WO2010110318A1 - Therapeutic agent for arteriosclerotic diseases comprising nucleic acid - Google Patents

Abstract

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

Description

Arteriosclerotic disease therapeutic agent containing nucleic acid

The present invention relates to a therapeutic agent for arteriosclerotic diseases, a composition for treating arteriosclerotic diseases, and the like.

Arteriosclerosis is a condition in which an artery is thickened and hardened, and various pathologies caused by it are called arteriosclerosis.A disease caused by arteriosclerosis is an arteriosclerotic disease (for example, ischemic heart disease (angina pectoris).・ Myocardial infarction), cerebrovascular disorder (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage).
In the early lesions of arteriosclerosis, the endothelium is damaged for some reason, and monocytes adhere to the cell surface. Monocytes enter the subendothelium, differentiate into macrophages, take up cholesterol, become foam cells, and form fatty streak. Furthermore, vascular smooth muscle cells migrate to the intima and secrete fiber components such as collagen to form fibrous plaques (disorder reaction hypothesis, Ross, R .: Nature 362: 801, 1993). At a site where arteriosclerosis occurs (arteriosclerotic site), neointimal proliferation due to neovascularization, macrophage accumulation, and smooth muscle cell migration / proliferation is observed. In addition, it is known that vascular remodeling occurs in response to vascular load accompanying arteriosclerosis.
In addition, percutaneous transluminal coronary angioplasty (PTCA) has been widely performed as a treatment for ischemic heart diseases such as acute myocardial infarction and angina pectoris. However, it is known that stenosis occurs again in about 30-40% of patients within several months after PTCA. On the other hand, percutaneous coronary stenting, in which a stenotic part is expanded with a balloon catheter and then a metal stent is placed, has been developed and the restenosis rate has been reduced. (See N Engl J Med. 1994 Aug 25; 331 (8): 489-95). Furthermore, the rate of restenosis after in-stent restenosis site expansion is also a serious problem. In the stent restenosis site, neointimal proliferation is observed due to neovascularization, macrophage accumulation, and smooth muscle cell migration / proliferation. (See Circulation. 1998; 98: 224-233)

In arteriosclerotic sites and stent restenosis sites, genes related to neovascular proliferation or neointimal proliferation are expressed as genes related to arteriosclerotic diseases or stent restenosis (Patent Documents 1, 2 and Non-patent documents 1, 2, and 3), by suppressing the expression of genes related to neovascular growth or neointimal proliferation, it is possible to treat or prevent arteriosclerotic diseases or suppress stent restenosis Expected.
However, in order to suppress the expression of genes related to arteriosclerotic diseases and stent restenosis, it is necessary to deliver drugs to arteriosclerotic sites and stent restenosis sites. For example, when nucleic acids are used as drugs Has a very low in vivo stability and must be delivered with high selectivity to atherosclerotic sites and stent restenosis sites. However, there has been no report on such delivery means.

Meanwhile, nucleic acid-encapsulated liposomes (liposomes encapsulating nucleic acids in liposomes) have been reported as means for delivering nucleic acids into cells (see Patent Documents 3 to 5 and Non-patent Document 4). In Patent Document 3, as a method for producing a liposome encapsulating nucleic acid or the like, for example, a cationic lipid is dissolved in chloroform in advance, then mixed with an oligodeoxynucleotide (ODN) aqueous solution and methanol, and then centrifuged. Transfer the cationic lipid / ODN complex to the chloroform layer, then remove the chloroform layer, and add polyethyleneglycolized phospholipid, neutral lipid and water to form a water-in-oil (W / O) emulsion. Then, a method for producing ODN-encapsulated liposomes by treatment by the reverse phase evaporation method has been reported.In Patent Document 4 and Non-Patent Document 4, ODN is dissolved in an aqueous citric acid solution at pH 3.8, and lipid (in ethanol) is In addition, the ODN-encapsulated liposomes were prepared by reducing the ethanol concentration to 20 v / v%, sizing filtered, excess ethanol was removed by dialysis, and the sample was further adjusted to pH 7.5. Dialysed to remove ODN adhering to the liposome surface is reported a method of producing the ODN encapsulated liposomes, liposomes were each encapsulating active ingredients such as nucleic acid has been produced.

On the other hand, Patent Document 5 reports that a liposome in which an active ingredient such as a nucleic acid is encapsulated is produced by a method of coating fine particles with a lipid bilayer in a liquid. In this method, by reducing the concentration of the polar organic solvent in the aqueous solution containing the polar organic solvent in which the microparticles are dispersed and the lipid is dissolved, the microparticles are coated with the lipid bilayer membrane, and the coating is not performed in the liquid. For example, fine particles (coated fine particles) coated with a lipid bilayer having a size suitable for fine particles for intravenous injection or the like are produced with excellent efficiency. Patent Document 5 exemplifies a complex formed by an electrostatic interaction composed of a water-soluble drug and a cationic lipid, as an example of fine particles. The particle size of the coated fine particles coated with the composite particles varies depending on the coated composite particles, but the coated fine particles obtained by coating the ODN-lipid complex have a small particle size and can be used as an injection. In fact, it has been reported that the coated microparticles show a high blood retention when administered intravenously and accumulate in tumor tissues in large amounts.
However, none of Patent Documents 3 to 5 and Non-Patent Document 4 have reports on selective delivery of nucleic acids to atherosclerotic sites or stent restenosis sites.

Special table 2003-509427 gazette Special Table 2005-525802 Special Table 2002-508765 Special table 2002-501511 gazette International Publication No. 02/28367 Pamphlet

An object of the present invention is to provide a therapeutic agent for arteriosclerotic diseases containing a nucleic acid.

