Corneal endothelium ECM therapeutic drug

Abstract

To provide a treatment or preventive drug for diseases, disorders or conditions relating to abnormalities in the extracellular matrix (ECM) of the corneal endothelium, said drug containing a TGF β-signal inhibitor.SOLUTION: More specifically, said diseases, disorders and conditions refer to those relating to Fuchs' corneal endothelial dystrophy. Such disorders include photophobia, blurred vision, vision disorders, eye pain, tearing, hyperaemia, pain, bullous keratopathy, eye discomfort, contrast reduction, glare, edema of the corneal stroma, bullous keratopathy and corneal opacity. One preferable TGF β-signal inhibitor is 4-[4-(1,3-benzodioxole-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a disease, disorder, or condition associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium. The present invention relates to techniques, methods, and agents for treating or preventing the above-mentioned diseases. [Background technology]

[0002] Visual information is perceived when light enters the cornea, the transparent tissue at the front of the eye, reaches the retina, excits retinal nerve cells, and the generated electrical signals are transmitted via the optic nerve to the visual cortex in the cerebrum. A transparent cornea is necessary for good vision. Corneal transparency is maintained by maintaining a constant water content through the pumping and barrier functions of the corneal endothelial cells.

[0003] At birth, human corneal endothelial cells are present at a density of approximately 3,000 cells per square millimeter, but once damaged, their regeneration capacity is extremely limited. Fuchs' endothelial corneal dystrophy is a disease in which endothelial cells on the inner surface of the cornea become abnormal, causing corneal edema, and the cause is unknown. In Fuchs' endothelial corneal dystrophy, extracellular matrix such as collagen is deposited in a portion of the posterior surface of Descemet's membrane at the posterior part of the cornea, resulting in thickening of the corneal guttae and Descemet's membrane. Thickening of the corneal guttae and Descemet's membrane causes photophobia and blurred vision in patients with Fuchs' endothelial corneal dystrophy, significantly impairing their quality of life. There is said to be no effective treatment for Fuchs' endothelial corneal dystrophy other than corneal transplantation, but there is a shortage of corneal donors in Japan, and while there are approximately 2,600 patients waiting for corneal transplants, only about 1,700 corneal transplants are performed in the country each year.

[0004] Regarding Fuchs' corneal endothelial dystrophy, there have been reports of culturing corneal endothelial cells derived from patients with Fuchs' cornea (Non-Patent Documents 1 and 3) and immortalizing them (Non-Patent Document 2). However, there have been no reports of cells that are suitable for screening therapeutic agents or agents to prevent progression of the disease while maintaining the characteristics of the disease, such as excessive production of extracellular matrix. As a result, there are limitations to the development of therapeutic agents, and currently no therapeutic agents are in clinical use, and the only option is to rely on corneal transplants.

[0005] Furthermore, Patent Document 1 discloses a TGF-β1 inhibitor peptide for treating corneal fibrosis and / or haze. Patent Document 2 discloses an antibody that binds to TGF-β1, 2, and 3. Patent Document 3 discloses that an Nrf2 agonist or activator can be used to treat corneal endothelial disorders. Patent Document 4 discloses a peptide that can bind to transforming growth factor TGF-β1 (TGF-β1) and that acts as a potent inhibitor of TGF-β1 biological activity by directly binding to the cytokine. Patent Document 5 discloses a scar formation inhibitor containing a BMP-7 polypeptide. Patent Document 6 generally describes corneal disorders as diseases for which TGFβ inhibition is effective for treatment or prevention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special publication 2013-520405 [Patent Document 2] International Publication No. 2012 / 167143 Brochure [Patent Document 3] International Publication No. 2012 / 009171 Brochure [Patent Document 4] Special Publication No. 2007-525204 [Patent Document 5] Special Publication No. 2006-508169 [Patent Document 6] International Publication No. 2004 / 018430 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Zaniolo K, et al. Exp Eye Res.;94(1):22-31. 2012 [Non-patent document 2] Azizi B, et al. Invest Ophthalmol Vis Sci. 2;52(13):9291-9297. 2011 [Non-patent document 3] Kelliher C. et al. Exp Eye Res Vol.93(6), 880-888, 2011 Summary of the Invention [Means for solving the problem]

[0008] The present inventors have discovered that inhibiting the transforming growth factor-β (TGF-β) pathway can suppress the deposition of extracellular matrix (ECM) such as collagen, which is seen in conditions such as Fuchs' endothelial corneal dystrophy, and have discovered a technology that can treat or prevent ECM-related disorders, leading to the completion of the present invention. Accordingly, the present invention provides the following: (1) A therapeutic or preventive agent for a disease, disorder, or condition associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, comprising a TGFβ signal inhibitor. (2) The therapeutic or preventive agent according to item 1, wherein the disease, disorder or condition is a disorder related to Fuchs' endothelial corneal dystrophy. (3) The therapeutic or preventive agent according to item 1 or 2, wherein the disease, disorder, or condition includes at least one selected from the group consisting of photophobia, blurred vision, visual impairment, eye pain, tearing, redness, pain, bullous keratopathy, ocular discomfort, decreased contrast, glare, corneal stromal edema, bullous keratopathy, and corneal opacity in Fuchs' endothelial corneal dystrophy. (4) The TGFβ signal inhibitor is selected from the group consisting of 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide, BMP-7, anti-TGF-β antibody, anti-TGF-β receptor antibody, siRNA for TGF-β, siRNA for TGF-β receptor, shRNA for TGF-β, shRNA for TGF-β receptor, aptamer for TGF-β receptor, aptamer for TGF-β receptor, antisense oligonucleotide for TGF-β, 6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone, 3-(6-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone, and the like. 4. The agent for treatment or prevention according to any one of items 1 to 3, comprising at least one of 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, 6-(4-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine, 2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine, 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, a pharmaceutically acceptable salt or solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof. (5) The TGF-β signal inhibitor is 4-[4-(1,3-benzodioxole-5- 5. The agent for treatment or prevention according to any one of items 1 to 4, comprising [5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide or a pharmaceutically acceptable salt thereof. (6) The treatment or prophylaxis according to any one of items 1 to 5, wherein the corneal endothelium is that of a primate. (7) The treatment or prophylaxis according to any one of items 1 to 6, wherein the corneal endothelium is human. (8) The agent for treatment or prevention according to any one of items 1 to 7, which further comprises a medicinal ingredient. (9) The agent for treatment or prevention according to any one of items 1 to 8, which is an eye drop. (10) A TGFβ signal inhibitor for treating or preventing disorders associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium. (10A) The TGFβ signal inhibitor has the characteristics of any one of the inhibitors described in (1) to (9). The TGFβ signal inhibitor according to (10), having the following characteristics: (11) A method for treating or preventing a disorder associated with an abnormality in the extracellular matrix (ECM) of the corneal endothelium in a subject, the method comprising administering to the subject an effective amount of a TGFβ signal inhibitor.

[0009] It is contemplated that the present invention may provide one or more of the above-described features in combinations other than those explicitly described. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0010] The present invention provides a technology that can be implemented as a pharmaceutical drug or eye drops, etc., that can treat or prevent diseases associated with extracellular matrix (ECM) abnormalities, such as Fuchs' endothelial corneal dystrophy, for which the only treatment option available until now was corneal transplantation. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 1 shows the upregulation of Snail1 and ZEB1 expression in corneal endothelial cells derived from patients with Fuchs endothelial corneal dystrophy. Figure 1 shows the results of real-time PCR analysis of gene expression levels related to epithelial-mesenchymal transition (EMT), which is involved in extracellular matrix production. iFCEDs were cultured from corneal endothelial cells obtained with written consent and approval from the ethics committee from three patients who had undergone endothelial keratoplasty (DMEK) due to bullous keratopathy diagnosed with Fuchs endothelial corneal dystrophy. Cultured corneal endothelial cells from patients with Fuchs endothelial corneal dystrophy were transfected with SV40 and hTERT genes via lentivirus to generate immortalized cell lines. As a control, corneal endothelial cells cultured from research corneas imported from the Seattle Eye Bank were immortalized in the same manner to generate immortalized cell lines (iHCECs). iHCECs and iFECDs were maintained in DMEM + 10% FBS. In each graph, the left shows iHCECs, and the right shows iFECDs. A shows the relative expression of Snail1, B shows the relative expression of Snail2, and C shows the relative expression of ZEB1. Real-time PCR revealed significantly increased expression of Snail1 and ZEB1 in iFECDs compared to iHCECs. [Figure 2]Figure 2 shows that TGF-beta increases the expression of Snail1, ZEB1, and extracellular matrix proteins. To confirm whether increased expression of Snail1 and ZEB1 is related to extracellular matrix production, we stimulated iHCECs with TGF-beta, which is known to promote Snail1 and ZEB1 expression. White indicates iHCECs, and black indicates iFECDs. A indicates the relative expression of Snail1, B indicates the relative expression of ZEB1, C indicates the relative expression of type I collagen, D indicates the relative expression of type IV collagen, E indicates the relative expression of type VIII collagen, and F indicates the relative expression of fibronectin. We confirmed that TGF-beta significantly promoted the expression of Snail1 and ZEB1 in iFECDs (A, B). Therefore, we analyzed the gene expression levels of extracellular matrix proteins by real-time PCR, and found that the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin was significantly promoted. [Figure 3]Figure 3 shows how TGF-beta promotes protein production in an in vitro matrix protein evaluation model. Figure 3 shows the results of an investigation into whether extracellular matrix production by iFECD is promoted by TGF-beta. iHCECs and iFECDs were cultured on Transwell plates in DMEM in a serum-free environment for 1 week. After confluence, they were fixed and subjected to HE staining. (The left panel shows HE-stained micrographs; the upper panel shows iHCECs, and the lower panel shows iFECDs. The left panel shows the control, and the right panel shows the results of TGF-beta stimulation.) The right panel shows the thickness measurements. In the graph, the left panel shows the control, and the right panel shows the results of TGF-beta stimulation. White indicates iHCECs, and black indicates iFECDs. * and # indicate statistical significance with p<0.05. Both iHCECs and iFECDs produced significantly thicker extracellular matrix upon TGF-beta stimulation. Furthermore, in the presence of TGF-beta, iFECDs produced significantly thicker extracellular matrix than iHCECs. These findings indicate that corneal endothelial cells from patients with Fuchs' corneal endothelial dystrophy have high expression levels of Snail1 and ZEB1, and produce significantly higher amounts of extracellular matrix in response to TGFβ stimulation than corneal endothelial cells from healthy individuals. [Figure 4]Figure 4 shows that ZEB1 or Snail1 negatively regulates the gene expression of extracellular matrix component proteins. Figure 4 shows the results of silencing Snail1 and ZEB1 using siRNA to demonstrate that increased expression of Snail1 and ZEB1 is responsible for extracellular matrix production. White indicates iHCEC, and black indicates iFECD. AE indicates the results of Snail1 siRNA, and FJ indicates the results of Snail1 siRNA. A indicates the results of ZEB1, F indicates the results of SNAIL1, B and G indicate the results of type I collagen, C and H indicate the results of type IV collagen, D and I indicate the results of type VIII collagen, and E and J indicate the results of fibronectin. * indicates statistical significance at p<0.01. The suppression of Snail1 and ZEB1 expression by siRNA was confirmed (A, F). Silencing of Snail1 or ZEB1 expression by siRNA significantly suppressed the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin. [Figure 5] Figure 5 shows how ZEB1 or Snail1 negatively regulates the expression of in vitro matrix components. Figure 5 also shows the results of immunostaining to examine the expression of type I collagen, type IV collagen, and fibronectin. The left side shows control siRNA, the center side shows ZEB siRNA, and the right side shows the results with SNAIL1 siRNA. The top two rows show type I collagen, the middle two rows show type IV collagen, and the bottom two rows show fibronectin. The upper row of each row shows iHCEC, and the lower row shows iFECD. We confirmed that silencing Snail1 or ZEB1 expression with siRNA also suppressed the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin at the protein level. [Figure 6]Figure 6 demonstrates that suppression of ZEB1 or Snail1 suppresses excessive extracellular matrix production in Fuchs corneal endothelial dystrophy cells. Figure 6 also shows the results of HE staining of iHCECs and iFECDs cultured in serum-free DMEM on Transwell plates after one week at confluence. The cells were then fixed and stained with HE. (The left panel shows HE-stained micrographs; the upper panel shows iHCECs, and the lower panel shows iFECDs. The left panel shows the siRNA control, the center panel shows siRNA ZEB1, and the right panel shows the results with siRNA SNAIL.) The right panel shows thickness measurements. The left panel shows the siRNA control, the center panel shows siRNA ZEB1, and the right panel shows the results with siRNA SNAIL. White indicates iHCECs, and black indicates iFECDs. * indicates statistical significance at p<0.01. Suppression of Snail1 or ZEB1 expression by siRNA suppressed excessive extracellular matrix production in iFECDs, returning them to normal levels. [Figure 7] Figure 7 shows that TGFβ signaling inhibition suppresses the expression of Snail1, ZEB1, and extracellular matrix proteins. Figure 7 shows the results of TGFβ signaling inhibition using the TGFβ signaling inhibitor SB431542 (0 μM, 1 μM, 3 μM, 10 μM). White indicates iHCECs, and black indicates iFECDs. A indicates Snail1, B indicates ZEB1, D indicates type I collagen, E indicates type IV collagen, F indicates type VIII collagen, and G indicates fibronectin. * indicates statistical significance at p<0.01. Real-time PCR confirmed significant reductions in the expression of Snail1 and ZEB1 following TGFβ signaling inhibition. Furthermore, real-time PCR analysis of the gene expression levels of extracellular matrix proteins in iFCEDs revealed significant suppression of the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin. [Figure 8]Figure 8 shows how TGFβ signaling inhibition can control the expression of in vitro matrix constituent proteins. Similar to Figure 7, Figure 8 shows the results of immunostaining to examine the expression of type I collagen, type IV collagen, and fibronectin. The left side shows the control, and the right side shows the results of stimulation with SB431542. The top two rows show type I collagen, the middle two rows show type IV collagen, and the bottom two rows show fibronectin. The upper row of each row shows iHCEC, and the lower row shows iFECD. We confirmed that TGFβ signaling inhibition with SB431542 also suppressed the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin at the protein level. [Figure 9] Figure 9 shows that inhibition of TGFβ signaling can suppress excessive extracellular matrix production in Fuchs' corneal endothelial dystrophy cells. Figure 9 also shows the results of HE staining of iHCECs and iFECDs cultured in serum-free DMEM on a Transwell plate after one week at confluence, which were then fixed (left panel, upper row shows iHCECs, lower row shows iFECDs. The left panel shows the control, and the right panel shows the results of SB431542 stimulation). The graph on the right shows the thickness measurements. The left panel shows the control, and the right panel shows the results of SB431542 stimulation, with white representing iHCECs and black representing iFECDs. * indicates statistical significance at p<0.01. Inhibition of TGFβ signaling with SB431542 suppressed excessive extracellular matrix production in iFECDs, returning them to normal levels. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will prevail.

