JPWO2004096277A1 - Preventive or therapeutic agent for intraocular neovascular diseases - Google Patents

Abstract

The present invention provides a preventive or therapeutic agent for intraocular neovascular diseases, comprising a crystallin inhibitor as an active ingredient. Specific examples of the crystallin inhibitor include double-stranded RNA such as siRNA composed of an RNA strand having a nucleotide sequence of CCUGAAGGAGACGUGAUUGTT (SEQ ID NO: 1) and its complementary strand, antisense oligonucleotides, and the like. The pharmaceutical composition of the present invention has an effect of suppressing angiogenesis in the eye and is useful for intraocular angiogenesis diseases such as diabetic retinopathy and retinopathy of prematurity.

Description

The present invention relates to a preventive or therapeutic agent for intraocular neovascular diseases.
More specifically, the present invention relates to a preventive or therapeutic agent for intraocular neovascular diseases, which contains a crystallin inhibitor.

Angiogenesis generally progresses with digestion and destruction of the vascular basement membrane by protease, migration and proliferation of vascular endothelial cells and differentiation of vascular endothelial cells, and reconstruction of blood vessels. As angiogenic diseases that develop due to such angiogenesis in the eye, various diseases such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, and age-related macular degeneration are known. For example, diabetic retinopathy is the largest disease causing blindness in which angiogenesis is observed in the optic disc, retina, iris or corner, and is one of the ophthalmic diseases in which angiogenesis is the most important factor in the prognosis. Retinal vein occlusion is a serious thrombotic disease in which retinal neovascular membranes are observed and vitreous hemorrhage is repeated. Retinopathy of prematurity is a disease in which angiogenesis is observed in the retina of premature infants due to environmental changes such as stress, oxygen administration, respiratory distress syndrome, exchange transfusion, sepsis, etc. It may lead to visual impairment such as. Age-related macular degeneration is a disease in which retinal pigment epithelial cells become weak with aging and angiogenesis occurs mainly from the choroid under the retina, which may impair visual function.
As the treatment method, retinal photocoagulation, panretinal cryocoagulation, vitreous surgery, laser ciliary body destruction, ciliary cryocoagulation, and radiation irradiation are performed. Further, in age-related macular degeneration, antioxidants such as vitamin E, zinc, interferon α2a and interferon β are administered, and ibuprofen has been reported to be effective in retinopathy of prematurity (for example, See Reference 1 below). However, the mechanism of angiogenesis in the eye has not yet been elucidated, and no established pharmacotherapy exists.
Recently, it has been reported that crystallin has been detected in drusen of patients with age-related macular degeneration, but there is no mention of the relationship between crystallin and the formation of angiogenesis (for example, see Reference 2 below). Currently, it is desired to develop a novel prevention or treatment method for the above-mentioned intraocular neovascular diseases.
Literature 1: JP 2002-326929 A Literature 2: Crabb JW. Eleven, "Proc. Natl. Acad. Sci. USA", November 2002, Vol. 99, No. 23, p. 14619-14621