The present invention relates to the following (1) to (60).
(1) (i) an RNA comprising a 15 to 30 base sequence of mRNA of a gene related to atherosclerotic disease or stent restenosis and a base sequence complementary to the sequence; and (ii) an internal RNA. A composition comprising a liposome encapsulated in a gel.
(2) The composition according to (1), wherein the liposome is a liposome having a size that can be administered intravenously.
(3) The composition according to (1) or (2), wherein the RNA is RNA having an action of suppressing expression of the gene using RNA interference (RNAi).
(4) Genes related to atherosclerotic disease or stent restenosis are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor Receptor, mitogen-activated protein kinase (MAP kinase; MAPK) signaling-related factor, platelet-derived growth factor, platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor (KLF) ), Survivin, an Ets transcription factor, a nuclear factor and a hypoxia-inducing factor, the composition according to any one of (1) to (3).
(5) The composition according to any one of (1) to (3), wherein the mRNA of a gene associated with atherosclerotic disease or stent restenosis is mRNA for KLF.
(6) The composition according to any one of (1) to (3), wherein the mRNA of a gene associated with arteriosclerotic disease or stent restenosis is KLF5 mRNA.
(7) The composition according to any one of (1) to (6), wherein the mRNA is human mRNA.
(8) The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a constituent, and a lipid bilayer coating the composite particle,
The components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. , (1) to (7).
(9) The composition according to (8), wherein the polar organic solvent is an alcohol.
(10) The composition according to (8), wherein the polar organic solvent is ethanol.
(11) The lead particles are lead particles containing a cationic substance, and the lipid bilayer membrane is a lipid double substance comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component. The composition according to any one of (8) to (10), which is a film.
(12) The liposome encapsulating RNA is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent component, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (1) to (7), wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative. Composition.
(13) The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- (11) one or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, Or the composition according to (12).
(14) The composition according to any one of (11) to (13), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
(15) The composition according to any one of (11) to (14), wherein the neutral lipid is egg yolk phosphatidylcholine.
(16) (i) an RNA comprising a continuous 15-30 base sequence of mRNA of a gene related to arteriosclerotic disease or stent restenosis and a base sequence complementary to the sequence, and (ii) an internal RNA An arteriosclerotic disease treatment or stent restenosis inhibitor comprising a liposome encapsulated in.
(17) The therapeutic agent for arteriosclerotic disease or the stent restenosis inhibitor according to (16), wherein the liposome is a liposome having a size that can be intravenously administered.
(18) The therapeutic agent for arteriosclerotic diseases or the stent restenosis inhibitor according to (16) or (17), wherein the RNA is RNA having an action of suppressing the expression of the gene using RNA interference (RNAi).
(19) Genes related to atherosclerotic disease or stent restenosis are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor Receptor, MAP kinase signaling related factor, platelet derived growth factor, platelet derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, KLF, survivin, Ets transcription factor, nuclear factor and hypoxia inducible factor The atherosclerotic disease treatment or stent restenosis inhibitor according to any one of (16) to (18), which is a gene for any of the above.
(20) The arteriosclerotic disease treatment or stent restenosis inhibitor according to any one of (16) to (18), wherein the mRNA of a gene associated with atherosclerotic disease or stent restenosis is mRNA for KLF.
(21) The therapeutic agent for arteriosclerotic disease or the inhibitor for stent restenosis according to any one of (16) to (18), wherein the mRNA of a gene related to arteriosclerotic disease or stent restenosis is KLF5 mRNA.
(22) The arteriosclerotic disease treatment or stent restenosis inhibitor according to any one of (16) to (21), wherein the mRNA is human mRNA.
(23) A liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a component, and a lipid bilayer coating the composite particle;
The components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. The therapeutic agent for arteriosclerosis or the stent restenosis inhibitor according to any one of (16) to (22).
(24) The arteriosclerotic disease treatment or stent restenosis inhibitor according to (23), wherein the polar organic solvent is alcohol.
(25) The arteriosclerotic disease treatment or stent restenosis inhibitor according to (23), wherein the polar organic solvent is ethanol.
(26) A lipid particle comprising lead particles containing a cationic substance and a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance. The arteriosclerotic disease treatment or stent restenosis inhibitor according to any one of (23) to (25), which is a membrane.
(27) The RNA encapsulated liposome is a liposome composed of a lead particle containing a cationic substance, a composite particle containing the RNA as a constituent component, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (16) to (22), wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance. For treating arteriosclerotic diseases or inhibiting stent restenosis.
(28) The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- One or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, (26) Or (27) The arteriosclerotic disease treatment or stent restenosis inhibitor.
(29) The arteriosclerotic disease treatment or stent repair according to any one of (26) to (28), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine. Stenosis inhibitor.
(30) The arteriosclerotic disease treatment or stent restenosis inhibitor according to any of (26) to (29), wherein the neutral lipid is egg yolk phosphatidylcholine.
(31) (i) an RNA comprising a sequence of 15 to 30 bases of mRNA of a gene related to arteriosclerotic disease or stent restenosis and a base sequence complementary to the sequence; and (ii) the RNA inside A method for treating an arteriosclerotic disease or a method for suppressing stent restenosis, comprising administering a composition containing a liposome encapsulated in a mammal to a mammal.
(32) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to (31), wherein the liposome is a liposome having a size that can be intravenously administered.
(33) The method for treating arteriosclerotic diseases or the method for suppressing stent restenosis according to (31) or (32), wherein the RNA is RNA having an action of suppressing the expression of the gene using RNA interference (RNAi).
(34) Genes associated with atherosclerotic disease or stent restenosis are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor Receptor, MAP kinase signaling related factor, platelet derived growth factor, platelet derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, KLF, survivin, Ets transcription factor, nuclear factor and hypoxia inducible factor The method for treating arteriosclerotic disease or method for suppressing stent restenosis according to any one of (31) to (33), which is a gene for any of the above.
(35) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to any of (31) to (33), wherein the mRNA of a gene associated with atherosclerotic disease or stent restenosis is mRNA for KLF. .
(36) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to any of (31) to (33), wherein the mRNA of a gene associated with arteriosclerotic disease or stent restenosis is KLF5 mRNA.
(37) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to any of (31) to (36), wherein the mRNA is human mRNA.
(38) RNA encapsulated liposome is a liposome composed of a lead particle, a composite particle comprising the RNA as a constituent, and a lipid bilayer coating the composite particle,
The components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to any of (31) to (37).
(39) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to (38), wherein the polar organic solvent is alcohol.
(40) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to (38), wherein the polar organic solvent is ethanol.
(41) The lipid bilayer in which the lead particle is a lead particle containing a cationic substance, and the lipid bilayer membrane is a lipid, fatty acid derivative or aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance. The method for treating arteriosclerotic disease or method for suppressing stent restenosis according to any one of (38) to (40), which is a membrane.
(42) The liposome encapsulating RNA is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent component, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (31) to (37), wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance. For treating arteriosclerotic disease or inhibiting stent restenosis.
(43) Cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- (41) one or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, Or the method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to (42).
(44) The method for treating arteriosclerotic disease or stent according to any one of (41) to (43), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine Restenosis suppression method.
(45) The method for treating arteriosclerotic disease or the method for suppressing stent restenosis according to any of (41) to (44), wherein the neutral lipid is egg yolk phosphatidylcholine.
(46) (i) an RNA comprising a sequence of 15 to 30 bases of mRNA of a gene related to atherosclerotic disease or stent restenosis and a base sequence complementary to the sequence; and (ii) an internal RNA Use of a composition containing a liposome encapsulated in an arteriosclerotic disease treatment or a stent restenosis inhibitor.
(47) The use according to (46), wherein the liposome is a liposome of a size that can be administered intravenously.
(48) The use according to (46) or (47), wherein the RNA is RNA having an action of suppressing the expression of the gene using RNA interference (RNAi).
(49) Genes associated with atherosclerotic disease or stent restenosis are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor Receptor, MAP kinase signaling related factor, platelet derived growth factor, platelet derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, KLF, survivin, Ets transcription factor, nuclear factor and hypoxia inducible factor The use according to any of (46) to (48), which is a gene for any of the above.
(50) The use according to any one of (46) to (48), wherein the mRNA of a gene associated with arteriosclerotic disease or stent restenosis is mRNA for KLF.
(51) The use according to any one of (46) to (48), wherein the mRNA of a gene associated with arteriosclerotic disease or stent restenosis is KLF5 mRNA.
(52) The use according to any one of (46) to (51), wherein the mRNA is human mRNA.
(53) The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising the RNA as a constituent, and a lipid bilayer coating the composite particle,
The components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. , Use according to any of (46) to (52).
(54) The use according to (53), wherein the polar organic solvent is an alcohol.
(55) The use according to (53), wherein the polar organic solvent is ethanol.
(56) A lipid particle comprising lead particles containing a cationic substance and a lipid bilayer membrane comprising a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a neutral lipid and a water-soluble substance. The use according to any one of (53) to (55), which is a membrane.
(57) RNA encapsulated liposome is a liposome composed of a lead particle containing a cationic substance, a composite particle comprising the RNA as a constituent, and a lipid bilayer coating the composite particle,
The lipid bilayer membrane according to any one of (46) to (52), wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance. Use of.
(58) Cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)] -N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl) )]-N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N- One or more selected from dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol, (56) Or use according to (57).
(59) The use according to any one of (56) to (58), wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
(60) The use according to any one of (56) to (59), wherein the neutral lipid is egg yolk phosphatidylcholine.

A composition comprising a liposome encapsulating RNA containing a continuous 15-30 base sequence of mRNA of a gene related to arteriosclerotic disease or stent restenosis of the present invention and a base sequence complementary to the sequence, Administration to mammals suppresses the expression of genes related to arteriosclerotic disease or stent restenosis, for example, genes related to neovascular growth or neointimal proliferation, at sites of arteriosclerosis and stent restenosis can do. In addition, at the arteriosclerosis site or stent restenosis site, by suppressing the expression of genes related to neovascular growth or neointimal proliferation, the treatment or prevention of arteriosclerotic disease or the suppression of stent restenosis Can do.

When the composition obtained in Example 1 was administered, a phase contrast microscopic image (left) and a confocal laser microscopic image (right) of a frozen section of an arteriosclerotic site in an ApoE-deficient mouse, which is a model of atherosclerosis. ). When the composition obtained in Example 2 was administered, H / E-stained images (left) and confocal laser microscope images (right) of frozen sections of the carotid arteries in mice of the common carotid artery ligation model are shown. When the composition obtained in Comparative Example 1 was administered, H / E-stained images (left) and confocal laser microscope images (right) of frozen sections of the carotid arteries in mice of the common carotid artery ligation model are shown. Fig. 2 shows H / E-stained images (left) and confocal laser microscope images (right) of frozen sections of the carotid artery in mice with a common carotid artery ligation model when physiological saline was administered. It was shown that when the composition obtained in Example 3 was administered, neointimal formation was suppressed in the common carotid artery ligation model mouse. The vertical axis represents the sum of the areas of the intima and media. In addition, * is a significant difference p value between the group administered with the composition obtained in Example 3 and the group administered with the composition obtained in Comparative Example 2 as a result of statistical analysis (student t test). <0.04kg is recognized.

The gene related to arteriosclerotic disease or stent restenosis used in the present invention may be a gene related to atherosclerotic disease or stent restenosis that is expressed by producing mRNA at the arteriosclerotic site or stent restenosis site. Examples thereof include, but are not limited to, genes associated with neovascular growth or neointimal proliferation, preferably vascular endothelial growth factor (hereinafter abbreviated as VEGF), vascular endothelial growth factor receptor Body (vascular endothelial growth factor receptor, hereinafter abbreviated as VEGFR), fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor receptor, MAP kinase signaling related factor, platelet-derived growth factor, Platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor (KLF) (Abbreviated), survivin, Ets transcription factor, nuclear factor, genes encoding proteins such as hypoxia-inducing factor, etc., specifically VEGF gene, VEGFR gene, fibroblast growth factor gene, fibroblast growth factor Receptor gene, epidermal growth factor gene, epidermal growth factor receptor gene, MAP kinase signaling related factor gene, platelet-derived growth factor gene, platelet-derived growth factor receptor gene, hepatocyte growth factor gene, hepatocyte growth factor receptor Gene, KLF gene, survivin gene, Ets transcription factor gene, nuclear factor gene, hypoxia inducing factor gene, etc., preferably VEGF gene, VEGFR gene, KLF gene, etc., more preferably KLF gene Even more preferred is the KLF5 gene.

* KLF includes the KLF family. The family is a family of transcription factors characterized by a C-terminal zinc finger motif, KLF1, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KLF10, KLF11, KLF12 , KLF13, KLF14, KLF15 or KLF16. In mammals, the KLF family is important for the differentiation of various tissues and cells such as erythrocytes, vascular endothelial cells, smooth muscle, skin or lymphocytes, as well as cancer, cardiovascular disease, cirrhosis, kidney disease or immune disease It has been reported to play an important role in the pathogenesis of various diseases such as [The Journal of Biological Chemistry (2001), 276, 37, p.34355-34358, Genome Biology, 2003, Vol. 4, No. 2, p. 206].

KLF5 in the KLF family is also called BTEB2 (basic transcriptional element binding protein 2) or IKLF (intestinal-enriched Kruppel-like factor). Expression of KLF5 in vascular smooth muscle is controlled at the developmental stage, and high expression is observed in fetal vascular smooth muscle, whereas expression is not observed in normal adult vascular smooth muscle. In addition, KLF5 is highly expressed in intimal smooth muscle that has been born after exfoliation with a balloon catheter, and KLF5 is also observed in smooth muscle in lesions of arteriosclerosis and restenosis [Circulation, 2000, Vol.102, No.20, p.2528-2534].

VEGF is a growth factor specific for vascular endothelial cells discovered by Ferrara et al. In 1983. In the same year, a factor with vascular permeability was discovered by Senger, Dvorak et al. And named VPF (vascular permeability factor). Analysis of the amino acid sequence of the protein revealed that the two were identical. VEGF binds to endothelial cell receptors inside blood vessels to promote proliferation. VEGF not only creates blood vessels during fetal life, but also acts when creating pathological blood vessels. For example, if the cancer grows to some extent and becomes deficient in oxygen, VEGF and its receptors increase and angiogenesis occurs. It is also thought to cause cancerous ascites due to the vascular permeability enhancing action. As diabetes progresses, new blood vessels form in the retina, and VEGF also works there. In other words, it is a protein that creates new blood vessels. It can be said that it plays an important role in angiogenesis by its expression being induced by hypoxia. Moreover, the involvement of this factor is strongly suggested in explaining not only angiogenesis but also the mechanism of edema observed in tumors or inflammatory lesions.

