure compared to the group administered with the composition obtained in Comparative Example 1.
試験例7
　試験例1と同じ病態モデルラットを用いて、実施例2で得られた組成物を投与した場合に、右心室の肥大化を抑制することを確認した。
　試験例6と同様に実施例2で得られた組成物を投与した。MCT投与から21日後にラットを安楽死させた後、心室を摘出し右心室、左心室および心室中隔の重量をそれぞれ測定し、左心室と心室中隔の総重量に対する右心室の重量の相対比を算出した(Kimura H, Kasahara Y, Kurosu K, Sugito K, Takiguchi Y, Terai M, Mikata A, Natsume M, Mukaida N, Matsushima K, Kuriyama T. Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. Laboratory investigation; a journal of technical methods and pathology. May 1998;78(5):571-581.)。対照群も試験例6と同様に用意し試験した。
　図7に左心室と心室中隔の総重量に対する右心室の重量の相対比を棒グラフで示した。
　図7から、肺高血圧症モデルのMCTラットでは、Shamに対して右心室重量が増加しており(MCT)、比較例1で得られた組成物は、その右心室の重量増加を抑制していないことが分かる。一方、実施例2で得られた組成物は、MCT誘発性の相対的な右心室重量の増加を、比較例1で得られた組成物を投与した群に対して有意に抑えることが分かる。 Test Example 7
It was confirmed that when the composition obtained in Example 2 was administered using the same disease state model rat as in Test Example 1, the right ventricular hypertrophy was suppressed.
As in Test Example 6, the composition obtained in Example 2 was administered. After euthanizing the rat 21 days after MCT administration, the ventricle was removed, the right ventricle, left ventricle, and ventricular septum were weighed, and the right ventricular weight relative to the total weight of the left ventricle and ventricular septum The ratio was calculated (Kimura H, Kasahara Y, Kurosu K, Sugito K, Takiguchi Y, Terai M, Mikata A, Natsume M, Mukaida N, Matsushima K, Kuriyama T. Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor / monocyte chemoattractant protein-1. Laboratory investigation; a journal of technical methods and pathology. May 1998; 78 (5): 571-581.). A control group was also prepared and tested as in Test Example 6.
FIG. 7 is a bar graph showing the relative ratio of the right ventricular weight to the total weight of the left ventricle and the ventricular septum.
From FIG. 7, in the pulmonary hypertension model MCT rat, the right ventricular weight increased with respect to Sham (MCT), and the composition obtained in Comparative Example 1 suppressed the increase in the right ventricular weight. I understand that there is no. On the other hand, it can be seen that the composition obtained in Example 2 significantly suppresses MCT-induced increase in the right ventricular weight relative to the group administered with the composition obtained in Comparative Example 1.
試験例8
　試験例1と同じ病態モデルラットを用いて、実施例2で得られた組成物を投与した場合に、肺動脈壁の肥大化を抑制することを確認した。
　試験例6と同様に実施例2で得られた組成物を投与し、ラットを安楽死させた後に肺を摘出した。摘出した肺のうち右肺下部から4μm厚のパラフィン切片を作製した。これをエラスチカ・ワンギーソン染色(EV染色)し、肺血管壁の肥大化を結合組織の弾性線維を染めることで鑑別した。肺の切片あたり、20箇所の動脈壁の外殻の直径(外径)および内殻の直径(内径)を測定し、肥大化の度合いを以下の計算式から算出した〔% Wall thickness＝(内径×2)/(外径)×100〕( Ono S, Voelkel NF. PAF antagonists inhibit monocrotaline-induced lung injury and pulmonary hypertension. J Appl Physiol. Dec 1991; 71(6):2483-2492.参照)。対照群も試験例6と同様に用意し、試験した。
　図8に各ラットにおける肺動脈の肥大化をEV染色によって可視化した顕微鏡画像を示した。
　図9にこれら切片から肺動脈の肥大化の度合いを算出し［% Wall thickness］を棒グラフで示した。
　図8から、肺高血圧症モデルのMCTラット(MCT)の切片から、MCTラットではShamラットに対して肺動脈周囲において肺血管壁の肥大化が進行し、中央の血管部位の狭窄が生じていることが分かる。比較例1で得られた組成物は、その肥大化を抑制していないが、一方、実施例2で得られた組成物が、MCT誘発性の肺動脈の肥大化を抑制し、狭窄が減少していることが分かる。
　図9では、実施例2で得られた組成物が、比較例1で得られた組成物と比較して、肺高血圧症モデルのMCTラットにおける血管の肥大化を有意に抑制することを実測値から定量的に示した。 Test Example 8
Using the same disease state model rats as in Test Example 1, it was confirmed that when the composition obtained in Example 2 was administered, the pulmonary artery wall hypertrophy was suppressed.