An object of the present invention is to provide a novel preventive or therapeutic agent for intraocular neovascular diseases.
As a result of intensive studies to find a drug useful for the prevention or treatment of intraocular neovascular diseases, the present inventors are deeply involved in the formation of angiogenesis in the eye and inhibit the expression of this crystallin. As a result, the inventors have obtained a surprising finding that angiogenesis in intraocular neovascular diseases such as retinopathy of prematurity can be suppressed, and the present invention has been achieved.
That is, the present invention
[1] A prophylactic or therapeutic agent for intraocular neovascular diseases, comprising a crystallin inhibitor;
[2] The preventive or therapeutic agent according to the above [1], wherein the crystallin is crystallin β;
[3] The prophylactic or therapeutic agent according to the above [2], wherein the crystallin is crystallin β-B2.
[4] The preventive or therapeutic agent according to any one of [1] to [3], wherein the crystallin inhibitor is a double-stranded RNA having a crystallin inhibitory effect;
[5] The preventive or therapeutic agent according to [4] above, wherein the double-stranded RNA is siRNA;
[6] The preventive or therapeutic agent according to the above [5], wherein the siRNA is an RNA composed of an RNA strand having the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 7 and a complementary strand thereof;
[7] The prophylactic or therapeutic agent according to any one of [1] to [3], wherein the crystallin inhibitor is an antisense oligonucleotide to crystallin;
[8] The preventive or therapeutic agent according to any one of [1] to [3] above, wherein the crystallin inhibitor is a compound having a crystallin inhibitory activity or a sense oligonucleotide thereof;
[9] The crystallin inhibitory substance is a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, or a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof The prophylactic or therapeutic agent according to any one of the above [1] to [3];
[10] From a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector to crystallin, and a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof The preventive or therapeutic agent according to any one of [1] to [3] above, which is a host cell transformed with the selected recombinant vector;
[11] The intraocular neovascular disease is a disease selected from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, neovascular macular disease, neovascular glaucoma, iris rubeosis and Eales disease The preventive or therapeutic agent according to any one of [1] to [10] above;
[12] The preventive or therapeutic agent according to [11] above, wherein the intraocular neovascular disease is diabetic retinopathy;
[13] The preventive or therapeutic agent according to [11] above, wherein the intraocular neovascular disease is retinopathy of prematurity;
[14] The preventive or therapeutic agent according to [11] above, wherein the intraocular neovascular disease is retinal vein occlusion;
[15] The prophylactic or therapeutic agent according to [11] above, wherein the intraocular neovascular disease is age-related macular degeneration or neovascular macular disease;
[16] A method for preventing or treating intraocular neovascular diseases, comprising administering an effective amount of a crystallin inhibitor;
[17] The prophylactic or therapeutic method according to the above [16], wherein the crystallin is crystallin β;
[18] The method for prevention or treatment according to [17], wherein the crystallin is crystallin β-B2.
[19] The method for prevention or treatment according to any of [16] to [18], wherein the crystallin inhibitor is a double-stranded RNA having a crystallin inhibitory effect;
[20] The prevention or treatment method according to [19] above, wherein the double-stranded RNA is siRNA;
[21] The prevention or treatment method according to the above [20], wherein the siRNA is an RNA composed of an RNA strand having the nucleotide sequence shown in any one of SEQ ID NOS: 1 to 7 and its complementary strand;
[22] The prevention or treatment method according to any of [16] to [18], wherein the crystallin inhibitor is an antisense oligonucleotide to crystallin;
[23] The method for prevention or treatment according to any of [16] to [18], wherein the crystallin inhibitor is a compound having a crystallin inhibitory activity or a sense oligonucleotide thereof;
[24] A crystallin inhibitor is a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, or a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof The prevention or treatment method according to any one of [16] to [18],
[25] From a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, and a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof The preventive or therapeutic method according to any one of [16] to [18] above, which is a host cell transformed with a selected recombinant vector;
[26] The intraocular neovascular disease is a disease selected from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, neovascular macular disease, neovascular glaucoma, iris rubeosis and eales disease The prevention or treatment method according to any of [16] to [25] above;
[27] The prevention or treatment method according to the above [26], wherein the intraocular neovascular disease is diabetic retinopathy;
[28] The prevention or treatment method according to the above [26], wherein the intraocular neovascular disease is retinopathy of prematurity;
[29] The prevention or treatment method according to the above [26], wherein the intraocular neovascular disease is retinal vein occlusion;
[30] The prevention or treatment method according to the above [26], wherein the intraocular neovascular disease is age-related macular degeneration or neovascular macular disease;
[31] Use of a crystallin inhibitor for producing a pharmaceutical composition for prevention or treatment of an intraocular neovascular disease;
[32] The use according to [31] above, wherein the crystallin is crystallin β;
[33] The use according to the above [32], wherein the crystallin is crystallin β-B2.
[34] The use according to any of [31] to [33] above, wherein the crystallin inhibitor is a double-stranded RNA having a crystallin inhibitory effect;
[35] The use according to [34] above, wherein the double-stranded RNA is siRNA;
[36] The use according to [35] above, wherein the siRNA is an RNA composed of an RNA strand having the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 7 and its complementary strand;