On the other hand, VEGFR is possessed by vascular endothelial cells and cancer cells themselves, and when VGEF binds to the receptor, the receptor itself is phosphorylated (activated), and as a result, various commands such as proliferation and migration occur inside the cell. Communicated. It is known that by inhibiting phosphorylation of this receptor, intracellular transmission is inhibited and angiogenesis is inhibited.

The RNA used in the present invention includes a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of the mRNA of the gene, and a base sequence complementary to the sequence. RNA.
The RNA used in the present invention also includes DNA in which part or all of ribose is substituted with deoxyribose, that is, DNA. Furthermore, ribonucleotides and deoxyribonucleotides in RNA used in the present invention may be modified, for example, sugar-modified nucleotide analogs, phosphodiester bond-modified nucleotide analogs, and the like. The RNA used in the present invention also includes derivatives in which an oxygen atom or the like contained in a phosphate part, an ester part, or the like in the RNA is substituted with another atom such as a sulfur atom.
In the present invention, the ribonucleotide in the RNA used in the present invention is deoxyribonucleotide, the ribonucleotide and deoxyribonucleotide in the RNA used in the present invention are modified, the phosphate in the RNA used in the present invention Substituting oxygen atoms, etc., contained in the ester part, ester part, etc., with other atoms, such as sulfur atoms, improves the nuclease resistance compared to RNA or DNA, and stabilizes it. It may be formed for any purpose such as increasing affinity, increasing cell permeability, or visualizing.

The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to part or all of the chemical structure of the sugar of the nucleotide. For example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged Nucleic Acid (BNA), more specifically, 2′-position oxygen atom and 4′-position carbon atom via methylene Crosslinked locked artificial nucleic acid (Locked Nucleic Acid) (LNA), ethylene bridged structure type artificial nucleic acid (Ethylene bridged nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)], etc. Nucleic acid (PNA) [Acc. Chem. Res., 32, 624) (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

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)].

The RNA used in the present invention is preferably an RNA having an action of suppressing the expression of the gene using RNA interference (RNAi). Here, RNA that suppresses the expression of a target gene using RNA interference (RNAi) will be described using RNA that suppresses the expression of the KLF5 gene as an example. Other genes have similar structures and can be obtained by similar operations.

The RNA that suppresses the expression of the KLF5 gene is a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of KLF5 配 列 mRNA (hereinafter referred to as sequence X) and complementary to the sequence. It contains a base sequence (hereinafter referred to as complementary sequence X ′). RNA includes: (A) double-stranded RNA consisting of the strand of sequence X (sense strand) and complementary strand X ′ (antisense strand); (B) the strand of sequence X (sense strand) and complementary sequence X ′ 1 to 6, preferably 2 to 4 nucleotides are the same at the 3 ′ end of the strand of the sequence X or the complementary sequence X ′ of the double-stranded RNA comprising the strands (antisense strands) RNA that consists of differently added double-stranded RNAs that suppress the expression of the KLF5 gene (hereinafter referred to as RNA with a structure like (A) and (B) is called KLF5siRNA), and (C) RNA consisting of sequence X And RNA consisting of complementary sequence X ′ is an RNA having a hairpin structure that is connected by a spacer oligonucleotide and suppresses the expression of KLF5 gene, (D) RNA consisting of sequence X and RNA consisting of complementary sequence X ′ , Linked by spacer oligonucleotides, with 1 to 6, preferably 2 to 4 nucleotides added to the 3 ′ end A RNA having a hairpin structure capable of suppressing the expression of KLF5 gene RNA (hereinafter, (C) and RNA is referred to as a KLF5shRNA such as (D)) and the like. The nucleotide base added to these RNAs may be one or more of guanine, adenine, cytosine, thymine and uracil, and may be RNA or DNA, but uridylic acid (U) and deoxythymidylic acid ( Any one or two of dT) are preferred. The spacer oligonucleotide is preferably RNA of 6 to 12 bases, and the sequence at the 5 'end is preferably 2 U. An example of the spacer oligonucleotide is RNA having the sequence UUCAAGAGA. Either of the two RNAs connected by the spacer oligonucleotide may be on the 5 'side. In either case, the nucleotide sequence of the nucleotide added adjacent to the 3 ′ end side of the complementary sequence X ′ may be the base sequence complementary to the sequence of the nucleotide adjacent to the sequence X in the mRNA. Well, this structure is more preferred. The sequence X may be any sequence as long as it is a sequence of 15 to 30 bases, preferably 17 to 25 bases, more preferably 19 to 23 bases of KLF5 mRNA. Extract a partial base sequence of 21 bases starting with AA from the base sequence of KLF5 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%.

The RNA that suppresses the expression of the KLF5 gene differs in the intensity of suppression of the KLF5 gene expression depending on the sequence X. In some cases, the suppression is weak. The RNA of the present invention is prepared by introducing the RNA into a cell expressing the KLF5 gene, measuring the expression of the KLF5 gene, and selecting an RNA that strongly suppresses the expression of the KLF5 gene. Can be obtained.
Examples of RNA that suppresses expression of the KLF5 gene include RNAs No. 1 to No. 11 shown in Table 1.

The method for synthesizing the RNA used in the present invention is not particularly limited, and it can be synthesized by a method using a known chemical synthesis, an enzymatic transcription method or the like. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, etc., for example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do. As an enzymatic transcription method, transcription or synthesis can be performed using a plasmid or DNA having a target base sequence as a template and using a typical phage RNA polymerase, for example, T7 polymerase, T3 polymerase, SP6 RNA polymerase, or the like.

In addition, KLF5siRNA No. 1 in Table 1 can be prepared by, for example, requesting Japan Bioservice Co., Ltd., chemical synthesis, and annealing. KLF5 siRNA Nos. 2 to 11 in Table 1 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. For example, a cationic lipid / RNA complex is a hydrophobic organic solvent. Liposomes produced by dispersing in layers, adding polyethylene glycolated lipids, neutral lipids and water to form water-in-oil (W / O) emulsions and processing by reverse phase evaporation (see Patent Document 3) ), RNA is dissolved in an acidic electrolyte aqueous solution, lipid (in ethanol) is added, and RNA-encapsulated liposomes are prepared by lowering the ethanol concentration, and then the RNA attached to the liposome surface by dialysis by raising the pH of the sample (See Patent Document 4 and Non-Patent Document 4) produced by removing the particles, composite particles composed of lead particles and the RNA, and liposomes composed of lipid bilayer membranes encapsulating the composite particles (specially Reference 5 and the pamphlet of International Publication No. 2006/080118) and the like, and a liposome composed of a composite particle composed of a lead particle and the RNA and a lipid bilayer membrane encapsulating the composite particle are preferred, and the lipid More preferably, the components of the bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. preferable. The liposome A is preferably composed of a lead particle containing a cationic substance, a composite particle containing the RNA as a constituent component, and a lipid bilayer covering the composite particle. And liposomes comprising lipid derivatives, fatty acid derivatives or aliphatic hydrocarbon derivatives of water-soluble substances as a constituent, and the lipid bilayer constituent is soluble in a specific polar organic solvent. More preferably, the components of the double membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration.
In the present invention, the term “dispersing” means dispersing without dissolving.

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. Is preferred. The lipid may be any of simple lipids, complex lipids or derived lipids, such as phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids, sterols, and cationic lipids, but are not limited thereto. Not. Preferable examples include phospholipids and cationic lipids.

Examples of the phospholipid in the lipid constituting the lead particles include phosphatidylcholine (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine, Oleoylphosphatidylcholine), phosphatidylethanolamine (specifically distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DORE), dimyristoylphosphoethanolamine (DMPE)) , Palmitoyl oleoyl-phosphatidylethanolamine (POPE), 1 -stearoyl- 2 -oleoyl-phosphine Glycidylphosphoamine (specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyl oleoylphosphatidylglycerol (POPG), lysophosphatidylcholine, etc.), sphingophospholipid (specifically Is sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate, etc.), glycerophosphonolipid, sphingophosphonolipid, natural lecithin (specifically egg yolk lecithin, soybean lecithin, etc.) or hydrogenated phospholipid ( Specific examples include natural or synthetic phospholipids such as hydrogenated soybean phosphatidylcholine.

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.

Examples of the glycosphingolipid in the lipid constituting the lead particle 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. As the derivative, for example, —NH ₂ such as sphingan, icosasphingan or sphingosine —NHCO (CH ₂ ) _x CH ₃ (wherein x represents an integer of 0 to 18, among which 6, 12 or 18 is preferable. ) And the like.

Examples of the sterol in the lipid constituting the lead particle include cholesterol, dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brush casterol, ergocasterol, fucostosterol and the like.

As the cationic lipid in the lipid constituting the lead particle, among the polar lipids having amphipathic properties that have both hydrophilic and hydrophobic properties and having a lipid bilayer structure in water, It has a structure having a primary amine, secondary amine, tertiary amine, quaternary ammonium, a heterocyclic ring containing a nitrogen atom, etc., for example, N- [1- (2,3-dioleoyl Propyl)]-N, N, N-trimethylammonium chloride (DOTAP), N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine (DODAP), N- [1- ( 2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl -1-propanaminium trifluoroacetic acid (DOSPA), N- [1- (2,3-ditetradecyloxypropyl)]-N, N-dimethyl-N-hydroxy ester Ammonium bromide (DMRIE), N- [1- (2,3-dioleyloxypropyl)]-N, N-dimethyl-N-hydroxyethylammonium bromide (DORIE), 1,2-dilinoleyloxy -N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride or 3β- [N- ( N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like.

In liposome B, these lipids are used singly or in combination of two or more, preferably in combination of two or more. As a combination when used in combination of two or more, for example, hydrogenated soybean phosphatidylcholine, polyethyleneglycolized lipid (synonymous with polyethyleneglycolized lipid described later) and cholesterol, a combination of two or more components, distearoylphosphatidylcholine, polyethyleneglycolated Combination of two or more components selected from lipid and cholesterol, combination of EPC and DOTAP, combination of DOTAP and polyethylene glycolated lipid, combination of EPC, DOTAP and polyethylene glycolated lipid, combination of EPC, DOTAP, cholesterol and polyethylene glycolated lipid Etc.