The composition obtained in Example 2 was administered in the same manner as in Test Example 6, and the lungs were removed after euthanizing the rats. Among the excised lungs, 4 μm thick paraffin sections were prepared from the lower right lung. This was stained with Elastica-Wangieson staining (EV staining), and the hypertrophy of the pulmonary vascular wall was identified by staining the elastic fibers of the connective tissue. The outer shell diameter (outer diameter) and inner shell diameter (inner diameter) of 20 arterial walls per lung section were measured, and the degree of enlargement was calculated from the following formula [% Wall thickness = (inner diameter X2) / (outer diameter) x100] (see Ono S, Voelkel NF. PAF antagonists inhibit monocrotaline-induced lung injury and pulmonary hypertension. J Appl Physiol. Dec 1991; 71 (6): 2483-2492.). A control group was prepared and tested in the same manner as in Test Example 6.
FIG. 8 shows a microscopic image in which enlargement of the pulmonary artery in each rat is visualized by EV staining.
In FIG. 9, the degree of enlargement of the pulmonary artery was calculated from these sections, and [% Wall thickness] was shown as a bar graph.
From Fig. 8, from the section of the pulmonary hypertension model MCT rat (MCT), in the MCT rat, stenosis of the central vascular site has occurred in the pulmonary artery wall around the pulmonary artery compared to the Sham rat I understand. The composition obtained in Comparative Example 1 did not suppress the enlargement, while the composition obtained in Example 2 suppressed the MCT-induced pulmonary artery enlargement and reduced stenosis. I understand that
FIG. 9 shows that the composition obtained in Example 2 significantly suppresses vascular hypertrophy in MCT rats of the pulmonary hypertension model as compared with the composition obtained in Comparative Example 1. It showed quantitatively.

Claims (32)

  1. (i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、組成物。 (i) an RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and an RNA comprising a base sequence complementary to the sequence; and (ii) a liposome encapsulating the RNA therein. A composition containing.
  2. リポソームが、静脈内投与可能な大きさのリポソームである、請求項1記載の組成物。 2. The composition according to claim 1, wherein the liposome is a liposome of a size that can be administered intravenously.
  3. RNAが、RNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAである、請求項1または2記載の組成物。 3. The composition according to claim 1, wherein the RNA is RNA having an action of suppressing expression of the gene using RNA interference (RNAi).
  4. 肺血管壁組織の肥大に関連した遺伝子が、細胞増殖関連因子、分裂促進因子活性化タンパク質キナーゼ（MAPキナーゼ;MAPK）シグナル伝達関連因子およびアポトーシス関連因子のいずれかについての遺伝子である、請求項1～3のいずれかに記載の組成物。 2. The gene associated with hypertrophy of pulmonary vascular wall tissue is a gene for one of a cell growth-related factor, a mitogen-activated protein kinase (MAP kinase; MAPK) signaling-related factor, and an apoptosis-related factor. 4. The composition according to any one of to 3.
  5. 肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPキナーゼシグナル伝達関連因子についてのmRNAである、請求項1～3のいずれかに記載の組成物。 The composition according to any one of claims 1 to 3, wherein mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signal transduction-related factor.
  6. 肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPK/ERKキナーゼ(MEK)についてのmRNAである、請求項1～3のいずれかに記載の組成物。 The composition according to any one of claims 1 to 3, wherein the mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue is mRNA for MAPK / ERK kinase (MEK).
  7. mRNAがヒトのmRNAである、請求項1～6のいずれかに記載の組成物。 The composition according to any one of claims 1 to 6, wherein the mRNA is human mRNA.
  8. RNAを封入したリポソームが、リード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
    該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能である、請求項1～7のいずれかに記載の組成物。     The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA 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 claims 1 to 7.