[37] The use according to any of [31] to [33] above, wherein the crystallin inhibitor is an antisense oligonucleotide to crystallin;
[38] The use according to any of [31] to [33] above, wherein the crystallin inhibitor is a compound having a crystallin inhibitory activity or a sense oligonucleotide thereof;
[39] A crystallin inhibitor is a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, or a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof The use according to any one of [31] to [33];
[40] From a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, and a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof The use according to any one of [31] to [33] above, which is a host cell transformed with a selected recombinant vector;
[41] The intraocular neovascular disease is a disease selected from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, neovascular macular disease, neovascular glaucoma, iris rubeosis and Eales disease And the use according to any of [31] to [40] above;
[42] The use according to [41] above, wherein the intraocular neovascular disease is diabetic retinopathy;
[43] The use according to the above [41], wherein the intraocular neovascular disease is retinopathy of prematurity;
[44] The use according to the above [41], wherein the intraocular neovascular disease is retinal vein occlusion;
[45] The use according to [41] above, wherein the intraocular neovascular disease is age-related macular degeneration or neovascular macular disease;
[46] An siRNA composed of an RNA strand having the nucleotide sequence shown in any one of SEQ ID NOS: 1 to 7 and its complementary strand; and the like.
More specifically, first, the present inventors, in an in vivo test using a hypertensive mouse retinal neovascularization model, crystallin β-B2, crystallin β-A3 in retinal tissue in which angiogenesis was observed. It was confirmed by DNA microarray that various crystallin mRNAs such as crystallin β-A1 and crystallin γ-C were highly expressed. In addition, we confirmed by Western blotting that crystallin β protein production was enhanced in retinal tissues where angiogenesis was observed, and immunized the presence of crystallin β only at the site where neovascularization occurred in the retina. Confirmed by staining. Furthermore, in human diabetic retinopathy patients, it was confirmed by immunostaining that crystallin β was localized at the retinal neovascular site. Therefore, from the above results, it is recognized that crystallin, particularly crystallin β, is deeply involved in the formation of angiogenesis and its development.
Furthermore, in order to inhibit crystallin production in the eye, the present inventors made siRNA, which is a specific crystallin β inhibitor, and intraocularly administered it to a mouse model of retinal neovascularization with high oxygen load. As a result, it was observed that the number of neovascular nuclei observed on the vitreous side of the retinal tissue section was significantly reduced. Therefore, it has been found that a crystallin inhibitor can inhibit angiogenesis that occurs in the eye and is useful for the prevention or treatment of intraocular angiogenesis diseases that develop due to angiogenesis.
That is, by containing, as an active ingredient, a crystallin inhibitor including siRNA composed of an RNA chain having the nucleotide sequence shown by the following SEQ ID NOs: 1 to 7 and its complementary chain, prevention of intraocular neovascular disease or Agents useful for treatment can be provided.
In the present invention, an intraocular neovascular disease refers to an intraocular disease that develops by involving angiogenesis, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, angiogenesis. Examples include macular disease, neovascular glaucoma, iris rubeosis, and Eales disease.
In the present invention, a crystallin inhibitor refers to a substance having a crystallin inhibitory action, and the crystallin inhibitory action is not limited to the inhibition of the production or action of crystallin per se. Including inhibition of production or action. As the crystallin inhibitor, those for crystallin β are preferable, and those for crystallin β-B2 are more preferable. Specifically, (i) a double-stranded RNA having a crystallin inhibitory effect, (ii) an antisense oligonucleotide for crystallin, (iii) a compound having a crystallin inhibitory effect and its sense oligonucleotide, and (iv) a crystallin inhibitory effect (V) an antisense oligonucleotide expression recombinant vector for crystallin, (vi) a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof, (vii) a crystallin inhibitory action A double-stranded RNA expression recombinant vector having antisense, an antisense oligonucleotide expression recombinant vector for crystallin, a compound having a crystallin inhibitory activity, or a sense oligonucleotide expression recombinant vector thereof It can be exemplified transformed host cells such as by coater. The double-stranded structure in double-stranded RNA can be formed by a single self-complementary RNA strand or two separate complementary RNA strands. In the present invention, the double-stranded RNA is, for example, hybridized with a crystallin gene under stringent conditions (for example, 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 50 ° C. or 70 ° C. for 12 to 16 hours. Double-stranded RNA composed of RNA that is washed after hybridization) and its complementary RNA are included. Stringent conditions generally refer to conditions used by those skilled in the art to establish at least 90% sequence identity between a crystallin gene or portion thereof and RNA. Examples of hybridization and subsequent washing conditions include, for example, Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John & Sons, Publishers (1994); Sambrook et al. , Molecular Cloning, Cold Spring Harbor (1989). The above sequence identities are determined by sequence comparison or alignment algorithms known to those skilled in the art (Sequence Analysis Primer, Stockton Press (1991); Computational Molecular Biology, Oxford University Presence (1988); Biocompamping Press (1988); 1993); Computer Analysis in Molecular Biology, Academic Press (1987)). As the crystallin inhibitor in the present invention, siRNA (small which means a double-stranded RNA of 21 to 23 bases which specifically inhibits the expression of crystallin by partially or completely losing the function of the target gene by RNA interference. interfering RNA). Such siRNA is preferably a double-stranded RNA composed of the RNA strand shown in any one of the following SEQ ID NOs: 1 to 7 described in Example 1 and its complementary strand. In the present invention, an antisense oligonucleotide expression recombinant vector for crystallin refers to replicating a base sequence encoding an antisense oligonucleotide for crystallin and expressing the antisense oligonucleotide in a target tissue or host cell. Means a recombinant vector capable of A compound having a crystallin inhibitory effect or a sense oligonucleotide expression recombinant vector thereof means a recombinant vector capable of expressing a compound having a crystallin inhibitory effect or a sense oligonucleotide thereof in a target tissue or host cell. Moreover, as a compound which has a crystallin inhibitory effect, the low molecular compound etc. which have a crystallin inhibitory effect other than the protein containing a crystallin antibody are contained.