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.

Examples of lipid aggregates include spherical micelles, spherical reverse micelles, sausage-like micelles, sausage-like reverse micelles, plate-like micelles, plate-like reverse micelles, hexagonal I, hexagonal II or aggregates composed of two or more lipid molecules. .

Examples of the 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.

Examples of the nonionic surfactant in the emulsion particles constituting the lead particles include polyoxyethylene sorbitan monooleate (specifically polysorbate 80), polyoxyethylene polyoxypropylene glycol (specifically Pluronic F68). ), Sorbitan fatty acid esters (specifically sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.) or glycerin fatty acid Examples include esters.

Examples of the polymer micelle include natural polymers such as albumin, dextran, polyfect, chitosan, dextran sulfate or DNA, such as poly-L-lysine, polyethyleneimine, polyaspartic acid, styrene maleic acid copolymer, isopropyl Examples include micelles composed of one or more polymers such as acrylamide-acrylpyrrolidone copolymer, polyethylene glycol-modified dendrimer, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or salts thereof.

Here, the salts in the polymer include, for example, metal salts, ammonium salts, acid addition salts, organic amine addition salts, amino acid addition salts and the like. Examples of the metal salt include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of the ammonium salt include salts such as ammonium and tetramethylammonium. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate or phosphate, and organic acid salts such as acetate, maleate, fumarate or citrate. Examples of organic amine addition salts include addition salts such as morpholine and piperidine. Examples of amino acid addition salts include addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid or lysine.

Examples of the metal colloid include metal colloids containing gold, silver, platinum, copper, rhodium, silica, calcium, aluminum, iron, indium, cadmium, barium or lead.

In addition, the lead particles in the present invention preferably contain a lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers. It is more preferable to contain a lipid derivative or fatty acid derivative of a water-soluble polymer, and it is further preferable to contain a lipid derivative or fatty acid derivative of a water-soluble polymer. Lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers are those in which part of the molecule and other components of the lead particle, such as hydrophobic affinity, electrostatic It is a substance with a two-sided property that has the property of binding due to mechanical interaction, etc., and the other part has the property of binding with the solvent at the time of lead particle production, for example, hydrophilic affinity, electrostatic interaction, etc. Preferably there is. The lipid derivative or fatty acid derivative or surfactant of one or more substances selected from sugar, peptide, nucleic acid and water-soluble polymer may be contained as a component of the lead particle, and in addition to the component of the lead particle It may be used.

Examples of lipid derivatives or fatty acid derivatives of one or more substances selected from sugars, peptides and nucleic acids include sugars such as sucrose, sorbitol, and lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides, and glutathione peptides. For example, a nucleic acid such as DNA, RNA, plasmid, siRNA, or ODN and a lipid listed in the definition of the lead particle or a fatty acid such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. And the like.

Further, the sugar lipid derivatives or fatty acid derivatives include, for example, glyceroglycolipids or sphingoglycolipids mentioned in the definition of the lead particles.

Examples of the water-soluble polymer lipid derivative or fatty acid derivative include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, Chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol and the like or their derivatives and the lipids mentioned in the definition of lead particles, for example, stearic acid, palmitic acid, Examples include those formed by bonding with fatty acids such as myristic acid or lauric acid, and more preferred are lipid derivatives such as polyethylene glycol derivatives and polyglycerin derivatives, or fatty acid derivatives. Is, more preferably, a lipid derivative or a fatty acid derivative of a polyethylene glycol derivative.

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.

Examples of lipid derivatives or fatty acid derivatives of polyglycerin derivatives include polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine) and polyglycerin fatty acid esters, and more preferably polyglycerinized lipids. can give.

Examples of the surfactant include polyoxyethylene sorbitan monooleate (specifically, polysorbate 80), polyoxyethylene polyoxypropylene glycol (specifically, Pluronic F68), sorbitan fatty acid ester (specifically, sorbitan) Monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid ester or polyethylene glycol alkyl ether, etc. are preferred, Examples thereof include polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether.

The above-described lead particles preferably have a positive charge. The positive charge described here includes a charge in RNA used in the present invention, a charge that generates an electrostatic attraction with respect to intramolecular polarization, a surface polarization, and the like. In order for the lead particles to have a positive charge, the lead particles preferably contain a cationic substance, and the lead particles more preferably contain a cationic lipid.

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.

As a cationic substance, for example, a cationic substance [specifically, a cationic lipid (as defined above), a cationic polymer, etc.] among those exemplified in the definition of the lead particle, a value below the isoelectric point Examples thereof include proteins or peptides capable of forming a complex at a pH of, preferably cationic lipids.

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. Examples of the protein or peptide include albumin, orosomucoid, globulin, fibrinogen, pepsin, and ribonuclease T1.

The lead particles in the present invention can be produced by a known production method or a method according thereto, and may be produced by any production method. For example, a known liposome preparation method can be applied to the production of lead particles containing liposome B, which is one of the lead particles, as a constituent component. Known liposome preparation methods include, for example, Bangham et al.'S liposome preparation method [“J. Mol. Biol.”, 1965, Vol. 13, p.238- 252], ethanol injection method ["Journal of Cell Biology", 1975, Vol. 66, pp. 621-634], French press method ["FBS. Letters (FEBS Lett.) ”, 1979, Vol. 99, p.210-214], freeze-thaw method [“ Arch. Biochem. Biophys. ””, 1981 Year 212, p. 186-194], reversed-phase evaporation [“Proceedings of the National Academy of Sciences United States of America (Proc. Natl. Acad. Sci. USA) ”, 1978, Vol. 75, p.4194-4198] or pH gradient method (for example, Patent No. 2572). No. 554, Japanese Patent No. 2659136, etc.). As a solution for dispersing liposome B in the production of liposome B, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, or the like can be used. In the production of liposome B, for example, an antioxidant such as citric acid, ascorbic acid, cysteine or ethylenediaminetetraacetic acid (EDTA), for example, an isotonic agent such as glycerin, glucose or sodium chloride can be added. It is. Liposomes B can also be produced by dissolving lipids or the like in an organic solvent such as ethanol and distilling off the solvent, and then adding physiological saline or the like and stirring to form liposomes.

Also, for example, surface modification of the lead particles such as liposome B with a cationic substance, polymer, polyoxyethylene derivative, etc. can be arbitrarily performed [Radics, edited by F. Martin, “Stealth” • Liposomes ”(USA), CRC Press Inc., 1995, p. 93-102]. Examples of the polymer that can be used for the surface modification include dextran, pullulan, mannan, amylopectin, and hydroxyethyl starch. Examples of the polyoxyethylene derivative include polysorbate 80, Pluronic F68, polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, and PEG-DSPE. Surface modification of lead particles such as liposome B is one of the methods in which lead particles contain lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from sugars, peptides, nucleic acids and water-soluble polymers. It is.

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].

In addition, a method for producing a composite comprising a combination of two or more selected from lipid aggregates, liposome B, emulsion particles, polymer micelles, metal colloids and the like constituting the lead particles, for example, lipids, polymers, etc. in water May be mixed, and a granulation step, a sterilization step, and the like may be added if desired. In addition, the complex can be produced in various solvents such as acetone or ether.

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. Here, the neutral lipid refers to the cationic lipids mentioned in the cationic substance and the anionic lipids mentioned in the adhesion competitor described later when the lead particles have a positive charge. More preferably, neutral lipids include phospholipids, glyceroglycolipids or sphingoglycolipids. More preferred are phospholipids, and more preferred is EPC. These lipids can be used alone or in combination of two or more.

The components of the lipid bilayer membrane covering the composite particles are preferably soluble in a specific polar organic solvent, and preferably dispersible in a liquid containing the polar organic solvent at a specific concentration. . The concentration of the polar solvent in the liquid containing the polar solvent at a specific concentration is preferably a concentration at which the constituent components of the lipid bilayer membrane can be dispersed and the composite particles can be dispersed. Examples of the polar organic solvent include alcohols such as methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, and tert-butanol, glycols such as glycerin, ethylene glycol, and propylene glycol, and polyethylene glycol. Examples thereof include polyalkylene glycols, among which alcohol is preferable and ethanol is more preferable.
Examples of the solvent other than the polar organic solvent in the liquid containing the polar organic solvent in the present invention include water, liquid carbon dioxide, liquid hydrocarbon, halogenated carbon or halogenated hydrocarbon, and preferably water. can give. Moreover, an ion or a buffer component etc. may be included. One or more solvents can be used, but when two or more solvents are used, a compatible combination is preferred.

The lipid bilayer coating the composite particles preferably contains a lipid derivative of a water-soluble substance, a fatty acid derivative or an aliphatic hydrocarbon derivative, polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester or polyethylene glycol alkyl ether, More preferably, it contains a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance. Examples of the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance include one or more lipid derivatives or fatty acid derivatives, or sugars, peptides selected from the aforementioned sugars, peptides, nucleic acids and water-soluble polymers. An aliphatic hydrocarbon derivative of one or more substances selected from nucleic acids and water-soluble polymers, preferably lipid derivatives or fatty acid derivatives of the water-soluble polymers, more preferably the polyethylene glycolated lipids. More preferred is polyethylene glycol-phosphatidylethanolamine. In addition, as the aliphatic hydrocarbon derivative of the water-soluble substance in the present invention, a substance obtained by binding a water-soluble substance and, for example, an alcoholic residue of a long-chain aliphatic alcohol, polyoxypropylene alkyl or glycerin fatty acid ester, etc. Can also be raised.

Examples of the aliphatic hydrocarbon derivatives of sugars, peptides or nucleic acids include sugars such as sucrose, sorbitol or lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides or peptides such as glutathione, or DNA, RNA, plasmids, etc. , Aliphatic hydrocarbon derivatives of nucleic acids such as siRNA or ODN.

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.

When the lead particle is a fine particle comprising liposome B as a constituent, the liposome A is composed of a composite particle comprising liposome B and RNA used in the present invention and a lipid bilayer coating the composite particle. Thus, it is classified as a liposome in a narrow sense based on its configuration, and even when the lead particle is other than a fine particle containing liposome B as a constituent component, it is classified as a liposome in a broad sense because it is covered with a lipid bilayer membrane. In the present invention, the lead particles are more preferably fine particles containing liposome B as a constituent component.