  9. 極性有機溶媒がアルコールである、請求項8記載の組成物。 9. The composition of claim 8, wherein the polar organic solvent is an alcohol.
  10. 極性有機溶媒がエタノールである、請求項8記載の組成物。 9. The composition of claim 8, wherein the polar organic solvent is ethanol.
  11. リード粒子が、カチオン性物質を含むリード粒子であり、脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、請求項8～10のいずれかに記載の組成物。 The lead particle is a lead particle containing a cationic substance, and the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative as a constituent component. The composition according to any one of claims 8 to 10.
  12. RNAを封入したリポソームが、カチオン性物質を含むリード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
    該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、請求項1～7のいずれかに記載の組成物。     The liposome encapsulating RNA is a liposome composed of lead particles containing a cationic substance, composite particles containing RNA as a constituent, and a lipid bilayer coating the composite particles,
    The composition according to any one of claims 1 to 7, wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance as constituent components. object.
  13. カチオン性物質がN-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムおよび3β-［N-(N’,N’-ジメチルアミノエチル)カルバモイル］コレステロールから選ばれる一つ以上である、請求項11または12記載の組成物。 The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl)]- N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy- N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane 13. One or more selected from (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride, and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol. Composition.
  14. 水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体が、ポリエチレングリコール-ホスファチジルエタノールアミンである、請求項11～13のいずれかに記載の組成物。 The composition according to any one of claims 11 to 13, wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
  15. 中性脂質が卵黄ホスファチジルコリンである、請求項11～14のいずれかに記載の組成物。 The composition according to any one of claims 11 to 14, wherein the neutral lipid is egg yolk phosphatidylcholine.
  16. (i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、肺高血圧症治療剤。 (i) an RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and an RNA comprising a base sequence complementary to the sequence; and (ii) a liposome encapsulating the RNA therein. A therapeutic agent for pulmonary hypertension.
  17. リポソームが、静脈内投与可能な大きさのリポソームである、請求項16記載の肺高血圧症治療剤。 17. The therapeutic agent for pulmonary hypertension according to claim 16, wherein the liposome is a liposome having a size that can be intravenously administered.
  18. RNAが、RNA干渉(RNAi)を利用した前記遺伝子の発現抑制作用を有するRNAである、請求項16または17記載の肺高血圧症治療剤。 18. The therapeutic agent for pulmonary hypertension according to claim 16 or 17, wherein the RNA is RNA having an action of suppressing the expression of the gene utilizing RNA interference (RNAi).
  19. 肺血管壁組織の肥大に関連した遺伝子が、細胞増殖関連因子、MAPキナーゼシグナル伝達関連因子およびアポトーシス関連因子のいずれかについての遺伝子である、請求項16～18のいずれかに記載の肺高血圧症治療剤。 The pulmonary hypertension according to any one of claims 16 to 18, wherein the gene associated with hypertrophy of pulmonary vascular wall tissue is a gene for any one of a cell growth-related factor, a MAP kinase signal transduction-related factor, and an apoptosis-related factor. Therapeutic agent.
  20. 肺血管壁組織の肥大に関連した遺伝子のmRNAがMAPキナーゼシグナル伝達関連因子のについてのmRNAである、請求項16～18のいずれかに記載の肺高血圧症治療剤。 The therapeutic agent for pulmonary hypertension according to any one of claims 16 to 18, wherein mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is mRNA for a MAP kinase signal transduction-related factor.
  21. 肺血管壁組織の肥大に関連した遺伝子のmRNAがMEKのmRNAである、請求項16～18のいずれかに記載の肺高血圧症治療剤。 The therapeutic agent for pulmonary hypertension according to any one of claims 16 to 18, wherein mRNA of a gene associated with hypertrophy of pulmonary vascular wall tissue is MEK mRNA.
  22. mRNAがヒトのmRNAである、請求項16～21のいずれかに記載の肺高血圧症治療剤。 The therapeutic agent for pulmonary hypertension according to any one of claims 16 to 21, wherein the mRNA is human mRNA.