The siRNA used in the present invention can be prepared, for example, using an RNA synthesis technique generally performed enzymatically or partially or totally organically (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989); Dna Cloning, Vol. I and II, DN Glover ed., (1985); Oligo Nucleotide Synthesis, M. Gait ed., C. , BD Hanies & SJ Higgins eds., (1984); Animal Cell Culture, RI Freshney ed., (1986). Immobilised Cells and Enzymes, IRL Press (1986); B.Perbal, A Practical Guide to Molecular Cloning (1984) etc.).
The method for using the crystallin inhibitory substance of the present invention includes the above-mentioned double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, a compound having a crystallin inhibitory action, or a sense oligonucleotide thereof Recombinant expression vectors and host cells transformed with these recombinant vectors are introduced into target tissues in vivo by a suitable method, and the desired double-stranded RNA, antisense nucleotide, various compounds, or sense oligonucleotides thereof Is used as a genetic preventive or therapeutic agent by expressing, double-stranded RNA having a crystallin inhibitory effect, an antisense oligonucleotide to crystallin, or a compound having a crystallin inhibitory activity or its sens It may include use as a drug prophylactic or therapeutic agents by which administration of a pharmaceutical composition comprising, as an active ingredient, an oligonucleotide orally or parenterally. Examples of parenteral administration methods include topical administration into the eye and intravenous administration.
Examples of vectors that can be used in the present invention include plasmid vectors, virus vectors (eg, retrovirus vectors, adenovirus vectors, herpes virus vectors, Sendai virus vectors, vaccinia virus vectors), liposome vectors (eg, cationic liposome vectors), and the like. Can be mentioned.
In a recombinant vector, in addition to a double-stranded RNA having a crystallin-inhibiting action, a base sequence encoding an antisense oligonucleotide for crystallin, etc., the desired double-stranded DNA is actually introduced into a target tissue or host cell. In order to express RNA, antisense oligonucleotide, etc., a base sequence (for example, promoter sequence, terminator sequence, enhancer sequence) that controls the expression and gene marker for selecting a microorganism, insect cell, or animal cultured cell ( For example, a neomycin resistance gene, a kanamycin resistance gene) and the like may be included.
Examples of host cells that can be used in the present invention include E. coli, yeast, insect cells, CHO cells, COS cells, mink lung epithelial cells (for example, Mv1Lu cells), lymphocytes, fibroblasts (NIH / 3T3 cells), There may be mentioned animal cells such as blood cells, kidney cells (eg 293 cells) and tumor cells (eg Hela cells).
As a method for introducing a recombinant vector into a target tissue or host cell, the HVJ liposome method (Kaneda, Experimental Medicine, Vol. 12, No. 2, p. 78 (1994); Morishita et al., Experimental Medicine, Vol. 12, No. 15, p.158 (1994)), a method in which a crystallin inhibitor is directly administered by injection or the like, a calcium phosphate method, a DEAE-dextran method, an electroporation method, a gene gun method (TM Klein et al. Bio / Technology 10, pp. 286-291 (1992)), administration by lipofection (Nabel et al., Science, Vol. 244, p. 1285 (1990)), vectors (eg, retroviral vectors, Adenovirus vector, herpesvirus vector Coater can be exemplified a method in which using a vaccinia virus vector, etc.).
When a pharmaceutical composition containing a crystallin inhibitory substance as an active ingredient is actually used in the prevention or treatment of intraocular neovascular diseases, various preparation forms are used depending on the usage. Examples of the dosage form include tablets, capsules, granules, powders, pills, fine granules, troches, injections, rectal administration, suppositories and the like, and are administered orally or parenterally. Is done.
These various pharmaceutical compositions containing a crystallin inhibitory substance include the above-mentioned crystallin inhibitory substance as an active ingredient, and usually used excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents. It can be produced by mixing or diluting or dissolving as appropriate with pharmaceutical additives such as an agent, preservative, wetting agent, emulsifying agent, dispersing agent, stabilizing agent, solubilizing agent and the like, and preparing according to a conventional method.
The dose of the crystallin inhibitor in pharmacological prophylaxis or treatment for intraocular neovascular diseases can be appropriately determined in consideration of usage, patient age, sex, symptom level, disease type, and the like. In addition, the dose of a crystallin inhibitor in genetic prevention or treatment for an intraocular neovascular disease can be appropriately determined in the same manner.
According to the present invention, double-stranded RNA having a crystallin-inhibiting action, an antisense oligonucleotide for crystallin, a compound having a crystallin-inhibiting action or a base sequence encoding the sense oligonucleotide, a vector containing the same, or The contact or introduction of the transformed host cells into the target tissue can suppress the onset or progression of intraocular neovascular diseases. Further, by administering orally or parenterally a double-stranded RNA having a crystallin inhibitory effect, an antisense oligonucleotide to crystallin, or a compound having a crystallin inhibitory activity or a sense oligonucleotide thereof, orally or parenterally, The onset or development of angiogenic diseases can be suppressed.