[0013] (definition) As used herein, "iFECD" (immobilized Fuchs' endothelial corneal dystrophy) is an abbreviation for immortalized cells of Fuchs' endothelial corneal dystrophy.

[0014] As used herein, "HCEC" (human corneal endothelial cells) is an abbreviation for human corneal endothelial cells. "iHCEC" is an abbreviation for immobilized human corneal endothelial cells.

[0015] As used herein, the term "transforming growth factor-β (also abbreviated as TGF-β)" is used in the same sense as in the art. It is a homodimeric multifunctional cytokine with a molecular weight of 25 kD that exhibits diverse biological activities, including being involved in the pathogenesis of various sclerotic diseases, rheumatoid arthritis, and proliferative vitreoretinopathy, deeply involved in hair loss, suppressing the activity of immunocompetent cells, preventing the breakdown of lung tissue and leading to emphysema by inhibiting the overproduction of proteases, and suppressing the proliferation of cancer cells. In humans, three isoforms, TGF-β1 to β3, exist. TGF-β is produced as an inactive latent form with a molecular weight of approximately 300 kD that cannot bind to receptors. It is activated on the surface of target cells or their surroundings to become an active form that can bind to receptors and exert its effects.

[0016] Without wishing to be bound by theory, it is believed that the action of TGF-β in target cells is mediated by the phosphorylation pathway of a series of signaling proteins called Smads. First, activated TGF-β binds to the type II TGF-β receptor on the surface of the target cell, forming a receptor complex consisting of two type II receptor molecules and two type I TGF-β receptor molecules. The type II receptor then phosphorylates the type I receptor. The phosphorylated type I receptor then phosphorylates Smad2 or Smad3. The phosphorylated Smad2 and Smad3 then form a complex with Smad4, translocate to the nucleus, bind to target sequences called CAGA boxes in the promoter regions of target genes, and, together with coactivators, induce transcriptional expression of the target genes.

[0017] The transforming growth factor-β (TGF-β) signaling pathway can regulate many cellular activities, such as cell proliferation and differentiation, growth arrest, apoptosis, and epithelial-mesenchymal transdifferentiation (EMT), by regulating its target genes. Members of the TGF-β family, including TGF-β itself (e.g., TGF-β1, TGF-β2, and TGF-β3), activins, and bone morphogenetic proteins (BMPs), are potent regulators of cell proliferation, differentiation, migration, and apoptosis.

[0018] TGF-β is an approximately 24 Kd protein produced by many cells, including B lymphocytes, T lymphocytes, and activated macrophages, as well as by many other cell types. Among the effects of TGF-β on the immune system are IL-2 receptor induction, inhibition of IL-1-induced thymocyte proliferation, and blockade of IFN-γ-induced macrophage activation. TGF-β has been implicated in a variety of pathological conditions (Border et al. (1992) J. Clin. Invest. 90:1), and it is well documented that it functions as either a tumor suppressor or tumor promoter.

[0019] TGF-β mediates its signaling through two serine / threonine kinase cell surface receptors, TGF-βRII and ALK5. TGF-β signaling is initiated by ligand-induced receptor dimerization, which allows TGF-βRII to phosphorylate the ALK5 receptor. This phosphorylation activates ALK5 kinase activity, and activated ALK5 then phosphorylates downstream effector Smad proteins (vertebrate homologs of MAD, or "Mothers against DPP" proteins), Smad2 or 3. The p-Smad2 / 3 complex with Smad4 enters the nucleus and activates the transcription of target genes.

[0020] Smad3 is a member of the R-Smad (receptor-activated Smad) subgroup of Smads and is a direct mediator of transcriptional activation by the TGF-β receptor. TGF-β stimulation leads to the phosphorylation and activation of Smad2 and Smad3, which form a complex with Smad4 (the "common Smad" or "co-Smad" in vertebrates), which accumulates with the nucleus and regulates the transcription of target genes. R-Smads are localized in the cytoplasm, and upon ligand-induced phosphorylation by the TGF-β receptor, they form complexes with co-Smads and translocate to the nucleus, where they regulate gene expression by associating with chromatin and cooperating transcription factors. Smad6 and Smad7 are inhibitory Smads ("I-Smads"), i.e., they are transcriptionally induced by TGF-β and function as inhibitors of TGF-β signaling (Feng et al. (2005) Annu. Rev. Cell. Dev. Biol. 21:659). Smad6 / 7 exert their inhibitory effects by preventing receptor-mediated activation of R-Smads; they associate with type I receptors, competitively preventing the recruitment and phosphorylation of R-Smads. Smad6 and Smad7 are known to recruit E3 ubiquitin ligases, leading to the ubiquitination and degradation of Smad6 / 7-interacting proteins.

[0021] In addition to the TGF-β signaling pathway, there is also a pathway mediated by BMP-7, etc., which is believed to function via ALK-1 / 2 / 3 / 6 and Smad1 / 5 / 8. For details of the TGF-β signaling pathway, see J. Massagu'e, Annu. Rev. Biochem. 1998. 67: 753-91; Vilar JMG, Jansen R, Sander C (2006) PLoS Comput Biol 2(1):e3; Leask, A., Abraham, DJ FASEB J.18, 816-827 (2004); Coert Margadant & Arnoud Sonnenberg EMBO reports (2010)11, 97-105; Joel Rosenbloom et al., Ann Intern Med. 2010; 152: 159-166, etc.

[0022] As used herein, the term "transforming growth factor (TGF)-β signaling inhibitor" refers to any factor that inhibits TGF signaling. When a factor antagonizes TGF-β, it may also be called an antagonist, but in the context of the present invention, TGF-β antagonists are encompassed by TGF-β signaling inhibitors. Because these inhibitors are common substances, the term "TGFβ signaling inhibitor" can be used interchangeably with "TGFβ signaling inhibitor."

[0023] Therefore, typical examples of TGF-β signal inhibitors used in the present invention include, but are not limited to, TGF-β antagonists, TGF-β receptor antagonists, or Smad3 inhibitors, ligand traps (antibodies against ligands, decoy receptors), antisense oligonucleotides, TGF-β receptor kinase inhibitors, peptide aptamers, siRNA, shRNA, etc. (see, e.g., Connolly E., et al. Int. J. Biol. Sci. 2012; 8(7): 964-978 Fig. 3).

[0024] Exemplary TGF-β signal inhibitors that can be used in the present invention include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)]-1H-imidazol-2-yl]benzamide), BMP-7, anti-TGF-β antibodies, anti-TGF-β receptor antibodies, siRNA for TGF-β, siRNA for TGF-β receptors, antisense oligonucleotides for TGF-β, 6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone, A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), and stemolecules. TM TLK inhibitor (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), Stemolecule TM Examples of suitable inhibitors include, but are not limited to, BMP inhibitors LDN-193189 (6-(4-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine), SD-208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine), LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), pharmaceutically acceptable salts or solvates thereof, or solvates of pharmaceutically acceptable salts thereof.