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 3, 4, 5, Non-Patent Document 4 or the like, or a method similar thereto, or, for example, RNA used in the present invention is attached to or encapsulated in lead particles. After the composite particles are manufactured, the composite particles and the coating layer component contain a polar organic solvent in which the coating layer component is soluble, the composite particles do not dissolve, and the coating layer component exists in a dispersed state. It can be produced by a production method including a step of dispersing in a liquid having a possible concentration and a step of coating the composite particles with the coating layer component.

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. In step 1, in order to suppress the aggregation of lead particles, the lead particles are preferably lead particles containing an aggregation inhibitor. Preferred examples of the aggregation inhibitor include lipid derivatives or fatty acid derivatives or surfactants of one or more substances selected from the sugars, peptides, nucleic acids, and water-soluble polymers. In addition, when the lead particle has a positive charge, the RNA used in the present invention and the adhesion competitor are coexisted in the liquid in which the lead particle is dispersed, and the adhesion competitor is attached to the lead particle together with the RNA. In addition, when the lead particles are lead particles containing an aggregation inhibitor, an adhesion competitor may be used to further suppress the aggregation of the lead particles. As a combination of any of the lead particles and RNA used in the present invention, it is preferable to select a combination in which the composite particles can be dispersed in a liquid containing a polar organic solvent. More preferably, the solubility is lower than that of the components of the lipid bilayer membrane used in Step 2 or 3, and the components of the lipid bilayer membrane can be dispersed in the liquid containing the polar organic solvent. It is more preferable to select a combination in which a liquid containing the polar organic solvent is present at a concentration capable of dispersing the composite particles.

Examples of adhesion competitors include anionic substances. The anionic substance includes a substance that adheres electrostatically to the constituent components of the lead particles by electrostatic attraction due to intramolecular charge, intramolecular polarization, and the like. An anionic substance as an adhesion competing agent is an anionic substance, but even an amphoteric substance having both an anionic group and a cationic group is affected by pH, binding to other substances, etc. Since the relative negative degree changes, it can be classified into anionic substances depending on the occasion.

Examples of the anionic substance include anionic lipids, anionic surfactants, anionic polymers, etc., and proteins, peptides, or nucleic acids that can form a complex at a pH higher than the isoelectric point, and preferably dextran sulfate. Dextran sodium sulfate, chondroitin sulfate, chondroitin sulfate sodium, hyaluronic acid, chondroitin, dermatan sulfate, heparan sulfate, heparin, keratan sulfate or dextran fluorescein anionic. These anionic substances can be used alone or in combination of two or more.

Examples of the anionic lipid include phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, and the like.
Examples of the anionic surfactant include acyl sarcosine, sodium alkyl sulfate, alkyl benzene sulfonate, and fatty acid sodium having 7 to 22 carbon atoms. Specific examples include sodium dodecyl sulfate, sodium lauryl sulfate, sodium cholate, sodium deoxycholate, or sodium taurodeoxycholate.

Examples of the anionic polymer include polyaspartic acid, styrene maleic acid copolymer, isopropylacrylamide-acrylpyrrolidone copolymer, polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid, polyethylene glycolated polylactic acid, dextran sulfate. Dextran sodium sulfate, chondroitin sulfate, chondroitin sulfate sodium, hyaluronic acid, chondroitin, dermatan sulfate, heparan sulfate, heparin, keratan sulfate or dextran fluorescein anionic.
The protein or peptide capable of forming a complex at a pH value equal to or higher than the isoelectric point is not particularly limited as long as it is a protein or peptide capable of forming a complex at a pH value equal to or higher than the isoelectric point of the substance. Examples include albumin, orosomucoid, globulin, fibrinogen, histone, protamine, ribonuclease or lysozyme.

Examples of the nucleic acid as the anionic substance include DNA, RNA, plasmid, siRNA, and ODN, and any nucleic acid having any length and sequence may be used as long as it does not exhibit physiological activity.

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.

More specifically, step 1 includes, for example, an operation for producing a liquid in which lead particles containing an aggregation-inhibiting substance are dispersed, and RNA used in the present invention is dispersed or dissolved in the liquid in which the lead particles are dispersed. Operation of containing (for example, an operation of adding and dispersing or dissolving RNA used in the present invention in a liquid in which the lead particles are dispersed, and RNA used in the present invention being dispersed or dissolved in a liquid in which the lead particles are dispersed) For example, an operation of adding the prepared liquid). Here, as the composite particles obtained by the step of dispersing or dissolving the RNA used in the present invention in the liquid in which the lead particles are dispersed, specifically, for example, liposome B containing a cationic lipid. Composite particles formed by adhering RNA used in the present invention to fine particles as constituent components, formed by adhering RNA used in the present invention to fine particles containing lipid aggregates containing cationic lipids Composite particles formed by adhering RNA used in the present invention to fine particles containing polymer micelles containing cationic polymers such as poly-L-lysine as constituents, and containing cationic polymers Used in the present invention for composite particles formed by attaching the RNA used in the present invention to fine particles comprising liposome B as a constituent, and polymer micelles containing cationic lipids as constituents Composite particles and the like that RNA to be is formed by deposition. Further, the operation of dispersing or dissolving the RNA used in the present invention in the liquid in which the lead particles are dispersed includes adding an adhesion competitor to the liquid in which the RNA used in the present invention is dispersed or dissolved. The lead particles are preferably added to a liquid in which the lead particles are dispersed. In this case, the RNA used in the present invention and the adhesion competitor are both attached to the lead particles to produce composite particles. Aggregation of the lead particles during the production of the composite particles and the aggregation of the composite particles after the production can be further suppressed.

The ratio of the lead particles to the liquid in which the lead particles are dispersed is not particularly limited as long as the RNA used in the present invention can adhere to the lead particles, but it is preferably about 1 μg / mL to 1 g / mL, and about 0.1 More preferably, it is ˜500 mg / mL.

Step 2) Step of coating composite particles with lipid bilayer (Part 1)
Operation for preparing a liquid (liquid A) containing the polar organic solvent in which the composite particles obtained in step 1 are dispersed and all or part of the components of the lipid bilayer are dissolved, and then the polarity in the liquid A By reducing the concentration of the organic solvent, liposome A can be produced by a production method including an operation of coating the composite particles with a lipid bilayer membrane. In this case, liposome A is obtained in the form of a dispersion (liquid B). The solvent in the liquid A is a solvent containing the polar organic solvent at a concentration of the polar organic solvent in which the components of the lipid bilayer membrane are soluble and the composite particles can be dispersed. In the liquid B having a reduced concentration, it is preferable that the constituent components of the lipid bilayer membrane can be dispersed and the composite particles can also be dispersed. When the solvent in the liquid A is a mixed liquid of a polar organic solvent and a solvent other than the polar organic solvent, for example, a solvent (liquid C) containing a solvent other than the polar organic solvent that can be mixed with the polar organic solvent is added. The concentration of the polar organic solvent can be reduced by selectively removing the polar organic solvent by evaporative distillation, semipermeable membrane separation, fractional distillation, or the like. Here, the liquid C is preferably a liquid containing a solvent other than the polar organic solvent, but the polar organic solvent may be included as long as it is lower than the concentration of the polar organic solvent in the liquid A.

Examples of the solvent other than the polar organic solvent in Step 2 include water, liquid carbon dioxide, liquid hydrocarbon, halogenated carbon, halogenated hydrocarbon, and the like, and preferably water. Moreover, the liquid A and the liquid C may contain an ion or a buffer component. These solvents can be used alone or in combination of two or more.

The combination of the polar organic solvent and the solvent other than the polar organic solvent is preferably a combination that can be mixed with each other. The solvent in the liquid A and the liquid B and the components of the composite particles and the lipid bilayer membrane for the liquid C It can be selected in consideration of solubility. On the other hand, it is preferable that the composite particles have low solubility in both the solvent in liquid A and liquid B and liquid C, and the solubility in both polar organic solvents and solvents other than polar organic solvents is also low. Preferably, the lipid bilayer component is preferably low in solubility in the solvent in solution B and in solution C, preferably high in solubility in the solvent in solution A, and The solubility in a polar organic solvent is preferably high, and the solubility in a solvent other than the polar organic solvent is preferably low. Here, “the solubility of the composite particles is low” means that each component such as the lead particles contained in the composite particles, the RNA used in the present invention, and the adhesion competing agent has low elution in a solvent, Even if the individual solubility of each component is high, it is sufficient that the elution property of each component is reduced by the binding between the components. For example, even if the solubility of any of the components contained in the lead particle in the solvent in the liquid A is high, if the lead particle has a positive charge, the charge in the RNA used in the present invention, the intramolecular polarization, etc. Thus, the elution of the components in the composite particles is suppressed, and the solubility of the composite particles in the solvent in the liquid A can be lowered. That is, the fact that the lead particles have a positive charge also has the effect of suppressing the elution of the components of the composite particles in the production of liposome A and improving the productivity and yield.

The concentration of the polar organic solvent in the liquid A is not particularly limited as long as the components of the lipid bilayer membrane are soluble and the composite particles can be dispersed. The solvent, the composite particles, and the configuration of the lipid bilayer membrane to be used Although it varies depending on the type of component, etc., it is preferably about 30 v / v% or more, more preferably about 60 to 90 v / v%. Further, the concentration of the polar organic solvent in the liquid B is particularly limited as long as it contains the polar organic solvent at a lower concentration than the liquid A, the constituent components of the lipid bilayer membrane can be dispersed, and the composite particles can also be dispersed. Although it is not a thing, Preferably it is about 50 v / v% or less.

The step of preparing the liquid A includes a step of preparing the liquid A by mixing polar organic solvents, composite particles and components of the lipid bilayer membrane, and if necessary, a solvent other than the polar organic solvent. The components of the polar organic solvent, the composite particle and the lipid bilayer membrane, and optionally the solvent other than the polar organic solvent are not particularly limited in the order of adding them unless the composite particles are dissolved. A liquid (liquid D) containing a polar organic solvent in which particles are dispersed is prepared, and the components of the lipid bilayer membrane are dissolved in a solvent containing a polar organic solvent that is the same as or different from the polar organic solvent in liquid D (Liquid E) is prepared, and liquid D and liquid E are mixed and prepared. When preparing liquid A by mixing liquid D and liquid E, it is preferable to mix gradually.