FIG. 1 is a western blot image showing the expression level of crystallin β protein in the isolated retinal neovascularization of a mouse loaded with high concentration. The vertical axis shows the weight of protein (kDa), the horizontal axis shows the number of days (days) from birth to retinal extraction, ROP P13 is 13 days after birth, ROP P14 is 14 days after birth, ROP P15 Means 15 days after birth, ROP P16 means 16 days after birth, ROP P17 means 17 days after birth, and ROP P18 means 18 days after birth.
FIG. 2 is a confocal laser fluorescence micrograph showing the expression site of crystallin β in new blood vessels in the mouse retina.
FIG. 3 is a confocal laser fluorescence micrograph showing the expression site of crystallin β in new blood vessels in the mouse retina.
FIG. 4 is a confocal laser fluorescence micrograph showing the expression site of crystallin β in a human diabetic retinopathy patient.

（試験例３）
網膜新生血管におけるクリスタリンβタンパク質の発現量の確認
試験例１記載の高酸素負荷群のマウスを用いて、生後１３〜１８日に眼球を摘出し、摘出網膜よりタンパク質を抽出し、ウエスタンブロット法にてクリスタリンβの発現量を確認した。その結果は、第１図に示す通りであり、網膜上の新生血管においては、生後１４及び１５日にクリスタリンβタンパク質が高発現していた。
（試験例４）
網膜新生血管におけるクリスタリンβの発現部位の確認
試験例１にて作製したパラフィン包埋切片を用いて、クリスタリンβ特異的ペプチド抗体（ＳｔｒｅｓＧｅｎＳＰＡ−２３０）及び血管内皮細胞特異的蛍光抗体（Ｇｒｉｆｆｏｎｉａ ｓｉｍｐｌｉｃｉｆｏｌｉａ ｌｅｃｔｉｎ）にてクリスタリンβの発現部位を共焦点レーザー蛍光顕微鏡で確認した。その結果は、第２図及び第３図に示す通りであり、クリスタリンβの発現が血管内皮細胞に局在的に認められた。特に、硝子体側に突出している新生血管部位（ｎｅｏｖａｓｃｕｌａｒ ｔｕｆｔ）に多く発現していた。
（試験例５）
ヒト糖尿病網膜症患者におけるクリスタリンβの発現部位の確認
ヒト糖尿病網膜症患者から摘出した網膜新生血管のパラフィン包埋切片を用いて、クリスタリンβ特異的ペプチド抗体（ＳｔｒｅｓＧｅｎＳＰＡ−２３０）及び血管内皮細胞特異的蛍光抗体（Ｇｒｉｆｆｏｎｉａ ｓｉｍｐｌｉｃｉｆｏｌｉａ ｌｅｃｔｉｎ）にてクリスタリンβの発現部位を共焦点レーザー蛍光顕微鏡で確認した。その結果は、第４図に示す通りであり、クリスタリンβの発現が新生血管部位に局在的に認められた。The content of the present invention will be described in more detail with reference to the following test examples and examples, but the present invention is not limited to the content.
(Test Example 1)
Preparation of high oxygen load mouse retinal neovascularization model C57BL / 6J mice were bred under 75% oxygen with their mother for 7 to 5 days after birth, and then returned to normal oxygen concentration (hereinafter referred to as high oxygen load group). The control group of mice was bred under normal oxygen concentration after birth. Under anesthesia, FITC (fluorescent isothiocyanate) -dextran 50 mg / mL was injected into the left ventricle, and then the eyeball was extracted. Retinal neovascularization was evaluated by preparing a flat-mount specimen and performing fluorescence fundus angiography. In addition, the isolated eyeball was fixed in 4% paraformaldehyde, and a paraffin-embedded section cut half-cut on a vertical plane passing through the nipple was prepared and stained with hematoxylin-eosin, and retinal neovascularization was also evaluated under an optical microscope. As a result, in the high oxygen load group, no new blood vessels were observed 12 days after birth, but new blood vessels were remarkably observed on days 15 and 17 after birth.
(Test Example 2)
Confirmation of mRNA expression level in retinal neovascularization Using the mouse described in Test Example 1, on the 15th day after birth, the eyeball was extracted from the high oxygen load group and the control group, and RNA was extracted from the extracted retina. Hybridization was performed using GeneChip MGU74Av2 (manufactured by Affymetrix), and the increase / decrease in RNA expression level between the two groups was compared. The results are as shown in Table 1 below. In the new blood vessels on the retina, mRNA of crystallin β-B2, crystallin β-A3 / A1, and crystallin γ-C is remarkably high expressed. confirmed.