[0025] Other TGF-β signaling inhibitors include monoclonal and polyclonal antibodies against one or more isoforms of TGF-β (U.S. Pat. No. 5,571,714; see also WO 97 / 13844 and WO 00 / 66631), TGF-β receptors, soluble forms of such receptors (e.g., soluble TGF-β type III receptors), or antibodies directed against TGF-β receptors (U.S. Pat. No. 5,693,607; U.S. Pat. No. 6,000,000). No. 1,969, U.S. Pat. No. 6,010,872, U.S. Pat. No. 6,086,867, U.S. Pat. No. 6,201,108; WO 98 / 48024; WO 95 / 10610; WO 93 / 09228; WO 92 / 00330), latency-associated peptide (WO 91 / 08291), large latent TGF-β (WO 94 / 09812), fetuin (U.S. Pat. No. 5,821,227), decorin as well as biglycan, fibromodulin, lumican, and and other proteoglycans such as endoglin (WO 91 / 10727; U.S. Pat. No. 5,654,270, U.S. Pat. No. 5,705,609, U.S. Pat. No. 5,726,149; U.S. Pat. No. 5,824,655; WO 91 / 04748; U.S. Pat. No. 5,830,847, U.S. Pat. No. 6,015,693; WO 91 / 10727; WO 93 / 09800; and WO 94 / 10187), somatostatin (WO 98 / 08529), mannoglobin (WO 98 / 08529), and riboflavin (WO 98 / 08529). Mannose-6-phosphate or mannose-1-phosphate (U.S. Pat. No. 5,520,926), prolactin (WO 97 / 40848), insulin-like growth factor II (WO 98 / 17304), IP-10 (WO 97 / 00691), Arg-Gly-Asp-containing peptides (Pfeffer, U.S. Pat. No. 5,958,411; WO 93 / 10808), plant, fungal, and bacterial extracts (EP-A-813875; JP 8-119984; and Matsunaga et al., U.S. Patent No. 5,693,610), antisense oligonucleotides (U.S. Patent No. 5,683,988; U.S. Patent No. 5,772,995; U.S. Patent No. 5,821,234; U.S. Patent No. 5,869,462; and WO 94 / 25588), proteins involved in TGF-β signaling, including Smads and MADs (EP-A-874046; WO 97 / 31020; WO 97 / 38729; WO 98 / 03663; WO 99 8 / 07735; WO 98 / 07849; WO 98 / 45467; WO 98 / 53068; WO 98 / 55512; WO 98 / 56913; WO 98 / 53830; WO 99 / 50296; U.S. Patent No. 5,834,248; U.S. Patent No. 5,807,708; and U.S. Patent No. 5,948,639), Ski and Sno (Vogel, 1999, Science 286:665; and Stroschein et al.(1999, Science, 286:771-774), one or more single-stranded oligonucleotide aptamers or expression plasmids encoding same suitable for inhibiting or interfering with the binding of TGF-β to its cognate receptor, as well as any mutant, fragment, or derivative of the above-identified molecules that retain the ability to inhibit the activity of TGF-β. TGF-β inhibitors may be TGF-β antagonists and may be human or humanized monoclonal antibodies (or fragments thereof, such as F(ab)2 fragments, Fv fragments, single-chain antibodies, and other forms or fragments of antibodies that retain the ability to bind TGF-β) that block TGF-β binding to its receptor. TGF-β receptors and TGF-β-binding fragments of TGF-β receptors, particularly soluble fragments, are useful TGF-β antagonists in the methods of the present invention. In one embodiment, a preferred inhibitor of TGF-β function is a soluble TGF-β receptor, particularly a TGF-β type II receptor (TGFBIIR) or a TGF-β type III receptor (TGFBIIIR or betaglycan), for example, comprising the extracellular domain of TGFBIIR or TGFBIIIR, preferably a recombinant soluble TGF-β receptor (rsTGFBIIR or rsTGFBIIIR). TGF-β receptors and TGF-β-binding fragments of TGF-β receptors, particularly soluble fragments, are useful TGF-β antagonists in the methods of the present invention. TGF-β receptors and the nucleic acids encoding them are well known in the art. The nucleic acid sequence encoding the TGF-β type 1 receptor is disclosed in GenBank Accession No. L15436 and U.S. Patent No. 5,538,892 (Donahoe et al.). The nucleic acid sequence of the TGF-β type 2 receptor is publicly available under GenBank accession numbers AW236001, AI35790, AI279872, AI074706, and AA808255. The nucleic acid sequence of the TGF-β type 3 receptor is also publicly available under GenBank accession numbers NM003243, AI887852, AI817295, and AI681599.

[0026] Still other TGF-β signaling inhibitors or antagonists and methods for their production are well known in the art, with many more currently under development. The specific TGF-β signaling inhibitor or antagonist used is not a limiting feature, as any effective TGF-β antagonist may be useful in the methods of the invention. Examples of such antagonists include monoclonal and polyclonal antibodies to one or more isotypes of TGF-β (U.S. Pat. No. 5,571,714 and WO 97 / 13844), TGF-β receptors, fragments thereof, derivatives thereof, and antibodies to TGF-β receptors (U.S. Pat. Nos. 5,693,607, 6,008,011, 6,001,969, and 6,010,872, and WO 92 / 00330, WO 93 / 09228, WO 95 / 10610, and WO 98 / 48024); latency associated peptide (WO 91 / 08291), large latent These include TGF-β (WO 94 / 09812), fetuin (U.S. Pat. No. 5,821,227), decorin, and other proteoglycans such as biglycan, fibromodulin, lumican, and endoglin (U.S. Pat. Nos. 5,583,103, 5,654,270, 5,705,609, 5,726,149, 5,824,655, 5,830,847, 6,015,693, and WO 91 / 04748, WO 91 / 10727, WO 93 / 09800, and WO 94 / 10187).

[0027] Further examples of such antagonists include somatostatin (WO 98 / 08529), mannose-6-phosphate or mannose-1-phosphate (U.S. Pat. No. 5,520,926), prolactin (WO 97 / 40848), insulin-like growth factor II (WO 98 / 17304), IP-10 (WO 97 / 00691), arginine (arg)-glycine (gly)-aspartic acid (asp)-containing peptides (U.S. Pat. No. 5,958,411 and WO 93 / 10808), plant, fungal and bacterial extracts (European Patent Application No. 813875, JP 8-119984 and U.S. Pat. No. 5,693,610), antisense oligonucleotides (U.S. Pat. Nos. 5,683,988, 5,772, 995, 5,821,234 and 5,869,462, and WO 94 / 25588), and Smads and MADs (European Patent Application EP 874046, WO 97 / 31020, WO 97 / 38729, WO 98 / 03663, WO 98 / 07735, WO 98 / 07849, WO 98 / 45467, WO 98 / 53068, WO 98 / 55512, WO 98 / 56913, WO 98 / 53830 and WO 99 / 50296, and U.S. Patent Nos. 5,834,248, 5,807,708 and 5,948,639), as well as Ski and Sno (G. Vogel, Science, 286:665 (1999) and Stroschein et al., Science, 286:771-74 (1999)), and a host of other proteins involved in TGF-β signaling, including fragments and derivatives of any of the above molecules that retain the ability to inhibit the activity of TGF-β.

[0028] Suitable TGF-β antagonists for use in the present invention also include those that have the functional properties of the aforementioned TGF-β antagonists, so long as their ability to inhibit the amount or activity of TGF-β is retained. Also included are variants, mutations, derivatives, and analogs. As used herein, "variant," "derivative," and "analog" refer to molecules that have a similar shape or structure to the parent compound and retain the ability to act as a TGF-β antagonist. For example, any of the TGF-β antagonists disclosed herein may be crystallized, and useful analogs may be rationally designed based on the resulting coordinates for the shape of the active site(s). Alternatively, one skilled in the art may modify functional groups of known antagonists without undue experimentation, or screen such modified molecules for increased activity, half-life, bioavailability, or other desirable characteristics. If the TGF-β antagonist is a polypeptide, fragments and variants of the polypeptide may be produced to increase ease of delivery, activity, half-life, etc. (e.g., humanized antibodies or functional antibody fragments as discussed above). Given the level of skill in the art of synthetic and recombinant polypeptide production, such variants may be achieved without undue experimentation. Those skilled in the art may also design novel inhibitors based on knowledge of the crystal structures and / or active sites of the TGF-β inhibitors described herein. Polypeptide inhibitors, such as soluble TGF-β receptors, can also be effectively introduced via gene transfer. Accordingly, certain embodiments of the methods of the present invention involve the use of a suitable vector for the expression of a TGF-β receptor or binding partner, preferably a soluble receptor or soluble binding partner. In a preferred embodiment, the administration of a soluble TGF-β antagonist can be achieved by gene transfer using a vector containing a cDNA encoding the soluble antagonist or a cDNA encoding the extracellular domain of the TGF-β type II receptor (rsTGFBIIR) or TGF-β type III receptor (rsTGFBIIIR), which causes in situ expression of the soluble TGF-β antagonist in cells transfected with the vector, inhibiting the activity of TGF-β and suppressing TGF-β-mediated fibrogenesis. Any suitable vector can be used.Preferred vectors include adenoviral vectors, lentiviral vectors, Epstein-Barr virus (EBV) vectors, adeno-associated virus (AAV) vectors, and retroviral vectors developed for gene transfer purposes. Other non-vector methods of gene transfer, such as lipid / DNA complexes, protein / DNA conjugates, and naked DNA transfer methods, can also be used. Additional suitable TGF-β antagonists developed for delivery via adenoviral gene transfer include, but are not limited to, a chimeric cDNA encoding the extracellular domain of the TGF-β type II receptor fused to an Ig Fc domain (Isaka et al., 1999, Kidney Int., 55:465-475), an adenoviral gene transfer vector for a dominant-negative mutant of the TGF-β type II receptor (Zhao et al., 1998, Mech. Dev., 72:89-100), and an adenoviral gene transfer vector for the TGF-β-binding proteoglycan decorin (Zhao et al., 1999, Am. J. Physiol., 277:L412-L422). Adenoviral-mediated gene transfer is highly efficient compared to other gene delivery modes.

[0029] TGF-β receptors and TGF-β-binding fragments, soluble fragments, etc. of TGF-β receptors are useful TGF-β antagonists in the present invention. TGF-β receptors and the nucleic acids encoding them are well known in the art. The nucleic acid sequence encoding the TGF-β type 1 receptor is disclosed in GenBank accession number L15436 and U.S. Patent No. 5,538,892 to Donahoe et al. The nucleic acid sequence of the TGF-β type 2 receptor is publicly available under GenBank accession numbers AW236001; AI35790; AI279872; AI074706; and AA808255. The nucleic acid sequence of the TGF-β type 3 receptor is also publicly available under GenBank accession numbers NM003243; AI887852; AI817295; and AI681599. In one exemplary embodiment, the TGF-β antagonist is an antibody that blocks TGF-β binding to its receptor or fragments thereof, such as F(ab)2 fragments, Fv fragments, single-chain antibodies, and other "antibody" forms that retain the ability to bind TGF-β. The antibody can be chimerized or humanized. As used herein, a chimerized antibody comprises the constant region of a human antibody and the variable region of a non-human antibody, such as a murine antibody. A humanized antibody comprises the constant region and framework variable region (i.e., the variable region other than the hypervariable region) of a human antibody and the hypervariable region of a non-human antibody, such as a murine antibody. Of course, the antibody can be any other type of antibody derivative, such as a human antibody selected or screened from a phage display system or generated from a xenomouse.

[0030] Knowledge about Smads is also increasing. The TGF-β signaling pathway is initiated when this molecule binds to and induces a heterodimeric cell surface complex consisting of type I (TbRI) and type II (TbRII) serine / threonine kinase receptors. This heterodimeric receptor then propagates the signal through phosphorylation of downstream target Smad proteins. As mentioned above, there are three functional classes of Smad proteins: receptor-regulated Smads (R-Smads), such as Smad2 and Smad3; coactivators (Co-Smads), also known as Smad4; and inhibitory Smads (I-Smads). Following phosphorylation by the heterodimeric receptor complex, the R-Smad forms a complex with the Co-Smad and translocates to the nucleus, where, in conjunction with other proteins, they regulate the transcription of target genes (Derynck, R., et al. (1998) Cell 95: 737-740; Massague, J. and Wotton, D. (2000) EMBO J. 19:1745). The nucleotide and amino acid sequences of human Smad3 are disclosed, for example, in GenBank Accession No. gi:42476202. The nucleotide and amino acid sequences of murine Smad3 are disclosed, for example, in GenBank Accession No. gi:31543221. As mentioned above, TGF-β stimulation leads to the phosphorylation and activation of Smad2 and Smad3, which form a complex with Smad4 (also called "common Smad" or "co-Smad"), which accumulates in the nucleus and regulates the transcription of target genes. Therefore, TGF-β signaling inhibition can also be achieved by inhibiting Smad2, 3, or co-Smad (Smad4). R-Smads are localized in the cytoplasm, and upon ligand-induced phosphorylation by the TGF-β receptor, they form complexes with co-Smads and translocate to the nucleus, where they regulate gene expression associated with chromatin and cooperating transcription factors. Therefore, TGF-β signaling inhibition can also be achieved by directly or indirectly inhibiting R-Smads.Smad6 and Smad7 are inhibitory Smads (I-Smads), i.e., they are transcriptionally induced by TGF-β and function as inhibitors of TGF-β signaling (Feng et al. (2005) Annu. Rev. Cell. Dev. Biol. 21: 659). Smad6 / 7 exert their inhibitory effects by preventing receptor-mediated activation of R-Smads. They associate with type I receptors, competitively preventing the recruitment and phosphorylation of R-Smads. Smad6 and Smad7 are known to recruit E3 ubiquitin ligases, which result in the ubiquitination and degradation of Smad6 / 7-interacting proteins. Therefore, Smad6 and Smad7 can function as TGF-β signaling inhibitors in the present invention.