  23. RNAを封入したリポソームが、リード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
    該脂質二重膜の構成成分が特定の極性有機溶媒に可溶であり、該脂質二重膜の構成成分および該複合粒子が、特定の濃度で該極性有機溶媒を含む液に分散可能である、請求項16～22のいずれかに記載の肺高血圧症治療剤。     The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA 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 therapeutic agent for pulmonary hypertension according to any one of claims 16 to 22.
  24. 極性有機溶媒がアルコールである、請求項23記載の肺高血圧症治療剤。 24. The therapeutic agent for pulmonary hypertension according to claim 23, wherein the polar organic solvent is alcohol.
  25. 極性有機溶媒がエタノールである、請求項23記載の肺高血圧症治療剤。 24. The therapeutic agent for pulmonary hypertension according to claim 23, wherein the polar organic solvent is ethanol.
  26. リード粒子が、カチオン性物質を含むリード粒子であり、脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、請求項23～25のいずれかに記載の肺高血圧症治療剤。 The lead particle is a lead particle containing a cationic substance, and the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative as a constituent component. The therapeutic agent for pulmonary hypertension according to any of claims 23 to 25.
  27. RNAを封入したリポソームが、カチオン性物質を含むリード粒子と該RNAを構成成分とする複合粒子および該複合粒子を被覆する脂質二重膜から構成されるリポソームであり、
    該脂質二重膜が、中性脂質および水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体を構成成分とする脂質二重膜である、請求項16～22のいずれかに記載の肺高血圧症治療剤。     The liposome encapsulating RNA is a liposome composed of lead particles containing a cationic substance, composite particles containing RNA as a constituent, and a lipid bilayer coating the composite particles,
    The lung according to any one of claims 16 to 22, wherein the lipid bilayer is a lipid bilayer comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance as a constituent component. Antihypertensive agent.
  28. カチオン性物質がN-［1-(2,3-ジオレオイルプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジオレオイルプロピル)］-N,N-ジメチルアミン、N-［1-(2,3-ジオレイルオキシプロピル)］-N,N,N-トリメチル塩化アンモニウム、N-［1-(2,3-ジテトラデシルオキシプロピル)］-N,N-ジメチル-N-ヒドロキシエチル臭化アンモニウム、1,2-ジリノレイルオキシ- N,N-ジメチルアミノプロパン(DLinDMA)、1,2-ジリノレニルオキシ-N,N-ジメチルアミノプロパン(DLenDMA)、塩化ジデシルジメチルアンモニウム、塩化ジステアリルジメチルアンモニウムおよび3β-［N-(N’,N’-ジメチルアミノエチル)カルバモイル］コレステロールから選ばれる一つ以上である、請求項26または27記載の肺高血圧症治療剤。 The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl)]- N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy- N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane 28. The one or more selected from (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride, and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol. For treating pulmonary hypertension.
  29. 水溶性物質の脂質誘導体、脂肪酸誘導体または脂肪族炭化水素誘導体が、ポリエチレングリコール-ホスファチジルエタノールアミンである、請求項26～28のいずれかに記載の肺高血圧症治療剤。 29. The therapeutic agent for pulmonary hypertension according to claim 26, wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
  30. 中性脂質が卵黄ホスファチジルコリンである、請求項26～29のいずれかに記載の肺高血圧症治療剤。 The pulmonary hypertension therapeutic agent according to any of claims 26 to 29, wherein the neutral lipid is egg yolk phosphatidylcholine.
  31. (i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、組成物を哺乳動物に投与する肺高血圧症の治療方法。 (i) an RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and an RNA comprising a base sequence complementary to the sequence; and (ii) a liposome encapsulating the RNA therein. A method for treating pulmonary hypertension, comprising administering the composition to a mammal.
  32. (i)肺血管壁組織の肥大に関連した遺伝子のmRNAの連続する15～30塩基の配列および該配列と相補的な塩基の配列を含むRNAおよび(ii)該RNAを内部に封入したリポソームを含有する、組成物の肺高血圧症治療剤の製造のための使用。 (i) an RNA comprising a 15 to 30 base sequence of mRNA of a gene related to hypertrophy of pulmonary vascular wall tissue and an RNA comprising a base sequence complementary to the sequence; and (ii) a liposome encapsulating the RNA therein. Use of the composition for production of a therapeutic agent for pulmonary hypertension.
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