(Test Example 3)
Confirmation of the expression level of crystallin β protein in retinal neovascularization Using the mice in the high oxygen load group described in Test Example 1, the eyeballs were excised from 13 to 18 days after birth, and the proteins were extracted from the excised retina and subjected to Western blotting. Thus, the expression level of crystallin β was confirmed. The result is as shown in FIG. 1. In the new blood vessels on the retina, the crystallin β protein was highly expressed at 14 and 15 days after birth.
(Test Example 4)
Confirmation of the expression site of crystallin β in retinal neovascularization Using the paraffin-embedded section prepared in Test Example 1, a crystallin β-specific peptide antibody (StresGenSPA-230) and a vascular endothelial cell-specific fluorescent antibody (Griffonia simplicifolia lectin) Then, the expression site of crystallin β was confirmed with a confocal laser fluorescence microscope. The results are as shown in FIGS. 2 and 3, and the expression of crystallin β was locally observed in vascular endothelial cells. In particular, it was highly expressed in the neovascular site protruding toward the vitreous body.
(Test Example 5)
Confirmation of the expression site of crystallin β in human diabetic retinopathy patients Using paraffin-embedded sections of retinal neovascularization removed from human diabetic retinopathy patients, crystallin β-specific peptide antibody (StresGenSPA-230) and vascular endothelial cell specific The expression site of crystallin β was confirmed with a confocal laser fluorescence microscope using a fluorescent antibody (Griffonia symphofolia lectin). The result is as shown in FIG. 4, and the expression of crystallin β was locally observed in the neovascular site.