[0031] Smad3 inhibitors that can be used in the present invention include, but are not limited to, antisense nucleotides, siRNAs, antibodies, and the like, as well as small molecule compounds such as 6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone, available from Calbiochem.

[0032] As used herein, the term "substances (e.g., nucleic acids) that suppress expression (such as TGF-β)" is not particularly limited as long as it is a substance that suppresses the transcription of mRNA of a target gene, a substance (e.g., nucleic acids) that degrades transcribed mRNA, or a substance (e.g., nucleic acids) that suppresses protein translation from mRNA. Examples of such substances include siRNA, antisense oligonucleotides, ribozymes, and nucleic acids such as expression vectors thereof. Among these, siRNA and its expression vectors are preferred, with siRNA being particularly preferred. In addition to the above, "substances that suppress gene expression" also include proteins, peptides, and other small molecules. In the present invention, a target gene is any gene involved in the TGF-β signaling pathway.

[0033] Methods using antisense technology to inhibit the expression of specific endogenous genes, such as TGF-β, targeted in the present invention, are well known to those skilled in the art. Antisense nucleic acids inhibit the expression of target genes through several factors, including: inhibition of transcription initiation by triplex formation; inhibition of transcription by hybridization with sites where RNA polymerase has created local open loop structures; inhibition of transcription by hybridization with RNA in the process of synthesis; inhibition of splicing by hybridization at intron-exon junctions; inhibition of splicing by hybridization with spliceosome formation sites; inhibition of nuclear-cytoplasmic transport by hybridization with mRNA; inhibition of splicing by hybridization with capping sites or poly(A) addition sites; inhibition of translation initiation by hybridization with translation initiation factor binding sites; inhibition of translation by hybridization with ribosome binding sites near the initiation codon; inhibition of peptide chain elongation by hybridization with the translated region or polysome binding sites of mRNA; and inhibition of gene expression by hybridization with nucleic acid-protein interaction sites. In this way, antisense nucleic acids inhibit the expression of target genes by interfering with various processes such as transcription, splicing, or translation (Hirashima and Inoue, New Biochemistry Experimental Lectures 2: Nucleic Acid IV Gene Replication and Expression, edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1993, 319-347).

[0034] The antisense nucleic acid used in the present invention may inhibit the expression and / or function of a gene (nucleic acid) encoding a member of the TGF-β signaling pathway, etc., by any of the above-mentioned mechanisms. In one embodiment, designing an antisense sequence complementary to the untranslated region near the 5' end of the mRNA of a gene encoding the above-mentioned TGF-β, etc., is considered to be effective in inhibiting gene translation. Sequences complementary to the coding region or the 3' untranslated region can also be used. Thus, nucleic acids containing antisense sequences not only to the translated region of the gene encoding the above-mentioned TGF-β, etc., but also to sequences in the untranslated region, are included in the antisense nucleic acids used in the present invention. The antisense nucleic acid used is linked downstream of an appropriate promoter, preferably linked to a sequence containing a transcription termination signal on the 3' side. The nucleic acid prepared in this manner can be transformed into the desired animal (cell) using known methods. The sequence of the antisense nucleic acid is preferably complementary to a gene encoding the TGF-β, etc., or a portion thereof, contained in the animal (cell) to be transformed. However, it does not have to be completely complementary as long as it effectively suppresses gene expression. The transcribed RNA preferably has 90% or more, and most preferably 95% or more, complementarity to the transcript of the target gene. To effectively inhibit the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is preferably at least 12 bases but less than 25 bases. However, the antisense nucleic acid of the present invention is not necessarily limited to this length and may be, for example, 11 bases or less, 100 bases or more, or 500 bases or more. The antisense nucleic acid may be composed solely of DNA, or may contain nucleic acids other than DNA, such as locked nucleic acids (LNAs). In one embodiment, the antisense nucleic acid used in the present invention may be an LNA-containing antisense nucleic acid containing an LNA at the 5' end and an LNA at the 3' end. In addition, in embodiments using antisense nucleic acids in the present invention, antisense sequences can be designed based on the nucleic acid sequence of TGF-β or the like, using, for example, the method described in Hirashima and Inoue, "New Biochemistry Experiments 2: Nucleic Acid IV Gene Replication and Expression," edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1993, pp. 319-347.

[0035] Inhibition of TGF-β expression can also be achieved using ribozymes or DNA encoding ribozymes. Ribozymes are RNA molecules with catalytic activity. Ribozymes have a variety of activities, but research focusing on ribozymes as RNA-cleaving enzymes has made it possible to design ribozymes that cleave RNA site-specifically. Ribozymes can be over 400 nucleotides in size, such as group I intron-type ribozymes and the M1 RNA contained in RNase P. However, there are also ribozymes with active domains of approximately 40 nucleotides, known as hammerhead or hairpin-type ribozymes (Koizumi Makoto and Otsuka Eiko, Proteins, Nucleic Acids, and Enzymes, 1990, 35, 2191).

[0036] For example, the self-cleaving domain of the hammerhead ribozyme cleaves the 3' side of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is considered to be important for its activity, and it has been shown that cleavage can also be achieved with A15 or U15 instead of C15 (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to an RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequences UC, UU, or UA in the target RNA (Koizumi, M. et al., FEBS Lett, 1988, 239, 285.; Koizumi, M. and Otsuka, Eiko, Proteins, Nucleic Acids, and Enzymes, 1990, 35, 2191.; Koizumi, M. et al., Nucl. Acids Res., 1989, 17, 7059.).

[0037] Hairpin ribozymes are also useful for the purposes of the present invention. These ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ringspot virus (Buzayan, JM., Nature, 1986, 323, 349). It has also been shown that target-specific RNA-cleaving ribozymes can be produced from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl. Acids Res., 1991, 19, 6751; Kikuchi, H., Chemistry and Biology, 1992, 30, 112). Thus, by using ribozymes to specifically cleave the transcripts of genes encoding TGF-β and the like, expression of the genes can be inhibited.

[0038] Suppression of the expression of endogenous genes such as TGF-β can also be achieved by RNA interference (RNAi) using double-stranded RNA with a sequence identical or similar to the target gene sequence. RNAi is a technique that is currently attracting attention because, when double-stranded RNA (dsRNA) is directly introduced into cells, the expression of genes with sequences homologous to the dsRNA is suppressed. In mammalian cells, RNAi can be induced using short dsRNA (siRNA). RNAi has many advantages over knockout mice, including stable effects, ease of experimentation, and low cost. siRNA is described in detail elsewhere in this specification.

[0039] As used herein, "siRNA" refers to an RNA molecule having a double-stranded RNA portion consisting of 15 to 40 bases. It cleaves mRNA of a target gene that has a sequence complementary to the antisense strand of the siRNA, thereby suppressing the expression of the target gene. Specifically, the siRNA of the present invention is an RNA containing a double-stranded RNA portion consisting of a sense RNA strand with a sequence homologous to a continuous RNA sequence in mRNA of a TGF-β or the like, and an antisense RNA strand with a sequence complementary to the sense RNA sequence. The design and production of such siRNAs and the mutant siRNAs described below are within the skill of those skilled in the art. Selecting any continuous RNA region in mRNA, which is a transcription product of a sequence such as TGF-β, and preparing a double-stranded RNA corresponding to this region is within the scope of ordinary trial and error for those skilled in the art. Furthermore, selecting an siRNA sequence with a stronger RNAi effect from the mRNA sequence, which is a transcription product of the sequence, can also be performed by those skilled in the art using known methods. Furthermore, if one strand is known, one skilled in the art can easily determine the base sequence of the other strand (complementary strand). Those skilled in the art can prepare siRNAs using commercially available nucleic acid synthesizers. Alternatively, general contract synthesis services can be used to synthesize desired RNAs.

[0040] The length of the double-stranded RNA portion is 15 to 40 bases, preferably 15 to 30 bases, more preferably 15 to 25 bases, even more preferably 18 to 23 bases, and most preferably 19 to 21 bases. It is understood that these upper and lower limits are not limited to these specific values ​​and may be any combination of the listed values. The terminal structure of the sense or antisense strand of the siRNA is not particularly limited and can be selected appropriately depending on the purpose. For example, it may have a blunt end or a protruding end (overhang), with a protruding 3' end being preferred. siRNAs with overhangs consisting of several bases, preferably 1 to 3 bases, and more preferably 2 bases, at the 3' end of the sense or antisense RNA strand are often preferred because they have a significant effect in suppressing target gene expression. The type of base in the overhang is not particularly limited and may be either a base constituting RNA or a base constituting DNA. A preferred overhang sequence is dTdT (2 bp of deoxy T) at the 3' end. For example, preferred siRNAs include, but are not limited to, those in which dTdT (2 bp of deoxy T) is attached to the 3'-end of both the sense and antisense strands of all siRNAs.

[0041] Furthermore, siRNAs in which one to several nucleotides have been deleted, substituted, inserted, and / or added in one or both of the sense and antisense strands of the siRNA can also be used. Here, "one to several nucleotides" is not particularly limited, but is preferably one to four nucleotides, more preferably one to three nucleotides, and most preferably one to two nucleotides. Specific examples of such mutations include, but are not limited to, 0 to three nucleotides in the 3'-overhang portion, nucleotide sequences in the 3'-overhang portion altered to other nucleotide sequences, nucleotides in which the lengths of the sense and antisense RNA strands differ by one to three nucleotides due to nucleotide insertion, addition, or deletion, and nucleotide substitutions in the sense and / or antisense strands. However, it is necessary that the sense and antisense strands in these mutant siRNAs can hybridize, and that these mutant siRNAs have the same gene expression suppression ability as siRNAs without the mutation.

[0042] Furthermore, siRNA may be a molecule with a closed structure at one end, such as an siRNA (Short Hairpin RNA; shRNA) with a hairpin structure. shRNA is an RNA that contains a sense strand RNA of a specific sequence of a target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand sequence, and a linker sequence connecting the two strands, and the sense strand portion and the antisense strand portion hybridize to form a double-stranded RNA portion.

[0043] When used clinically, it is desirable that siRNA does not exhibit so-called off-target effects. Off-target effects refer to the suppression of expression of genes other than the target gene that share partial homology with the siRNA used. To avoid off-target effects, it is possible to confirm in advance that there is no cross-reactivity of candidate siRNAs using DNA microarrays or other methods. Furthermore, it is possible to avoid off-target effects by using publicly known databases provided by organizations such as NCBI to confirm whether there are any genes other than the target gene that contain a portion highly homologous to the sequence of the candidate siRNA.

[0044] To prepare the siRNA of the present invention, known methods such as chemical synthesis and recombinant DNA technology can be used as appropriate. In synthetic methods, double-stranded RNA can be synthesized by standard methods based on sequence information. In methods using recombinant DNA technology, expression vectors encoding sense and antisense strand sequences are constructed, and the vectors are introduced into host cells, followed by transcription to obtain the sense and antisense strands, respectively. Alternatively, desired double-stranded RNA can be prepared by expressing an shRNA that contains a sense strand of a specific sequence of a target gene, an antisense strand consisting of a sequence complementary to the sense strand, and a linker sequence connecting the two strands, forming a hairpin structure.