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.

The method for preparing liquid F can take any form. For example, after preparing a dispersion of composite particles and a solution or dispersion of the components of the lipid bilayer membrane, liquid F may be prepared by mixing both solutions. Liquid F may be prepared by preparing a dispersion of either one of the components, and adding and dispersing one of the remaining components of the composite particles in the solid state or the lipid bilayer membrane to the dispersion. When mixing the composite particle dispersion and the lipid bilayer component solution or dispersion, the composite particle dispersion medium may contain a polar organic solvent in advance. The component solvent or dispersion medium may be a liquid containing a polar organic solvent or a liquid composed only of a polar organic solvent. On the other hand, when preparing a dispersion of either the composite particles or the components of the lipid bilayer membrane, and adding the remaining one of the solid-state composite particles or the components of the lipid bilayer to the dispersion, The dispersion is preferably a liquid containing a polar organic solvent. In addition, when the composite particles are not dissolved after the preparation of the liquid F and the components of the lipid bilayer are dispersed, the polar organic particles are not dissolved and the components of the lipid bilayer are dispersed. A polar organic solvent may be added within the solvent concentration range, the polar organic solvent may be removed, or the concentration may be decreased. On the other hand, the composite particles are not dissolved after preparing the liquid F. However, when the components of the lipid bilayer membrane are dissolved, the composite particles are not dissolved and the components of the lipid bilayer membrane are dispersed. The polar organic solvent may be removed or the concentration reduced within the range of the polar organic solvent concentration. In addition, the components of the composite particles and lipid bilayer membrane are mixed in advance in a solvent other than the polar organic solvent, and the range of polar organic solvent concentration in which the composite particles do not dissolve and the components of the lipid bilayer membrane are dispersed A polar organic solvent may be added. In that case, each of the components of the composite particle and the lipid bilayer membrane may be dispersed in a solvent other than the polar organic solvent, and after mixing both dispersions, the polar organic solvent may be added. Either one of the components of the lipid bilayer membrane was dispersed in a solvent other than the polar organic solvent, and the remaining one of the solid-state composite particles or the components of the lipid bilayer membrane was added to the dispersion and dispersed. Later, a polar organic solvent may be added. In addition, the component of the composite particles and the lipid bilayer membrane is dispersed, and a liquid containing a polar organic solvent is allowed to stand or mix for a time sufficient for the composite particles to be coated with the lipid bilayer membrane. Is preferred. After dispersing the components of the composite particles and the lipid bilayer membrane in a liquid containing a polar organic solvent, the time for standing or mixing the components of the composite particles and the lipid bilayer membrane with the polar organic solvent There is no limitation unless it is instantaneously terminated after being dispersed in the liquid containing, but can be arbitrarily set according to the components of the lipid bilayer membrane and the type of liquid containing the polar organic solvent, It is preferable to set a time during which the yield of the obtained liposome A is a steady amount, for example, about 3 seconds to 30 minutes. When the components of the composite particle and the lipid bilayer are dispersed in a liquid containing a polar organic solvent, the coating of the lipid bilayer on the composite particle is started, and the lipid bilayer on the composite particle is quickly The coating of the membrane may be completed. For example, after preparing a solution of lipid bilayer components, mix the composite particle dispersion and the solution of lipid bilayer components. When preparing F, if the solubility of the lipid bilayer components in liquid F is low, the lipid bilayer components are complexed almost simultaneously with the dispersion in the liquid containing the polar organic solvent. Sometimes the coating of the lipid bilayer on the particles is complete.

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%.

In the present invention, “the component of the lipid bilayer membrane is soluble in the polar organic solvent” means that when the component of the lipid bilayer membrane has the property of being dissolved in the polar organic solvent, a solubilizer or the like is used. When the components of the lipid bilayer membrane have the property of being dissolved in a polar organic solvent, the components of the lipid bilayer membrane can form emulsions or micelles in the polar organic solvent and become emulsion or emulsion The case where it has is included.
In addition, “the components of the lipid bilayer membrane are dispersed” means that all of the components of the lipid bilayer membrane are aggregated or micelles and are emulsified or emulsified. Part of the constituents forms aggregates or micelles to become an emulsion or emulsion, and the remaining part is dissolved, part of the constituents of the lipid bilayer membrane forms aggregates or micelles, etc. It includes a state where the emulsion is emulsified or emulsified, and the remaining part is precipitated, and does not include a state where all the components of the lipid bilayer are dissolved.

In the present invention, “composite particles are dispersed” means a state in which the composite particles are suspended, emulsified or emulsified, and a part of the composite particles are suspended, emulsified or emulsified, and the remaining part. Including a state in which a part of the composite particles is emulsified or emulsified and a remaining part is precipitated, and does not include a state in which all of the composite particles are dissolved. “Composite particles do not dissolve” has the same meaning as “composite particles are dispersed”.

The concentration of the composite particles in the polar organic solvent-containing aqueous solution used in the method for producing liposome A in the present invention is not particularly limited as long as the composite particles can be covered with a lipid bilayer membrane, but is about 1 μg / mL to 1 g. / mL, preferably about 0.1 to 500 mg / mL. The concentration of the constituent components of the lipid bilayer membrane used is not particularly limited as long as the composite particles can be coated, but is preferably about 1 μg / mL to 1 g / mL, preferably about 0.1 to 400 mg / mL. More preferably.

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.

In addition, the size of the liposome A in the present invention is preferably an injectable size, for example. Specifically, the average particle size is preferably about 10 nm to 1000 nm, more preferably about 50 nm to 300 nm, and further preferably about 70 nm to 200 nm.

Furthermore, the liposome A obtained above can be modified with substances such as proteins such as antibodies, saccharides, glycolipids, amino acids, nucleic acids, various low molecular compounds or high molecular compounds, and the coated composite particles obtained from these Also included in liposome A. For example, for application to targeting, the liposome A obtained above can be further subjected to surface modification of the lipid bilayer with proteins such as antibodies, peptides or fatty acids [D. D. Lasic ), Edited by F. Martin, "Stealth Liposomes" (USA), CRC Press Inc, 1995, p. 93-102]. In addition, the liposome A can be optionally subjected to surface modification with, for example, a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative, and the water-soluble substance lipid derivative, fatty acid derivative or The aliphatic hydrocarbon derivative is synonymous with a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as a constituent component of the lipid bilayer membrane.

By administering the composition of the present invention to mammals (including humans) having an arteriosclerotic site or a stent restenosis site, the RNA used in the present invention can be used for the gene associated with atherosclerotic disease or stent restenosis. It can be delivered to an arteriosclerosis site or a stent restenosis site as an expression site, and the expression of the gene is suppressed. By suppressing the expression of genes related to arteriosclerotic disease or stent restenosis, neointimal proliferation due to neovascularization, macrophage accumulation and smooth muscle cell migration / proliferation, and vascular remodeling are suppressed. Atherosclerotic disease is treated or prevented or stent restenosis is suppressed or prevented.
That is, the present invention also provides a method for treating or preventing an arteriosclerotic disease or a method for suppressing stent restenosis, wherein the composition of the present invention described above is administered to a mammal. The administration target is preferably a person suffering from arteriosclerosis or a person who has undergone PTCA treatment, and more preferably a person who has undergone PTCA treatment.
In addition, a composition obtained by replacing RNA in the composition of the present invention with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, and the like can also be delivered to sites of arteriosclerosis and stent restenosis. And can be used as an arteriosclerotic disease treatment or a stent restenosis inhibitor.

In addition, the composition of the present invention, and the composition in which the RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, etc., are delivered directly or indirectly. It can also be used as a diagnostic agent for diagnosing whether it is suffering from arteriosclerosis or stent restenosis has occurred.
In addition, the composition of the present invention, and the composition in which the RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, and the like, for example, biological components such as blood components ( Also used as a preparation for stabilizing the RNA, peptide, protein or nucleic acid in the blood, gastrointestinal tract, etc., reducing side effects or increasing drug accumulation at arteriosclerotic sites or stent restenosis sites it can.

The composition of the present invention, and a composition in which RNA in the composition of the present invention is replaced with peptides and proteins such as coenzymes and antibodies, nucleic acids such as oligonucleotides and plasmids, etc. are treated for treating arteriosclerotic diseases or suppressing stent restenosis When used as an agent, it is desirable to use the most effective route for treatment, such as buccal, respiratory tract, rectal, subcutaneous, intramuscular or intravenous administration, or parenteral or oral administration. Preferably intravenous administration or intramuscular administration, more preferably intravenous administration.
The dose varies depending on the disease state, age, route of administration, etc. of the administration subject, but for example, it may be administered so that the daily dose converted to RNA is about 0.1 μg to 1000 mg.

Examples of suitable dosage forms for intravenous administration or intramuscular administration include injections, and the dispersion of liposome A prepared by the above-described method can be used as it is, for example, in the form of injections. The dispersion can be used after removing the solvent by, for example, filtration, centrifugation, etc., or the dispersion can be used by lyophilization, or an excipient such as mannitol, lactose, trehalose, maltose or glycine can be used. The added dispersion can be lyophilized for use.
In the case of injections, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, etc. are mixed into the liposome A dispersion or the liposome A from which the solvent is removed or lyophilized. It is preferred to use the prepared composition of the invention. Further, for example, an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA, or an isotonic agent such as glycerin, glucose or sodium chloride can be added. Moreover, it can also be cryopreserved by adding a cryopreservation agent such as glycerin.

The arteriosclerotic disease treatment or stent restenosis inhibitor of the present invention includes the composition of the present invention intended for use in the treatment or prevention of arteriosclerotic disease or stent restenosis. Among the compositions, liposome A is composed of a composite particle containing lead particles and the RNA as constituents, and a lipid bilayer covering the composite particles, and the constituent of the lipid bilayer is a specific polar organic solvent A liposome capable of being dispersed in a liquid containing the polar organic solvent at a specific concentration, or a lead particle containing a cationic substance and the RNA. Composed of a composite particle as a constituent component and a lipid bilayer membrane covering the composite particle, wherein the constituent component of the lipid bilayer membrane is soluble in a polar organic solvent, Composite grain It is preferable that the child contains liposomes that can be dispersed in a liquid containing the polar organic solvent at a specific concentration.
The present invention also provides the use of the composition of the present invention described above for the treatment of arteriosclerotic diseases or the production of a stent restenosis inhibitor.