また、同様にして、下記配列番号２〜７で示されたＲＮＡから相当するｓｉＲＮＡを作製することができる（ｓｉＲＮＡＮｏ．２〜７）。

Preparation of siRNA against crystallin β After preparing the RNA shown in SEQ ID NO: 1 below and its complementary RNA using an RNA synthesis technique generally carried out enzymatically or partially or totally organically, The siRNA (siRNA No. 1) was produced by annealing.

Similarly, siRNA corresponding to the RNAs shown in SEQ ID NOs: 2 to 7 below can be prepared (siRNA Nos. 2 to 7).

Confirmation test of inhibitory effect on retinal neovascularization Using the mice of the high oxygen load group described in Test Example 1, siRNA No. 1 described in Example 1 on days 12, 14 and 16 after birth. 1 (5 μg) was dissolved in physiological saline and intravenously injected (siRNA administration group). In the control group, only physiological saline was intravenously injected. After the retina was removed on the 17th day after birth, the excised eyeball was fixed in 4% paraformaldehyde, and a paraffin-embedded section cut in a vertical plane passing through the nipple was prepared, stained with hematoxylin-eosin, and retinal neovascularization under an optical microscope. The number of nuclei was measured and the effect of inhibiting angiogenesis was evaluated. The results are as shown in Table 2. A significant angiogenesis inhibitory effect was confirmed by the administration of siRNA.

  The present invention relates to the prevention or treatment of intraocular neovascular diseases using a crystallin inhibitor as an active ingredient. The present invention can provide a preventive or therapeutic agent for various intraocular neovascular diseases such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, and age-related macular degeneration.

[Sequence Listing]

Claims (46)