[0045] As long as siRNA has the activity of suppressing the expression of a target gene, the nucleic acid constituting the siRNA may be a natural nucleic acid or a modified nucleic acid in whole or in part.

[0046] The siRNA of the present invention does not necessarily have to be a single set of double-stranded RNA corresponding to the target sequence, but may also be a mixture of multiple sets of double-stranded RNA corresponding to a region containing the target sequence ("multiple" is not particularly limited, but preferably refers to a small number, such as 2 to 5). Those skilled in the art can appropriately prepare siRNA as a nucleic acid mixture corresponding to the target sequence using a commercially available nucleic acid synthesizer and DICER enzyme. Alternatively, general contract synthesis services can be used to synthesize desired RNA. The siRNA of the present invention also includes so-called "cocktail siRNA." Furthermore, not all nucleotides in the siRNA of the present invention need to be ribonucleotides (RNA). That is, in the present invention, one or more ribonucleotides constituting the siRNA may be corresponding deoxyribonucleotides. The term "corresponding" here refers to the same base species (adenine, guanine, cytosine, thymine (uracil)) despite the different sugar moiety structures. For example, a deoxyribonucleotide corresponding to a ribonucleotide containing adenine refers to a deoxyribonucleotide containing adenine.

[0047] Furthermore, DNA (vectors) capable of expressing the RNA of the present invention are also included in preferred embodiments of nucleic acids capable of suppressing the expression of TGF-β and the like. For example, DNA (vectors) capable of expressing the double-stranded RNA of the present invention are DNAs having a structure in which DNA encoding one strand of the double-stranded RNA and DNA encoding the other strand of the double-stranded RNA are linked to promoters so that they can be expressed. The DNA of the present invention can be appropriately prepared by those skilled in the art using common genetic engineering techniques. More specifically, the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.

[0048] In the present invention, modified nucleic acids may be used as nucleic acids that suppress the expression of target genes. Modified nucleic acids refer to nucleic acids in which modifications have been made to the nucleosides (base moieties, sugar moieties) and / or internucleoside linkages, resulting in a structure different from that of natural nucleic acids. Examples of the "modified nucleosides" that constitute modified nucleic acids include abasic nucleosides; arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and other nucleosides with sugar modifications; peptide nucleic acids (PNAs), phosphate-linked peptide nucleic acids (PHONAs), locked nucleic acids (LNAs), and morpholino nucleic acids. Nucleosides with the above sugar modifications include nucleosides with substituted pentasaccharides such as 2'-O-methylribose, 2'-deoxy-2'-fluororibose, and 3'-O-methylribose; 1',2'-deoxyribose; arabinose; substituted arabinose sugars; hexasaccharides, and alpha-anomeric sugar modifications. These nucleosides may also contain modified bases in which the base moiety is modified. Such modified bases include, for example, pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases.

[0049] Examples of the "modified internucleoside bond" that constitutes a modified nucleic acid include an alkyl linker, a glyceryl linker, an amino linker, a poly(ethylene glycol) bond, a methylphosphonate internucleoside bond; and unnatural internucleoside bonds such as methylphosphonothioate, phosphotriester, phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, and phosphoramidate.

[0050] Examples of nucleic acid sequences contained in the double-stranded siRNA of the present invention include siRNAs against TGF-β or other members of the TGF-β signaling pathway.

[0051] Alternatively, the nucleic acid or drug of the present invention can be introduced into phospholipid vesicles such as liposomes and then administered. Vesicles containing siRNA or shRNA can be introduced into specific cells using lipofection. The resulting cells can then be administered systemically, for example, intravenously or intraarterially. They can also be administered locally to the desired site, such as the eye. While siRNA exhibits highly specific post-transcriptional silencing effects in vitro, they are rapidly degraded in vivo by serum nuclease activity, limiting their duration of effect. Therefore, the development of more optimal and effective delivery systems has been sought. For example, OCHIYA, T et al., Nature Med., 5:707-710, 1999; Curr. Gene Ther., 1:31-52, 2001, reported that when mixed with nucleic acid to form a complex, atelocollagen, a biocompatible material, protects the nucleic acid from degradative enzymes in the body, making it an extremely suitable carrier for siRNA. While such forms can be used, the method of delivery of the nucleic acid or drug of the present invention is not limited to these. In this way, the nucleic acid is rapidly degraded in vivo by the action of nucleases in serum, allowing for long-lasting effects. For example, Takeshita F. PNAS. (2003) 102(34) 12177-82 and Minakuchi Y Nucleic Acids Research (2004) 32(13) e109 report that atelocollagen derived from bovine skin forms a complex with nucleic acids, protecting them from degrading enzymes in the body, making it highly suitable as a carrier for siRNA. This technology can be used.

[0052] As used herein, the terms "drug," "agent," and "factor" (all of which correspond to the English term "agent") are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands), etc.), and composite molecules thereof. Typical examples of factors specific to a polynucleotide include, but are not limited to, polynucleotides that are complementary to the sequence of the polynucleotide with a certain degree of sequence homology (e.g., 70% or more sequence identity), and polypeptides such as transcription factors that bind to promoter regions. Typical examples of factors specific to a polypeptide include, but are not limited to, antibodies specifically directed against the polypeptide or derivatives or analogs thereof (e.g., single-chain antibodies), specific ligands or receptors when the polypeptide is a receptor or ligand, and substrates when the polypeptide is an enzyme.

[0053] As used herein, the term "disease, disorder, or condition associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium" refers to a disease, disorder, or condition of the corneal endothelium that is associated with abnormalities in the extracellular matrix (ECM). Examples of such diseases, disorders, or conditions include disorders related to Fuchs' endothelial corneal dystrophy, pterygium, allergic diseases, keratitis, corneal ulcers, etc.

[0054] As used herein, the term "disorder related to Fuchs' corneal endothelial dystrophy" refers to any disorder related to Fuchs' corneal endothelial dystrophy, of which those related to extracellular matrix (ECM) abnormalities are of particular interest to the present invention, but are not limited to these. Disorders related to Fuchs' corneal endothelial dystrophy that are related to such extracellular matrix (ECM) abnormalities include, but are not limited to, Examples of side effects include, but are not limited to, photophobia, blurred vision, impaired vision, eye pain, tearing, congestion, pain, bullous keratopathy, eye discomfort, decreased contrast, glare, edema of the corneal stroma, bullous keratopathy, and corneal opacity.

[0055] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM(1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MAet al.(1995).PCR Strategies, Academic Press; Ausubel, FM(1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJet al.(1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Gait, MJ(1985).Oligonucleotide Synthesis: A Practical Approach, IRLPress; Gait, MJ(1990).Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991).Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al.(1992).The Biochemistry of the Nucleic Acids, Chapman&Hall; Shabarova, Z. et al.(1994).Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al.(1996).Nucleic Acids in Chemistry and These are described in, for example, "Biology," Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press; and "Experimental Methods for Gene Transfer and Expression Analysis," a special edition of Experimental Medicine, Yodosha, 1997. Regarding corneal endothelial cells, reports by Nancy Joyce et al. {Joyce, 2004 #161} {Joyce, 2003 #7} are well known, but as mentioned above, long-term culture and subculture result in fibroblast-like transformation, and research into efficient culture methods is currently underway. These are incorporated herein by reference in their entirety (perhaps in their relevant parts).

[0056] DESCRIPTION OF THE PREFERRED EMBODIMENTS Although the following description of preferred embodiments is given, it should be understood that these embodiments are merely examples of the present invention and that the scope of the present invention is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present invention by referring to the following preferred examples.

[0057] (Treatment or prevention of diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, including TGFβ signal inhibitors) In one aspect, the present invention provides a therapeutic or preventive agent for diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, comprising a TGFβ signaling inhibitor. The present invention discovered that ECM abnormalities in diseases, disorders, or conditions associated with ECM in the corneal endothelium could unexpectedly be reduced or eliminated by administering a TGFβ signaling inhibitor. Therefore, the use of such TGFβ signaling inhibitors for the treatment or prevention of diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium could be said to be an application that could not have been predicted from conventional knowledge.

[0058] In a preferred embodiment, the disease, disorder, or condition targeted by the present invention is a disorder related to Fuchs' endothelial corneal dystrophy. Currently, there are no fundamental treatment methods or techniques for Fuchs' endothelial corneal dystrophy, and treatment of Fuchs' endothelial corneal dystrophy has relied on corneal transplantation. Because the present invention can treat extracellular matrix (ECM) abnormalities, which are the cause of one of the key abnormalities or disorders in Fuchs' endothelial corneal dystrophy, it is understood that the present invention is useful for treating or preventing Fuchs' endothelial corneal dystrophy.

[0059] In one specific embodiment, the disease, disorder, or condition targeted by the present invention comprises photophobia, blurred vision, visual impairment, eye pain, tearing, redness, pain, bullous keratopathy, ocular discomfort, decreased contrast, glare, corneal stromal edema, and corneal opacity in Fuchs' endothelial corneal dystrophy.

[0060] The subjects for administration (transplantation) of the medicament or method of the present invention include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.), but primates are preferred, with humans being particularly preferred. Corneal endothelial therapy in primates has not yet achieved satisfactory results, and in this sense, the present invention provides a groundbreaking treatment and medicament.

[0061] The TGF-β signaling pathway is broadly classified into the Smad2 / 3 system, which is mediated by ALK4, 5, or 7, and the Smad1 / 5 / 8 system, which is mediated by ALK1, 2, 3, or 6. Both systems are well known to be involved in fibrosis (J. Massagu'e, Annu. Rev. Biochem. 1998.67:753-91; Vilar JMG, Jansen R, Sander C (2006) PLoS Comput Biol 2(1):e3; Leask, A., Abraham, DJ FASEB J.18, 816-827 (2004); Coert Margadant & Arnoud Sonnenberg EMBO reports (2010) 11, 97-105; Joel Rosenbloom et al., Ann Intern Med. 2010;152:159-166.) It is also known that BMP-7 can suppress TGF-β signaling and inhibit fibrosis (in addition to the above references, Ralf Weiskirchen, et al., Frontiers in Bioscience 14, 4992-5012, June 1, 2009; Elisabeth M Zeisberg et al., Nature Medicine 13, 952-961 (2007); Michael Zeisberg et al., Nature Medicine 9, 964-968 (2003)). However, these studies only describe the involvement of TGF-β in the very specific disease of syphilitic stromal keratitis or in artificially created severe lesions that actually involve a membranous structure composed of extracellular matrix such as collagen, but it is difficult to predict therapeutic efficacy from these findings. It has also been shown that fibrosis in severe corneal lesions is mediated by IL-1β and is mediated by activation of p38 MAPK. On the other hand, it has been shown in rabbits that severe inflammation caused by excessive cryotrauma is accompanied by activation of p38 MAPK, and that this fibrosis can be partially suppressed with an inhibitor. These findings demonstrate that p38 MAPK activation occurs in situations where extremely severe inflammation occurs in the body and involves a membranous tissue composed of extracellular matrix. However, they do not state that TGF-β signaling inhibitors are effective in treating or preventing diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, such as Fuchs' endothelial dystrophy, nor do they provide any suggestion regarding maintaining a normal state. Thus, it has previously been thought that it is difficult to culture corneal endothelial cells while maintaining normal function, and previous reports have ultimately failed to treat or prevent diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, such as Fuchs' endothelial dystrophy. Furthermore, it was not thought that inhibiting the TGF-β signaling pathway could treat or prevent diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, such as Fuchs' endothelial dystrophy.