Next, the present invention will be specifically described with reference to examples and test examples. However, the present invention is not limited to these examples and test examples. The RNA used in Example 1 was modified with cyanine 5 (Cy5) at the 5 ′ end of each single-stranded RNA of KLF5siRNA No. 4 in the KLF5 siRNA in Table 1 and added to each 3 ′ end. RNA [Cy5-AAGCUCACCUGAGGACUCAdTdT (SEQ ID NO: 23), Cy5-UGAGUCCUCAGGUGAGCUUdTdT (SEQ ID NO: 24)] obtained by replacing UU with dTdT was prepared by annealing from Hokkaido System Science (hereinafter referred to as Cy5-labeled). KLF-5 siRNA). The RNA used in Example 2 and Comparative Example 1 was modified with Cy5 at the 5 ′ end of RNA containing a 19-base sequence of mRNA of the bcl-2 gene and a base sequence complementary to the sequence. , RNA [Cy5-GUGAAGUCAACAUGCCUGCdTdT (SEQ ID NO: 25), Cy5-GCAGGCAUGUUGACUUCACdTdT (SEQ ID NO: 26)] in which UU added to each 3 ′ end was replaced with dTdT (hereinafter referred to as Cy5-labeled bcl-2 siRNA) Each was obtained from Hokkaido System Science and prepared by annealing. In addition, RNA used in Comparative Example 2 is Luciferase [see Photochemistry and Photobiology (Photochem. Photobiol.), 1969, Volume 10, No.3, p.153-170] ] RNA [5'-CUUACGCUGAGUACUUCGAdTdT-3 'in which UU added to the 3' end of each RNA containing 19 consecutive nucleotide sequences of the mRNA of the gene and the complementary nucleotide sequence is replaced with dTdT (SEQ ID NO: 27) and 5′-UCGAAGUACUCAGCGUAAGdTdT-3 ′ (SEQ ID NO: 28)] (hereinafter referred to as “LucRNAsiRNA”), each of which was obtained from Japan Easy Co., Ltd. and prepared by annealing. In addition, the RNA used in Example 3 and Comparative Example 3 is an RNA [5′-AAGCUCACCUGAGGACUCAdTdT (SEQ ID NO: 29)] containing a 19-base sequence of mRNA of the KLF5 gene and a base sequence complementary to the sequence. 5′-UGAGUCCUCAGGUGAGCUUUdTdT (SEQ ID NO: 30)] (hereinafter referred to as KLF5 siRNA), each obtained from Hokkaido System Science Co., Ltd. and prepared by annealing.

DOTAP (manufactured by Avanti Polar Lipids) / PEG-DSPE (manufactured by NOF Corporation, the same shall apply hereinafter) / distilled water was mixed to 30 mg / 12 mg / 1 mL, and the mixture was shaken and stirred with a vortex mixer. The resulting dispersion was applied to a 0.4 μm polycarbonate membrane filter (Whatman) four times at room temperature, 10 times to a 0.1 μm polycarbonate membrane filter (Whatman), and then 24 times to a 0.05 μm polycarbonate membrane filter (Whatman). Lead particles were prepared. To 0.5 mL of the obtained lead particle dispersion, 0.25 mL of an 8 mg / mL aqueous solution of Cy5-labeled KLF5 siRNA was added, and 1 mL of ethanol was added to prepare composite particles. To the obtained dispersion of composite particles, 0.25 mL of a solution obtained by mixing EPC (manufactured by NOF Corporation) / PEG-DSPE / ethanol as a component of the lipid bilayer so as to be 120 mg / 25 mg / 1 mL. Then, 23 mL of distilled water was gradually added to adjust the concentration of ethanol to 5 v / v% or less to prepare liposomes. The obtained liposome dispersion was subjected to ultracentrifugation (1 hour, 110,000 × g, 25 ° C.), the supernatant was removed, and physiological saline was added and redispersed to obtain a liposome dispersion. 50 parts by weight of PEG-DSPE with respect to 120 parts by weight of EPC was dissolved in a small amount of ethanol (about 1/25 volume of the liposome dispersion) (PEG-DSPE ethanol solution). The liposome dispersion and the PEG-DSPE ethanol solution were each heated at 70 ° C. for 2 minutes. Subsequently, the liposome dispersion was added to the PEG-DSPE ethanol solution, mixed, heated at 70 ° C. for 2 minutes, and then cooled with water to obtain a composition.

Test example 1
ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler. Thromb Vasc. Biol., Jan 1994; 14: 133-140) and the following method was used to confirm that the composition obtained in Example 1 was accumulated at the site of arteriosclerosis.
ApoE-deficient mice (Andrew S. Plump, Jonathan D. Smith, Tony Hayek, Katriina Aalto-Setala, Annemarie Walsh, Judy G. Verstuyft, Edward M. Rubin and Jan L. Breslow. Cell 1992 71: 343-353) was given a high fat diet (Clea Japan, High Fat Diet 32) for 7 weeks. 100 μL of the composition obtained in Example 1 (corresponding to 150 μg of Cy5-labeled KLF5 siRNA) was administered from the mouse tail vein, euthanized 24 hours after the composition was administered, the aorta was excised, and a frozen section was prepared. Atherosclerosis sites were observed with a confocal laser microscope (LSM510 Meta; Carl Zeiss).
The left side of FIG. 1 shows a photograph of a frozen section obtained by phase contrast microscopy 24 hours after administration of the composition obtained in Example 1. In addition, a photograph of the frozen section observed with a confocal laser microscope is shown on the right of FIG.
From the left of FIG. 1, an arteriosclerotic site is observed in the blood vessel. Further, from the right side of FIG. 1, the red color of Cy5-labeled KLF5 siRNA (white portion in the figure) can be observed, and it can be seen that Cy5-labeled KLF5 siRNA is distributed at the site of arteriosclerosis.

DOTAP / PEG-DSPE / distilled water was mixed to 40 mg / 16 mg / 1 mL, and the mixture was shaken and stirred with a vortex mixer. The resulting dispersion was passed through a 0.4 μm polycarbonate membrane filter at room temperature 4 times, through a 0.1 μm polycarbonate membrane filter 10 times, and further through a 0.05 μm polycarbonate membrane filter 24 times to prepare lead particles. A composite particle was prepared by adding 0.0832 mL of a 24 mg / mL aqueous solution of Cy5-labeled bcl-2 siRNA to 0.2496 mL of the resulting lead particle dispersion. Obtained by mixing 0.32 mL of the resulting composite particle dispersion with 15 mg / 3.125 mg / 0.625 mL / 0.375 mL of EPC / PEG-DSPE / ethanol / distilled water, which is a component of the lipid bilayer membrane. The solution was added to 1.28 mL (ethanol concentration was approximately 62.5 v / v%). Next, 0.4 mL of distilled water was gradually added to adjust the ethanol concentration to approximately 40 v / v%. Next, add 0.128 mL of the solution obtained by mixing EPC / PEG-DSPE / ethanol / distilled water to 62.5 mg / 62.5 mg / 0.4 mL / 0.6 mL, and add 14.896 mL of distilled water. Liposomes were prepared. The resulting liposome dispersion is ultracentrifugated (1 hour, 110,000 × g, 25 ° C.), the supernatant is removed, and physiological saline is added to the precipitate for redispersion, and physiological saline is further added. The concentration was adjusted to obtain a composition.

Comparative Example 1
Cy5-labeled bcl-2 siRNA was dissolved in physiological saline to obtain a composition.

Test example 2
Using the common carotid artery ligation model (Lindner V, Fingerle J, Reidy MA Mouse model of arterial injury.Mouse model of arterial injury.Circ Res (1993) 73: 792-796), the composition obtained in Example 2 was used. When administered, it was confirmed that RNA specifically reached the neointimal site after vascular injury.
The common carotid artery of C57BL / 6J mice was ligated, and after 3 weeks, the composition obtained in Example 2 and the composition obtained in Comparative Example 1 were administered at 100 μL per mouse (corresponding to 150 μg of Cy5-labeled bcl-2 siRNA) did. Twenty-four hours after administration, the mice were euthanized, the carotid artery was removed, frozen sections were prepared, and Cy5-derived fluorescence was observed with hematoxylin and eosin staining (H / E staining) and a confocal laser microscope. As a control, physiological saline was administered at 100 μL per mouse, and the same test was performed.
2 to 4 on the left are H / E-stained images of frozen sections 24 hours after administration of the composition obtained in Example 2, the composition obtained in Comparative Example 1, and physiological saline, respectively. 4 The fluorescence observation image by the confocal laser microscope is shown on the right.
The shape of the blood vessel is shown on the left side of FIGS. 2 to 4, and on the right side of FIG. 2, the red fluorescence of Cy5-labeled siRNA (white part in the figure) is strongly recognized. The composition obtained in Example 2 is RNA It can be seen that is accumulated at the neointimal site. On the other hand, on the right side of FIG. 3, the red fluorescence of Cy5-labeled siRNA (the white part in the figure) is not observed, and the composition obtained in Comparative Example 1 does not accumulate RNA at the neointimal site. I understand. From the right side of FIG. 4, it was confirmed that there was no autofluorescence.