A prophylactic or therapeutic agent for intraocular neovascular diseases, comprising a crystallin inhibitor. The preventive or therapeutic agent according to claim 1, wherein the crystallin is crystallin β. The preventive or therapeutic agent according to claim 2, wherein the crystallin is crystallin β-B2. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the crystallin inhibitor is a double-stranded RNA having a crystallin inhibitory action. The preventive or therapeutic agent according to claim 4, wherein the double-stranded RNA is siRNA. The preventive or therapeutic agent according to claim 5, wherein the siRNA is an RNA composed of an RNA strand having a nucleotide sequence represented by any of SEQ ID NOs: 1 to 7 and a complementary strand thereof. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the crystallin inhibitor is an antisense oligonucleotide to crystallin. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the crystallin inhibitor is a compound having a crystallin inhibitory action or a sense oligonucleotide thereof. The crystallin inhibitor is a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, or a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof. Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3. The crystallin inhibitor is selected from a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, and a compound having a crystallin inhibitory action or its sense oligonucleotide expression recombinant vector The preventive or therapeutic agent according to any one of claims 1 to 3, which is a host cell transformed with a recombinant vector. The intraocular neovascular disease is a disease selected from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, neovascular macular disease, neovascular glaucoma, iris rubeosis and Eales disease. The preventive or therapeutic agent according to any one of 1 to 10. The prophylactic or therapeutic agent according to claim 11, wherein the intraocular neovascular disease is diabetic retinopathy. The prophylactic or therapeutic agent according to claim 11, wherein the intraocular neovascular disease is retinopathy of prematurity. The prophylactic or therapeutic agent according to claim 11, wherein the intraocular neovascular disease is retinal vein occlusion. The preventive or therapeutic agent according to claim 11, wherein the intraocular neovascular disease is age-related macular degeneration or angiogenic macular disease. A method for preventing or treating intraocular neovascular diseases, comprising administering an effective amount of a crystallin inhibitor. The method for prevention or treatment according to claim 16, wherein the crystallin is crystallin β. The method for prevention or treatment according to claim 17, wherein the crystallin is crystallin β-B2. The method for prevention or treatment according to any one of claims 16 to 18, wherein the crystallin inhibitor is a double-stranded RNA having a crystallin inhibitory action. The prevention or treatment method according to claim 19, wherein the double-stranded RNA is siRNA. 21. The prevention or treatment method according to claim 20, wherein the siRNA is an RNA composed of an RNA strand having a nucleotide sequence represented by any of SEQ ID NOs: 1 to 7 and a complementary strand thereof. The method for prevention or treatment according to any one of claims 16 to 18, wherein the crystallin inhibitor is an antisense oligonucleotide to crystallin. The method for prevention or treatment according to any one of claims 16 to 18, wherein the crystallin inhibitor is a compound having a crystallin inhibitory action or a sense oligonucleotide thereof. The crystallin inhibitor is a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, or a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof. Item 19. The prevention or treatment method according to any one of Items 16 to 18. The crystallin inhibitor is selected from a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, and a compound having a crystallin inhibitory action or its sense oligonucleotide expression recombinant vector The method for prevention or treatment according to any one of claims 16 to 18, which is a host cell transformed with a recombinant vector. The intraocular neovascular disease is a disease selected from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, neovascular macular disease, neovascular glaucoma, iris rubeosis and Eales disease. The prevention or treatment method according to any of 16 to 25. 27. The prevention or treatment method according to claim 26, wherein the intraocular neovascular disease is diabetic retinopathy. 27. The prevention or treatment method according to claim 26, wherein the intraocular neovascular disease is retinopathy of prematurity. 27. The prevention or treatment method according to claim 26, wherein the intraocular neovascular disease is retinal vein occlusion. 27. The prevention or treatment method according to claim 26, wherein the intraocular neovascular disease is age-related macular degeneration or angiogenic macular disease. Use of a crystallin inhibitor for producing a pharmaceutical composition for prevention or treatment of an intraocular neovascular disease. 32. Use according to claim 31, wherein the crystallin is crystallin beta. 33. Use according to claim 32, wherein the crystallin is crystallin [beta] -B2. The use according to any of claims 31 to 33, wherein the crystallin inhibitor is a double-stranded RNA having a crystallin inhibitory action. 35. Use according to claim 34, wherein the double stranded RNA is siRNA. The use according to claim 35, wherein the siRNA is an RNA composed of an RNA strand having the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 7 and a complementary strand thereof. 34. Use according to any of claims 31 to 33, wherein the crystallin inhibitor is an antisense oligonucleotide to crystallin. The use according to any one of claims 31 to 33, wherein the crystallin inhibitor is a compound having a crystallin inhibitory activity or a sense oligonucleotide thereof. The crystallin inhibitor is a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, or a compound having a crystallin inhibitory action or a sense oligonucleotide expression recombinant vector thereof. The use according to any one of Items 31 to 33. The crystallin inhibitor is selected from a double-stranded RNA expression recombinant vector having a crystallin inhibitory action, an antisense oligonucleotide expression recombinant vector for crystallin, and a compound having a crystallin inhibitory action or its sense oligonucleotide expression recombinant vector 34. Use according to any of claims 31 to 33, which is a host cell transformed with a recombinant vector. The intraocular neovascular disease is a disease selected from diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, age-related macular degeneration, neovascular macular disease, neovascular glaucoma, iris rubeosis and Eales disease. Use in any one of 31-40. 42. Use according to claim 41, wherein the intraocular neovascular disease is diabetic retinopathy. 42. Use according to claim 41, wherein the intraocular neovascular disease is retinopathy of prematurity. 42. Use according to claim 41, wherein the intraocular neovascular disease is retinal vein occlusion. 42. Use according to claim 41, wherein the intraocular neovascular disease is age-related macular degeneration or angiogenic macular disease. An siRNA composed of an RNA strand having the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 7 and its complementary strand.

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