[0062] The TGF-β signal inhibitor used in the present invention may be any agent as long as it can inhibit the TGF-β signaling pathway. As is well known, the TGF-β signaling pathway to be inhibited may be one directly related to TGF-β and TGF-β receptors, or any signal-related factor, such as BMP-7, as long as it ultimately exerts an effect similar to that of the TGF-β signaling pathway (or the opposite effect in the case of inhibitors, antagonists, etc.).

[0063] In the present invention, a single TGF-β signal inhibitor may be included, or several types may be included in combination as necessary.

[0064] In one embodiment, the TGF-β signaling inhibitor comprises at least one TGF-β antagonist, TGF-β receptor antagonist, or Smad3 inhibitor, any of the components exemplified elsewhere herein, their pharmaceutically acceptable salts or solvates, or solvates of their pharmaceutically acceptable salts. The TGF-β antagonist, TGF-β receptor antagonist, and Smad3 inhibitor may be any of those described elsewhere herein.

[0065] In one embodiment, the TGF-β signal inhibitor that can be used in the present invention is SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), BMP-7, an anti-TGF-β antibody, an anti-TGF-β receptor antibody, an siRNA for TGF-β, an siRNA for TGF-β receptor, an antisense oligonucleotide for TGF-β, 6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3, 4-Tetrahydroisoquinolone, A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), Stemolecule TMTLK inhibitor (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), Stemolecule TM The antibody may include, but is not limited to, at least one of the BMP inhibitors LDN-193189 (6-(4-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine), SD-208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine), LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), other components exemplified herein, pharmaceutically acceptable salts or solvates thereof, or solvates of pharmaceutically acceptable salts thereof. The antibody may be, but is not limited to, a neutralizing antibody. Without wishing to be bound by theory, both SB431542, which exerts its effect via Smad2 / 3 (associated with ALK4, 5 and 7), and BMP-7, which exerts its effect via Smad1 / 5 / 8 (associated with ALK1, 2, 3 and 6), have been observed to be effective in treating or preventing diseases, disorders or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, such as Fuchs' endothelial corneal dystrophy. Therefore, it is understood that the effects of the present invention can be achieved with a TGF-β signal inhibitor of either of these pathways.

[0066] In a preferred embodiment, the TGF-β signaling inhibitor used in the present invention comprises SB431542 (4-[4-(1,3-benzodioxol-5-yl)2-pyridinyl)-1H-imidazol-2-yl]benzamide). This is because it has been shown to improve diseases, disorders, or conditions associated with abnormalities in the extracellular matrix (ECM) of the corneal endothelium, such as Fuchs' endothelial corneal dystrophy. In a preferred embodiment, SB431542 is present at a concentration of about 0.1 μM to about 10 μM upon use, preferably about 1 μM to about 10 μM upon use, and even more preferably about 1 μM upon use.

[0067] The concentration of the TGFβ signal inhibitor used in the present invention is usually about 0.1 to 100 μmol / l, preferably about 0.1 to 30 μmol / l, and more preferably about 1 μmol / l. When several types are used, this can be changed appropriately. Other concentration ranges include, for example, usually about 0.001 to 100 μmol / l, preferably about 0.01 to 75 μmol / l, about 0.05 to 50 μmol / l, about 1 to 10 μmol / l, about 0.01 to 10 μmol / l, about 0.05 to 10 μmol / l, about 0.075 to 10 μmol / l, about 0.1 to 10 μmol / l, and about 0 .5~10μmol / l, approx. 0.75~10μmol / l, approx. 1.0~10μmol / l, approx. 1.25~10μmol / l, approx. 1.5~10μmol / l, approx. 1.75~10μmol / l, approx. 2.0~10μmol / l, approx. 2.5~10μmol / l, approx. 3.0~10μmol / l, about 4.0-10μmol / l, about 5.0-10μmol / l, about 6.0-10μmol / l, about 7.0-10μmol / l, about 8.0-10μmol / l, about 9.0-10μmol / l, about 0.01-50μmol / l, about 0.05-5.0μmol / l, about 0.075 ~5.0μmol / l, approximately 0.1~5.0μmol / l, approximately 0.5~5.0μmol / l, approximately 0.75~5.0μmol / l, approximately 1.0~5.0μmol / l, approximately 1.25~5.0μmol / l, approximately 1.5~5.0μmol / l, approximately 1.75~5.0μmol / l, approximately 2.0~ 5.0μmol / l, approximately 2.5~5.0μmol / l, approximately 3.0~5.0μmol / l, approximately 4.0~5.0μmol / l, approximately 0.01~3.0μmol / l, approximately 0.05~3.0μmol / l, approximately 0.075~3.0μmol / l, approximately 0.1~3.0μmol / l, approximately 0.5~ 3.0 μmol / l, about 0.75 to 3.0 μmol / l, about 1.0 to 3.0 μmol / l, about 1.25 to 3.0 μmol / l, about 1.5 to 3.0 μmol / l, about 1.75 to 3.0 μmol / l, about 2.0 to 3.0 μmol / l, about 0.01 to 1.0 μmol / l, about 0.05 to 1.0 μmol / l, about 0.075 to 1.0 μmol / l, about 0.1 to 1.0 μmol / l, about 0.5 to 1.0 μmol / l, about 0.75 to 1.0 μmol / l, about 0.09 to 35 μmol / l, about 0.09 to 3.2 μmol / l, and more preferably about 0.05 to 1.Examples of the concentration include, but are not limited to, about 0 μmol / L, about 0.075 to 1.0 μmol / L, about 0.1 to 1.0 μmol / L, about 0.5 to 1.0 μmol / L, and about 0.75 to 1.0 μmol / L.

[0068] In a preferred embodiment, the TGF-β signal inhibitor used comprises 4-[4-(1,3-benzodioxol-5-yl)2-pyridinyl)-1H-imidazol-2-yl]benzamide or a pharmaceutically acceptable salt thereof.

[0069] In another preferred embodiment, the TGF-β signal inhibitor used in the present invention includes BMP-7. This is because it has been shown that fibrosis is suppressed, proteins responsible for normal function are maintained, and the inhibitor can withstand transplantation into primates. In a preferred embodiment, BMP-7 is contained so as to be present at a concentration of about 10 ng / ml to about 1000 ng / ml when used, and more preferably at a concentration of about 100 ng / ml to about 1000 ng / ml when used. Even if BMP-7 is contained so as to be present at a concentration of about 100 ng / ml when used, it is not necessary to contain BMP-7 at a concentration of about 10 It may be present at a concentration of 0.000 ng / ml.

[0070] The therapeutic or preventive agent of the present invention may contain additional pharmaceutical ingredients. Representative examples of such pharmaceutical products include Rho kinase inhibitors and steroids. Without wishing to be bound by theory, the inclusion of a Rho kinase inhibitor enhances the adhesion of corneal endothelial cells, thereby preventing cell shedding and enabling the formation of a corneal endothelial cell layer with good cell morphology and high cell density, thereby enhancing the effect of the TGFβ signaling inhibitor. In the present invention, one type of Rho kinase inhibitor can be contained alone, or several types can be contained in combination as needed.

[0071] Examples of Rho kinase inhibitors that can be used in the present invention include those described in the following documents: U.S. Patent No. 4,678,783, Japanese Patent No. 3,421,217, WO 95 / 28387, WO 99 / 20620, WO 99 / 61403, WO 02 / 076976, WO 02 / 076977, WO 2002 / 083175, WO 02 / 100833, WO 03 / 059913, WO 03 / 062227, WO 2004 / 009555, WO 2004 / 022541, WO 2004 / 022542, WO 2004 / 022543, WO 2004 / 022544, WO 2004 / 022545, WO 2004 / 022546, WO 2004 / 022547, WO 2004 / 022548, WO 2004 / 022549 ... 2004 / 108724, WO 2005 / 003101, WO 2005 / 039564, WO 2005 / 034866, WO 2005 / 037197, WO 2005 / 037198, WO 2005 / 035501, WO 2005 / 035503, WO 2005 / 035506, WO 2005 / 080394, WO 2005 / 103050, WO 2006 / 057270, WO 2007 / 026664, and the like. Such compounds can be produced by the methods described in the respective documents, and examples thereof include 1-(5-isoquinolinesulfonyl)homopiperazine or a salt thereof (e.g., fasudil (1-(5-isoquinolinesulfonyl)homopiperazine)), (R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide or a salt thereof (e.g., Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate)), and the like.

[0072] The concentration of the Rho kinase inhibitor in the present invention is usually about 1 to 100 μmol / l, preferably about 5 to 20 μmol / l, and more preferably about 10 μmol / l. When several types are used, the concentration can be appropriately changed. Other concentration ranges include, for example, usually about 0.001 to 100 μmol / l, preferably about 0.01 to 75 μmol / l, about 0.05 to 50 μmol / l, about 1 to 10 μmol / l, about 0.01 to 10 μmol / l, about 0.05 to 10 μmol / l, about 0.075 to 10 μmol / l, about 0.1 to 10 μmol / l, and about 0.5 to 10 μmol / l. , about 0.75-10μmol / l, about 1.0-10μmol / l, about 1.25-10μmol / l, about 1.5-10μmol / l, about 1.75-10μmol / l, about 2.0-10μmol / l, about 2.5-10μmol / l, about 3.0-10μmol / l, about 4.0-10μm ol / l, approximately 5.0~10μmol / l, approximately 6.0~10μmol / l, approximately 7.0~10μmol / l, approximately 8.0~10μmol / l, approximately 9.0~10μmol / l, approximately 0.01~50μmol / l, approximately 0.05~5.0μmol / l, approximately 0.075~5.0μmol / l, approximately 0 .1~5.0μmol / l, approx. 0.5~5.0μmol / l, approx. 0.75~5.0μmol / l, approx. 1.0~5.0μmol / l, approx. 1.25~5.0μmol / l, approx. 1.5~5.0μmol / l, approx. 1.75~5.0μmol / l, approx. 2.0~5.0μmol / l, approx. 2.5 ~5.0μmol / l, approx. 3.0~5.0μmol / l, approx. 4.0~5.0μmol / l, approx. 0.01~3.0μmol / l, approx. 0.05~3.0μmol / l, approx. 0.075~3.0μmol / l, approx. 0.1~3.0μmol / l, approx. 0.5~3.0μmol / l, approx. 0.75 about 1.0 to 3.0 μmol / l, about 1.25 to 3.0 μmol / l, about 1.5 to 3.0 μmol / l, about 1.75 to 3.0 μmol / l, about 2.0 to 3.0 μmol / l, about 0.01 to 1.0 μmol / l, about 0.05 to 1.0 μmol / l, about 0.075 to 1.0 μmol / l, about 0.1 to 1.0 μmol / l, about 0.5 to 1.0 μmol / l, about 0.75 to 1.0 μmol / l, about 0.09 to 35 μmol / l, and about 0.09 to 3.2 μmol / l, and more preferably about 0.05 to 1.0 μmol / l and about 0.075 to 1.0 μmol / l.Examples of the concentration include, but are not limited to, about 0 μmol / L, about 0.1 to 1.0 μmol / L, about 0.5 to 1.0 μmol / L, and about 0.75 to 1.0 μmol / L.

[0073] The present invention can be administered as eye drops.

[0074] The dosage and frequency of administration vary depending on the symptoms, age, body weight, and administration form. Generally, for adults, when used as eye drops, a formulation containing about 0.0001 to 0.1 w / v%, preferably about 0.003 to 0.03 w / v%, of the active ingredient can be administered 1 to 10 times, preferably 1 to 6 times, more preferably 1 to 3 times per day, at a dose of about 0.01 to 0.1 mL per administration. When the pharmaceutical agent of the present invention is injected into the anterior chamber, a concentration of 1 / 10 to 1 / 1000 of the above-mentioned concentration can be used. Those skilled in the art can appropriately select the type and concentration of the TGFβ signal inhibitor, Rho kinase inhibitor, etc., depending on the condition of the disease.