DOTAP / PEG-DSPE / 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 applied to a 1 μm polycarbonate membrane filter 5 times at 70 ° C., 10 times to a 0.4 μm polycarbonate membrane filter, 10 times to a 0.2 μm polycarbonate membrane filter, 10 times to a 0.1 μm polycarbonate membrane filter, and 0.05 times more. Lead particles were prepared by passing 18 times through a μm polycarbonate membrane filter. Composite particles were prepared by adding 1.3728 mL of a 24 mg / mL aqueous solution of KLF5 siRNA to 4.12 mL of the obtained lead particle dispersion. The obtained composite particle dispersion 5.28mL is obtained by mixing EPC / PEG-DSPE / ethanol / distilled water, which is a component of the lipid bilayer membrane, to 15mg / 3.125mg / 0.625mL / 0.375mL. 21.12mL (ethanol concentration is approximately 62.5v / v%), then gradually add 6.6mL of distilled water and add 62.5mg / 62.5mg / 0.4mL / EPC / PEG-DSPE / ethanol / distilled water. Liposomes were prepared by adding the solution obtained by mixing to 0.6 mL to make the ethanol concentration approximately 20 v / v%. The obtained liposome dispersion was subjected to tangential flow filtration (TFF) using distilled water as replacement water until the ethanol concentration was 1 v / v% or less. The TFF flowed the stock solution from the tank parallel to the membrane surface, and only the substances smaller than the pores on the membrane surface were discharged as the filtrate, and the liposomes larger than the pores and the residual liquid returned to the tank. Since the liquid in the tank was gradually reduced by filtration, the same amount of distilled water as the reduced water was supplied siphonically at the same time. By this method, only ethanol was removed while the concentration of the obtained liposome dispersion was kept constant. Subsequently, the replacement water was used as physiological saline to increase the salt concentration, and then concentrated by stopping the supply of replacement water to obtain a composition.

Comparative Example 2
The KLF5 siRNA in Example 3 was replaced with Luc siRNA to obtain a composition in the same manner.

Comparative Example 3
KLF5 siRNA was dissolved in physiological saline to obtain a composition.

Test example 3
A disease model was prepared in the same manner as in Test Example 2, and the effect of the composition obtained in Example 3 on neointimal formation after vascular injury was evaluated by the following method.
From the point where the internal carotid artery and vertebral artery branch from the common carotid artery of the C57BL / 6J mouse, ligate the 500 μm common carotid artery side, 100 μL of the composition obtained in Example 3 every 2 days (equivalent to 150 μg of KLF5 siRNA) for 2 weeks (N = 6). Two weeks after the first administration, the mice were euthanized, the carotid artery was removed, frozen sections were prepared, and H / E staining was performed.
In addition, as a control, the composition obtained in Comparative Example 3 was administered in an amount of 100 μL (corresponding to 150 μg of KLF5 siRNA) instead of the composition obtained in Example 3, and the same test was performed (n = 7). Furthermore, instead of the composition obtained in Example 3, 100 μL (corresponding to 150 μg of LucsiRNA) was administered each time, and the composition obtained in Comparative Example 2 was similarly tested (n = 7).
After administration of the compositions obtained in Example 3, Comparative Example 2 and Comparative Example 3, a H / E-stained image of the frozen section 2 weeks later was photographed and observed, and image analysis (ImageJ) was performed on the section. The area of the intima and media was measured.
As a result of observation, when the composition obtained in Example 3 was administered, the stenosis of the carotid artery due to the neointima was reduced compared to the case where the composition obtained in Comparative Example 2 and Comparative Example 3 was administered. It was confirmed that
FIG. 5 shows a bar graph representing the sum of the area of the inner membrane and the inner membrane. Compared with the group administered with the composition obtained in Comparative Example 2 and Comparative Example 3 in the group administered with the composition obtained in Example 3, the sum of the areas of the intima and intima decreased, The possibility of the composition of the invention as a therapeutic or prophylactic agent in arteriosclerotic diseases has been revealed. In contrast to the group administered with the composition obtained in Comparative Example 2 and Comparative Example 3, the inhibition rate of the group administered with the composition obtained in Example 3 was 26% and 22%, respectively, p = 0.04 and 0.30. there were.

Claims (32)

1. (i) an RNA comprising a continuous 15-30 base sequence of mRNA of a gene related to arteriosclerotic disease or stent restenosis and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside A composition comprising liposomes.
2. 2. The composition according to claim 1, wherein the liposome is a liposome of a size that can be administered intravenously.
3. 3. The composition according to claim 1, wherein the RNA is RNA having an action of suppressing expression of the gene using RNA interference (RNAi).
4. Genes associated with atherosclerotic disease or stent restenosis are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor receptor, Mitogen-activated protein kinase (MAP kinase; MAPK) signaling-related factor, platelet-derived growth factor, platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, Kruppel-like factor (KLF), survivin 4. The composition according to claim 1, which is a gene for any one of Ets transcription factor, nuclear factor and hypoxia-inducible factor.
5. The composition according to any one of claims 1 to 3, wherein mRNA of a gene associated with arteriosclerotic disease or stent restenosis is mRNA for KLF.
6. The composition according to any one of claims 1 to 3, wherein the mRNA of a gene associated with arteriosclerotic disease or stent restenosis is KLF5 mRNA.
7. The composition according to any one of claims 1 to 6, wherein the mRNA is human mRNA.
8. The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a constituent component, and a lipid bilayer coating the composite particle,
   The components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. The composition according to any one of claims 1 to 7.
9. 9. The composition of claim 8, wherein the polar organic solvent is an alcohol.
10. 9. The composition of claim 8, wherein the polar organic solvent is ethanol.
11. The lead particle is a lead particle containing a cationic substance, and the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative as a constituent component. The composition according to any one of claims 8 to 10.
12. The liposome encapsulating RNA is a liposome composed of lead particles containing a cationic substance, composite particles containing RNA as a constituent, and a lipid bilayer coating the composite particles,
    The composition according to any one of claims 1 to 7, wherein the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of a water-soluble substance as constituent components. object.
13. The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl)]- N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy- N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane 13. One or more selected from (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride, and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol. Composition.
14. The composition according to any one of claims 11 to 13, wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
15. The composition according to any one of claims 11 to 14, wherein the neutral lipid is egg yolk phosphatidylcholine.
16. (i) an RNA comprising a continuous 15-30 base sequence of mRNA of a gene related to arteriosclerotic disease or stent restenosis and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside An arteriosclerotic disease treatment or stent restenosis inhibitor comprising a liposome.
17. 17. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to claim 16, wherein the liposome is a liposome having a size that can be intravenously administered.
18. 18. The arteriosclerotic disease treatment or stent restenosis inhibitor according to claim 16 or 17, wherein the RNA is an RNA having an action of suppressing the expression of the gene utilizing RNA interference (RNAi).
19. Genes associated with atherosclerotic disease or stent restenosis are vascular endothelial growth factor, vascular endothelial growth factor receptor, fibroblast growth factor, fibroblast growth factor receptor, epidermal growth factor, epidermal growth factor receptor, MAP kinase signaling related factor, platelet-derived growth factor, platelet-derived growth factor receptor, hepatocyte growth factor, hepatocyte growth factor receptor, KLF, survivin, Ets transcription factor, nuclear factor and hypoxia-inducible factor The arteriosclerotic disease treatment or stent restenosis inhibitor according to any one of claims 16 to 18, which is a gene of
20. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to any one of claims 16 to 18, wherein mRNA of a gene related to arteriosclerotic disease or stent restenosis is mRNA for KLF.
21. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to any one of claims 16 to 18, wherein mRNA of a gene related to arteriosclerotic disease or stent restenosis is mRNA of KLF5.
22. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to any one of claims 16 to 21, wherein the mRNA is human mRNA.
23. The liposome encapsulating RNA is a liposome composed of a lead particle, a composite particle comprising RNA as a constituent component, and a lipid bilayer coating the composite particle,
    The components of the lipid bilayer membrane are soluble in a specific polar organic solvent, and the components of the lipid bilayer membrane and the composite particles can be dispersed in a liquid containing the polar organic solvent at a specific concentration. The arteriosclerotic disease treatment or stent restenosis inhibitor according to any one of claims 16 to 22.
24. 24. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to claim 23, wherein the polar organic solvent is alcohol.
25. 24. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to claim 23, wherein the polar organic solvent is ethanol.
26. The lead particle is a lead particle containing a cationic substance, and the lipid bilayer membrane is a lipid bilayer membrane comprising a neutral lipid and a water-soluble substance lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative as a constituent component. The arteriosclerotic disease treatment or stent restenosis inhibitor according to any one of claims 23 to 25.
27. The liposome encapsulating RNA is a liposome composed of lead particles containing a cationic substance, composite particles containing RNA as a constituent, and a lipid bilayer coating the composite particles,
    The artery according to any one of claims 16 to 22, wherein the lipid bilayer is a lipid bilayer comprising a neutral lipid and a lipid derivative, a fatty acid derivative or an aliphatic hydrocarbon derivative of a water-soluble substance as constituent components. A sclerosing disease treatment or stent restenosis inhibitor.
28. The cationic substance is N- [1- (2,3-dioleoylpropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-dioleoylpropyl)]-N, N-dimethylamine, N- [1- (2,3-dioleyloxypropyl)]-N, N, N-trimethylammonium chloride, N- [1- (2,3-ditetradecyloxypropyl)]- N, N-dimethyl-N-hydroxyethylammonium bromide, 1,2-dilinoleyloxy- N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N, N-dimethylaminopropane 28. The one or more selected from (DLenDMA), didecyldimethylammonium chloride, distearyldimethylammonium chloride, and 3β- [N- (N ′, N′-dimethylaminoethyl) carbamoyl] cholesterol. For treating arteriosclerotic diseases or inhibiting stent restenosis.
29. 29. The agent for treating arteriosclerotic disease or inhibiting stent restenosis according to claim 26, wherein the lipid derivative, fatty acid derivative or aliphatic hydrocarbon derivative of the water-soluble substance is polyethylene glycol-phosphatidylethanolamine.
30. The therapeutic agent for arteriosclerotic disease or the stent restenosis inhibitor according to any one of claims 26 to 29, wherein the neutral lipid is egg yolk phosphatidylcholine.
31. (i) an RNA comprising a continuous 15-30 base sequence of mRNA of a gene related to arteriosclerotic disease or stent restenosis and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside A method for treating an arteriosclerotic disease or a method for suppressing stent restenosis, comprising administering a composition containing a liposome to a mammal.
32. (i) an RNA comprising a continuous 15-30 base sequence of mRNA of a gene related to arteriosclerotic disease or stent restenosis and a base sequence complementary to the sequence, and (ii) enclosing the RNA inside Use of a composition containing a liposome for the treatment of an arteriosclerotic disease or a stent restenosis inhibitor.

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