WO2010110314A1 - Agent thérapeutique pour hypertension pulmonaire comprenant un acide nucléique - Google Patents

Agent thérapeutique pour hypertension pulmonaire comprenant un acide nucléique 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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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/ja
Publication of WO2010110314A1 publication Critical patent/WO2010110314A1/fr

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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.

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Abstract

La présente invention concerne un agent thérapeutique pour hypertension pulmonaire comportant les éléments suivants : (i) un ARN comprenant une séquence composée de 15 à 30 nucléotides contigus, contenus dans un mRNA pour un gène associé à l'hypertrophie d'un tissu de vaisseau sanguin pulmonaire et une séquence nucléotide complémentaire à la séquence susmentionnée ; (ii) un liposome renfermant un ARN ; et d'autres éléments. Un exemple dudit liposome devant renfermer ledit ARN est un liposome qui comprend une particule composite, composée d'une particule tête de série comprenant une substance cationique et un ARN, et une membrane bicouche lipidique qui recouvre ladite particule composite. Ladite membrane bicouche lipidique comprend, par exemple, un dérivé lipidique composé d'un lipide neutre et d'une substance soluble dans l'eau, un dérivé d'acide gras, ou un dérivé d'hydrocarbure aliphatique.
PCT/JP2010/055080 2009-03-27 2010-03-24 Agent thérapeutique pour hypertension pulmonaire comprenant un acide nucléique WO2010110314A1 (fr)

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Publication number Priority date Publication date Assignee Title
JP2015518874A (ja) * 2012-06-08 2015-07-06 エスリス ゲーエムベーハーｅｔｈｒｉｓ ＧｍｂＨ メッセンジャーｒｎａの肺送達
JP2015520195A (ja) * 2012-06-08 2015-07-16 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド 非肺標的細胞へのｍＲＮＡの経肺送達
JP2017206567A (ja) * 2012-06-08 2017-11-24 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド 非肺標的細胞へのｍＲＮＡの経肺送達
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JP2019065052A (ja) * 2012-06-08 2019-04-25 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド 非肺標的細胞へのｍＲＮＡの経肺送達
US11090264B2 (en) 2012-06-08 2021-08-17 Translate Bio, Inc. Pulmonary delivery of mRNA to non-lung target cells

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