[0075] In another aspect, the present invention provides a TGFβ signaling inhibitor for treating or preventing a disorder associated with an abnormality in the extracellular matrix (ECM) of the corneal endothelium. The term "TGFβ signaling inhibitor" may be used interchangeably with "TGFβ signaling inhibitor." In this application, any of the embodiments described herein for the abnormality in the extracellular matrix (ECM) of the corneal endothelium and the TGFβ signaling inhibitor may be used.

[0076] In another aspect, the present invention provides a method for treating or preventing a disorder associated with an abnormality in the extracellular matrix (ECM) of the corneal endothelium in a subject, the method comprising the step of administering to the subject an effective amount of a TGFβ signaling inhibitor. In this method, any of the embodiments described herein for the abnormality in the extracellular matrix (ECM) of the corneal endothelium and the TGFβ signaling inhibitor can be used.

[0077] The subjects for administration (transplantation) of the medicament or method of the present invention include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.), but primates are preferred, with humans being particularly preferred. Corneal endothelial therapy in primates has not yet achieved satisfactory results, and in this sense, the present invention provides a groundbreaking treatment and medicament.

[0078] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0079] The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Example]

[0080] Below, we describe an example of successful culturing of the corneal endothelial cells of the present invention. Where applicable, handling of biological samples and the like complied with the standards stipulated by the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, and the like, and, where applicable, was carried out in accordance with the Declaration of Helsinki or the ethical standards established based on the Declaration. Regarding the donation of eyes for research, written consent was obtained from the next of kin of all deceased donors. This study was conducted in collaboration with the University of Erlangen (Germany), SightLife TM Approved by the Eye Bank Ethics Committee (Seattle, WA) or equivalent.

[0081] In Fuchs' endothelial corneal dystrophy, corneal endothelial cells undergo cell death. When the remaining corneal endothelial cells are unable to compensate for the pumping and barrier functions, corneal transparency cannot be maintained, leading to corneal opacity and blindness. Furthermore, corneal endothelial cells in patients with Fuchs' endothelial corneal dystrophy are known to produce excessive extracellular matrix, resulting in the formation of guttae and thickening of Descemet's membrane. Guttae formation and thickening of Descemet's membrane cause light scattering, leading to decreased visual acuity, photophobia, and blurred vision, significantly impairing the quality of life of patients without Fuchs' endothelial corneal dystrophy. Using an immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs' endothelial corneal dystrophy as a model, we compared it with an immortalized corneal endothelial cell line (iHCEC) derived from a healthy donor to clarify the causes of extracellular matrix production and identify therapeutic targets.

[0082] (Preparation example: Creation of an immortalized corneal endothelial cell line (iFECD) model derived from a patient with Fuchs corneal endothelial dystrophy) In this example, an immortalized corneal endothelial cell line (iFECD) was prepared from corneal endothelial cells derived from a patient with Fuchs' corneal endothelial dystrophy.

[0083] (Culture method) Corneal endothelial cells were mechanically detached together with the basement membrane from research corneas purchased from the Seattle Eye Bank, and then recovered by peeling from the basement membrane using collagenase, followed by primary culture. The medium used was Opti-MEM I Reduced-Serum Medium, Liquid (Invitrogen, catalog number: 31985-070) supplemented with 8% FBS (Biowest, catalog number: S1820-500), 200 mg / ml CaCl₂·2H₂O (Sigma, catalog number: C7902-500G), 0.08% chondroitin sulfate (Sigma, catalog number: C9819-5G), 20 μg / ml ascorbic acid (Sigma, catalog number: A4544-25G), 50 μg / ml gentamicin (Invitrogen, catalog number: 15710-064), and 5 ng / ml EGF (Invitrogen, catalog number: PHG0311), which had been conditioned for 3T3 feeder cells. In addition, the cells were cultured in basal medium supplemented with SB431542 (1 μmol / L) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole < 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine) (1 μmol / L) (referred to as "SB203580 + SB431542 + 3T3 conditioned medium").

[0084] (How to obtain) Corneal endothelial cells were obtained from three human patients with a clinical diagnosis of Fuchs' endothelial corneal dystrophy who underwent corneal endothelial transplantation (Descemet's membrane endothelial keratoplasty, or DMEK) for bullous keratopathy, with written consent and approval from the ethics committee. During DMEK, the diseased corneal endothelial cells and the basement membrane, Descemet's membrane, were mechanically removed and immersed in Optisol-GS (Bausch & Lomb), a corneal preservative solution. Corneal endothelial cells were then enzymatically harvested using collagenase treatment and cultured in a mixture of SB203580, SB431542, and 3T3-conditioned medium. The SV40 large T antigen and hTERT gene were amplified by PCR and transfected into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc.). The lentiviral vectors were then transfected into 293T cells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) using a transfection reagent (Fugene HD; Promega Corp., Madison, WI) along with three helper plasmids (pLP1, pLP2, and pLP / VSVG; Life Technologies Inc.). After 48 h of infection, the virus-containing culture supernatant was collected and added to cultured corneal endothelial cells derived from a patient with Fuchs' corneal endothelial dystrophy using 5 μg / ml polybrene to transduce the SV40 large T antigen and hTERT gene. Phase-contrast microscopy images of an immortalized corneal endothelial cell line derived from a patient with Fuchs' corneal endothelial dystrophy (iFECD) were confirmed. As a control, corneal endothelial cells cultured from research corneas imported from the Seattle Eye Bank were immortalized in the same manner to generate an immortalized normal corneal endothelial cell line (iHCEC). Phase contrast microscopic images of immortalized corneal endothelial cell lines (iHCEC) and iFECD derived from healthy donors show that both iHCEC and iFECD have a single-layer polygonal morphology similar to normal corneal endothelial cells. iHCEC and iFECD were maintained and cultured in DMEM + 10% FBS. SB431542 was obtained from TOCRIS (catalog number: 1614).SB203580 was obtained from CALBIOCHEM (catalog number: 559389).

[0085] (Preparation Example 2: Confirmation of normal function of immortalized corneal endothelial cell line (iFECD)) In this example, the normal function of the immortalized corneal endothelial cell line (iFECD) was confirmed.

[0086] (Na + / K + -ATPase and ZO-1 immunostaining) First, to confirm the normal function of the immortalized corneal endothelial cell line (iFECD), Na + / K + Immunostaining with Na-ATPase and ZO-1 was performed to confirm the pump function and barrier function of corneal endothelial cells. + / K + The expression of ATPase and ZO-1 indicates the normality of the pump function and barrier function, respectively, of the corneal endothelial cells.

[0087] (Cell observation methods such as staining (histological examination)) Cell observation was performed using a phase-contrast microscope. After fixing the cells, functional markers such as ZO-1 and Na + / K + Immunostaining was performed using α-ATPase and the cells were observed under a fluorescent microscope. TM Chamber Slides TM (NUNC A / S, Roskilde, Denmark), fixed with 4% formaldehyde for 10 minutes at room temperature (RT), and incubated with 1% bovine serum albumin (BSA) for 30 minutes. TM Chamber Slides TMCultured cells on a 1000-well plate (NUNC A / S, Roskilde, Denmark) were fixed in 4% formaldehyde for 10 minutes at room temperature and then incubated with 1% bovine serum albumin (BSA) for 30 minutes. To examine the cell phenotype, we analyzed the tight junction-associated protein ZO-1 (Zymed Laboratories, Inc., South San Francisco, CA), the protein involved in the pump function, and the Na+ receptor agonist (NA receptor agonist). + / K + Immunohistochemical analysis was performed for ZO-1 and Na+ ATPase (Upstate Biotec, Inc., Lake Placid, NY) as markers related to cell function. + / K + -ATPase was used. + / K + -ATPase staining was performed using ZO-1 polyclonal antibody and Na + / K + The primary antibody was a 1:200 dilution of α-ATPase monoclonal antibody. The secondary antibody was a 1:2000 dilution of Alexa Fluor® 488-conjugated or Alexa Fluor® 594-conjugated goat anti-mouse IgG (Life Technologies). Cell nuclei were then stained with DAPI (Vector Laboratories, Inc., Burlingame, CA) or PI (Sigma-Aldrich). Slides were then observed under a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Welzlar, Germany).

[0088] The results showed that Na was expressed in all cells in both iHCEC and iFECD. + / K + The immortalized cell line expressed α-ATPase and ZO-1, and maintained normal function.

[0089] The morphological images of iHCEC and iFECD observed by transmission electron microscopy are also shown. iHCEC and iFECD were cultured on a Transwell plate in serum-free DMEM for one week, and then fixed in a confluent state. The morphology was observed by transmission electron microscopy, revealing a single layer of cells with no obvious morphological abnormalities.

[0090] Corneal endothelial cells in patients with Fuchs' endothelial corneal dystrophy are known to produce excessive extracellular matrix, leading to guttae formation and thickening of Descemet's membrane. Therefore, iHCECs and iFECDs were cultured in culture dishes and immunostained for the expression of extracellular matrix proteins, type I collagen, type IV collagen, and fibronectin. Compared with iHCECs, iFECDs showed increased expression of type I collagen, type IV collagen, and fibronectin. Furthermore, real-time PCR analysis of gene expression levels in cultured iHCECs and iFECDs revealed significantly increased expression of type I collagen and fibronectin, with a tendency toward increased expression of type IV collagen. We investigated whether iFECDs, like the corneal endothelium in patients with Fuchs' endothelial corneal dystrophy, produce excessive extracellular matrix. iHCECs and iFECDs were cultured on Transwell plates in DMEM serum-free media and fixed at confluence after 1 week, then subjected to HE staining. iFECDs produced significantly thicker extracellular matrix than iHCECs. Based on the above, we created a disease model cell line that exhibits the characteristic excessive extracellular matrix production seen in patients with Fuchs' endothelial corneal dystrophy. Because analysis using the disease model cell line is expected to contribute to elucidating the pathogenesis of Fuchs' endothelial corneal dystrophy, which remains largely unknown, we attempted to develop a therapeutic drug for Fuchs' endothelial corneal dystrophy using these cells.

[0091] Example 1: Analysis of expression levels of genes involved in epithelial-mesenchymal transition (EMT) involved in extracellular matrix production using real-time PCR In this example, the results of analyzing the expression levels of genes involved in epithelial-mesenchymal transition (EMT), which is involved in the production of extracellular matrix, in iHCECs and iFECDs using real-time PCR are shown.

[0092] (Real-time PCR) Real-time PCR: PCR was also performed using the Taqman method for Snail1, Snail2, or ZEB1 using the following method. Taqman probes were purchased from INVITROGEN. The mRNA levels of type I collagen, type IV collagen, and fibronectin were examined by real-time PCR. RNEasy (QIAGEN, catalog number: 74106) was used to extract total RNA from cells. The extracted RNA was reverse-transcribed (42°C, 60 minutes) using ReverTra Ace (TOYOBO, catalog number: TRT-101), and type I collagen, type IV collagen, and fibronectin were amplified using the TaqMan Fast Advanced mastermix (Applied Biosystems) reaction reagent with GAPDH as an internal standard. PCR was performed using the probes shown below (labeled primer sets available from Applied Biosystems) and StepOne PCR. TM This was performed using an Applied Biosystems real-time PCR system. Snail1 Hs00195591_ml SNAI1 Snail2 Hs00950344_ml SNAI2 ZEB1 Hs00232783_ml ZEB1 GAPDH TaqMan(R)predeveloped Assay Reagents Human GADPH (cat no.: 4333764F).

[0093] (result) The results are shown in Figure 1. As shown in Figure 1, when the expression levels of genes involved in epithelial-mesenchymal transition (EMT), which is involved in the production of extracellular matrix, were analyzed using real-time PCR, the expression of Snail1 and ZEB1 was found to be significantly increased in iFECD compared to iHCEC.

[0094] (TGFβ-induced increase in Snail1 and ZEB1 expression) To confirm whether the increased expression of Snail1 and ZEB1 is related to the production of extracellular matrix, we stimulated them with TGFβ, which is known to promote the expression of Snail1 and ZEB1. The method is as follows: iFECD and iHCEC were cultured in DMEM containing 10% fetal bovine serum, and then cultured overnight in DMEM without 10% fetal bovine serum. The expression of Snail1, ZEB1, type I collagen, type IV collagen, type VIII collagen, and fibronectin was examined by real-time PCR. The following probes were used for PCR reactions using StepOne TM This was performed using an Applied Biosystems real-time PCR system. Snail1 Hs00195591_ml SNAI1 ZEB1 Hs00232783_ml ZEB1 Type I collagen Hs00164004_ml COL1A1 Type IV collagen Hs00266327_ml COL4A1 Type VIII collagen Hs00697025_ml COL8A2 Fibronectin Hs01549976_ml FN1 GAPDH TaqMan(R)predeveloped Assay Reagents Human GADPH (cat no.: 4333764F).

[0095] The results are shown in Figure 2. We confirmed that TGFβ significantly promoted the expression of Snail1 and ZEB1 in iFECD (A, B). Therefore, we analyzed the gene expression levels of extracellular matrix component proteins by real-time PCR, and found that the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin was significantly promoted.

[0096] Example 2: Promotion of extracellular matrix production by iFECD by TGFβ In this example, an investigation was conducted to determine whether the extracellular matrix produced by iFECD is promoted by TGFβ.

[0097] iHCECs and iFECDs were cultured in DMEM serum-free on Transwell plates. After 1 week, they were fixed at confluence and subjected to HE staining. The procedure was as follows: Deparaffinization (e.g., with pure ethanol) was performed as needed, and the samples were immersed in Omni's hematoxylin for 10 minutes. They were then rinsed with running water and decolorized with ammonia water for 30 seconds. They were then rinsed with running water for 5 minutes, stained with 10x diluted HCl-eosin for 2 minutes, dehydrated, permeabilized, and mounted. iHCECs and iFECDs produced significantly thicker extracellular matrix upon TGFβ stimulation. Furthermore, in the presence of TGFβ, iFECDs produced significantly thicker extracellular matrix than iHCECs. These findings suggest that corneal endothelial cells from Fuchs' corneal endothelial dystrophy patients have higher expression levels of Snail1 and ZEB1, and produce significantly greater extracellular matrix in response to TGFβ stimulation than corneal endothelial cells from healthy controls.

[0098] Example 3: Effect of siRNA-mediated inhibition of Snail1 and ZEB1 on extracellular matrix production In this example, to demonstrate that increased expression of Snail1 and ZEB1 is responsible for extracellular matrix production, we used siRNA to suppress Snail1 and ZEB1 and examined the effects on extracellular matrix production. The experimental procedure was as follows.

[0099] (Method) iFECDs and iHCECs were seeded and transfected with Snail1 Stealth RNAi TM (Life Technologies Corp., Carlsbad, CA) or ZEB1 Stealth RNAi TM (Life Technologies Corp., Carlsbad, CA) and Lipofectamine TM The cells were incubated with RNAiMAX (Life Technologies Corp., Carlsbad, CA) at 37°C for 12 hours. Random sequence RNAi was used as a control. The cells were then passaged and used in the experiment. TM and ZEB1 Stealth RNAi TM The experiments were performed using StepOne and the results are shown as representative examples. Cells in which Snail1 or ZEB1 was knocked down with siRNA were seeded, and the expression of Snail1, ZEB1, type I collagen, type IV collagen, type VIII collagen, and fibronectin was examined by real-time PCR. The probes shown below were used for PCR reactions. TM This was performed using a real-time PCR system (Applied Biosystems).

[0100] (material) siRNA Snail1 siRNA (SNAI1 HSS143995*, SNAI1 HSS143996, SNAI1 HSS143997) ZEB1 siRNA (ZEB1 HSS110548*, ZEB1 HSS110549, ZEB1 HSS186235) However, siRNAs shown in the results are indicated with *.

[0101] Real-time PCR probes Snail1 Hs00195591_ml SNAI1 ZEB1 Hs00232783_ml ZEB1 Type I collagen Hs00164004_ml COL1A1 Type IV collagen Hs00266327_ml COL4A1 Type VIII collagen Hs00697025_ml COL8A2 Fibronectin Hs01549976_ml FN1 GAPDH TaqMan(R)predeveloped Assay Reagents Human GADPH (cat no.: 4333764F).

[0102] (result) The results are shown in Figure 4. We confirmed that siRNA suppressed the expression of Snail1 and ZEB1 (A, F). Inhibition of Snail1 or ZEB1 expression by siRNA significantly suppressed the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin. These results demonstrate that ZEB1 or Snail1 negatively regulates the gene expression of extracellular matrix component proteins.

[0103] (Regulation of type I collagen, type IV collagen, and fibronectin expression by immunohistochemistry) Next, immunostaining was performed to confirm that the expression of type I collagen, type IV collagen, and fibronectin was suppressed. The immunostaining procedure was similar to that of Preparation Example 2, but the antibodies used were changed to antibodies against type I collagen, type IV collagen, and fibronectin. Anti-collagen type I (Rabbit polyclonal) (ROCKLLANDTM antibodies and assays, Cat no.: 600-401-103S) Antibody to type IV collagen: collagen type IV (Rabbit polyclonal) (Abcam, Cat. no.: ab6586) Anti-fibronectin antibody: Anti-fibronectin (mouse monoclonal) (BD Biosciences, Cat. no.: 610077) For histological staining, cultured cells were transferred to Lab-Tek TM Chamber Slides TM (NUNC A / S, Roskilde, Denmark), fixed with 4% formaldehyde for 10 minutes at room temperature (RT), and incubated with 1% bovine serum albumin (BSA) for 30 minutes. TM Chamber Slides TM Cultured cells on slides (NUNC A / S, Roskilde, Denmark) were fixed in 4% formaldehyde for 10 minutes at room temperature and then incubated with 1% bovine serum albumin (BSA) for 30 minutes. To examine the expression of extracellular matrix, antibodies against type I collagen, type IV collagen, and fibronectin were used at a 1:200 dilution. Secondary antibodies were Alexa Fluor® 488-conjugated or Alexa Fluor® 594-conjugated goat anti-mouse IgG (Life Technologies) at a 1:2000 dilution. Cell nuclei were then stained with DAPI (Vector Laboratories, Inc., Burlingame, CA) or PI (Sigma-Aldrich). Slides were then observed under a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Welzlar, Germany).

[0104] (result) The results are shown in Figure 5. As shown in Figure 5, it was confirmed that suppression of Snail1 or ZEB1 expression by siRNA also suppressed the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin at the protein level.

[0105] Example 4: Suppression of excessive extracellular matrix production in iFECD by suppressing Snail1 or ZEB1 expression In this example, it was confirmed that excessive production of extracellular matrix in iFECD was suppressed by suppressing the expression of Snail1 or ZEB1.

[0106] Furthermore, iHCECs and iFECDs were cultured on Transwell plates in DMEM in a serum-free environment, and after one week, they were fixed in a confluent state and subjected to HE staining, which was carried out according to the procedure described in the above example.

[0107] (result) The results are shown in Figure 6. As shown in Figure 6, suppression of Snail1 or ZEB1 expression by siRNA suppressed excessive extracellular matrix production in iFECD, returning it to normal levels. Therefore, it was found that suppression of ZEB1 or Snail can suppress excessive extracellular matrix production in Fuchs' corneal endothelial dystrophy cells.

[0108] Example 5: Regulation of extracellular matrix abnormalities in corneal endothelium by TGFβ signal inhibitors Next, we investigated whether TGFβ signaling could be inhibited using SB431542, a TGFβ signaling inhibitor, to regulate extracellular matrix abnormalities in the corneal endothelium. SB431542 was obtained from TOCRIS (catalog number: 1614).

[0109] (Real-time PCR study) The gene expression levels were confirmed by real-time PCR. Real-time PCR was performed according to the above example. The following probes were used for type I collagen, type IV collagen, type VIII collagen, and fibronectin.

[0110] Type I collagen Hs00164004_ml COL1A1 Type IV collagen Hs00266327_ml COL4A1 Type VIII collagen Hs00697025_ml COL8A2 Fibronectin Hs01549976_ml FN1 GAPDH TaqMan(R)pre developed Assay Reagents Human GADPH (cat no.: 4333764F) (result) The results are shown in Figure 7. As shown in Figure 7, real-time PCR confirmed a significant decrease in the expression levels of Snail1 and ZEB1. Furthermore, when the gene expression levels of components of the extracellular matrix of iFCED were analyzed by real-time PCR, the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin was significantly suppressed by SB431542.

[0111] (Examination of the expression of type I collagen, type IV collagen, and fibronectin by immunostaining) Next, the expression of type I collagen, type IV collagen, and fibronectin was examined by immunostaining in the same manner as in the above example, except that the following antibodies for type I collagen, type IV collagen, and fibronectin were used. Anti-collagen type I (Rabbit polyclonal) (ROCKLLANDTM antibodies and assays, Cat no.: 600-401-103S) Antibody to type IV collagen: collagen type IV (Rabbit polyclonal) (Abcam, Cat. no.: ab6586) Anti-fibronectin antibody: Anti-fibronectin (mouse monoclonal) (BD Biosciences, Cat. no.: 610077) (result) The results are shown in Figure 8. As shown in Figure 8, it was confirmed that the expression of type I collagen, type IV collagen, type VIII collagen, and fibronectin was also suppressed at the protein level by inhibiting TGFβ signaling with SB431542.

[0112] Example 6: Effect of TGFβ signal inhibitor when fixed in a confluent state after 1 week In this example, the effect of a TGFβ signal inhibitor was confirmed when the cells were fixed in a confluent state after one week.

[0113] Furthermore, iHCECs and iFECDs were cultured serum-free on Transwell Permeable Supports: 0.4 μm, 6-well plates (Costar, Cat. no.: 3450). After 1 week, the cells were fixed in a confluent state and subjected to HE staining, as described in the above example.

[0114] (result) The results are shown in Figure 9. As shown in Figure 9, by inhibiting TGFβ signaling with SB431542, excessive production of extracellular matrix in iFECD was suppressed and returned to normal levels.

[0115] These results demonstrate that patients with Fuchs' corneal endothelial dystrophy produce excessive amounts of extracellular matrix (ECM) under TGFβ signaling, due to increased expression of Snail1 or ZEB1. Furthermore, we demonstrated that silencing EMT-related and protein production-related genes, such as Snail1 or ZEB1, with siRNA or other methods can suppress ECM production. Furthermore, we demonstrated that inhibition of TGFβ signaling can also suppress ECM production. These results suggest that inhibition of TGFβ signaling or EMT-related genes or signals, such as Snail1 or ZEB1, may suppress excessive ECM production in corneal endothelial cells of patients with Fuchs' corneal endothelial dystrophy, thereby potentially suppressing guttae formation and thickening of Descemet's membrane.

[0116] While the present invention has been illustrated by way of preferred embodiments thereof, it is understood that the scope of the present invention should be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and literature cited herein are incorporated by reference in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0117] A disease, disorder or condition associated with an abnormality in the extracellular matrix (ECM) of the corneal endothelium, particularly an ECM abnormality in Fuchs' endothelial corneal dystrophy, comprising a TGFβ signal inhibitor. In general, the provided technology is applicable to industries related to therapeutic or preventive drugs for photophobia (cell culture industry, pharmaceuticals, etc.).

Claims

[Claim 1] The compositions described in the specification.

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