Composition for inhibiting ocular tissue fibrosis

Abstract

To provide a composition for inhibiting fibrosis in ocular tissue.SOLUTION: A drug composition comprises a substance that inhibits fibrosis in a plurality of ocular tissues.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for inhibiting fibrosis of ocular tissue. [Background technology]

[0002] Fibrosis, a widespread disease affecting vital organs such as the lungs, liver, kidneys, and skin, occurs when vital organs, such as the lungs and liver, are damaged and undergo repair. This damage causes collagen fibers, such as type I collagen, to accumulate, resulting in the organ losing its elasticity and becoming hardened, unable to function normally. Fibrosis in solid organs (liver, heart, lungs, kidneys, gastrointestinal tract, etc.) is a serious, chronic disease that can lead to death if left untreated. Despite the need to improve prognosis, research into this disease has lagged significantly, and there are currently no effective medications. Previous research into organ fibrosis has focused primarily on the role of the tissue growth factor (TGF-β) in immune function and collagen production.

[0003] Tissue fibrosis occurs when extracellular matrix (ECM), primarily collagen, is produced and accumulated excessively in tissues. When tissue cells are damaged by various stresses, such as oxidative stress, hypoxia, inflammation, and apoptosis, the tissue attempts to repair itself by replacing the damaged tissue with extracellular matrix. However, when damage is severe or stresses such as chronic inflammation become chronic over a long period of time, the accumulation of extracellular matrix becomes excessive, leading to a pathological state in which the tissue's intrinsic function falls below the threshold required to maintain physiological balance.

[0004] Collagen-producing cells such as myofibroblasts are thought to be involved in the pathology of fibrosis. However, it is believed that the pathology actually involves a wide variety of cells and complex intercellular interactions. The molecular mechanisms underlying this pathology remain unclear, posing a major bottleneck in drug discovery. For example, one of the inventors reported in 2002 that macrophages (Mps), which gather at the affected site during the fibrotic phase, are involved in the onset of fibrosis. This suggests that drug development targeting Mps may enable the development of drugs for fibrosis, which has previously lacked effective treatments. Furthermore, the inventors have long been researching methods for inhibiting Mps function, which is involved in the onset of fibrosis, and technologies for suppressing chronic inflammation, which is closely related to tissue fibrosis.

[0005] Chronic inflammation is a condition characterized by persistent inflammation and marked fibrosis (tissue remodeling) at the site of inflammation, angiogenesis, and the accumulation of specific immune cells. Chronic inflammation has diverse causes and pathologies, and internal and external environmental stressors trigger inflammation as a defense mechanism mediated by the immune and endocrine systems. Persistent or recurrent inflammation can progress through an asymptomatic pre-disease state, eventually establishing an adaptive state characterized by abnormal chronic inflammation accompanied by functional impairment of cells and tissues. Persistence of this chronic inflammatory state leads to tissue fibrosis and irreversible organ dysfunction. At the tissue and organ level, this disease process disrupts tissue homeostasis through the involvement of various factors, including altered interactions between tissue-constituting cells and infiltrating immune cells, heterogeneous and diverse cell types with different activation states, nonphysiological metabolic responses, and disruptions in extracellular matrix and humoral factor networks.

[0006] The eye is an immunologically special, functionally closed organ and is known as an "immune privileged site." Immune privilege is a self-defense mechanism inherently possessed by the body. Immune privilege can be interpreted as a homeostatic mechanism that exists to protect the function of organs where normal immune inflammatory responses would actually worsen tissue and functional damage. However, once inflammation exceeds its limits, the immune privilege mechanism is lost, and ocular inflammation worsens. Damaged ocular tissues have difficulty recovering their function.

[0007] Immune responses are broadly classified into "acquired immunity," which is centered on T lymphocytes, and "innate immunity," which responds earlier. Innate immune cells are important inflammatory cells in various eye diseases. In recent years, it has become increasingly recognized that Mps, NKT cells, and γδ T cells, which are responsible for so-called "innate immunity," are essential for maintaining ocular homeostasis and transparency.

[0008] Among the clinical pathologies of choroidal neovascular diseases, such as age-related macular degeneration (AMD), much research on choroidal neovascularization (CNV) has focused on its formation mechanism. However, in actual clinical pathology, the inflammatory response associated with the scar healing process in the macular area that occurs after bleeding from CNV is crucial. In recent years, new treatments have been developed to inhibit the CNV formation process, such as intravitreal administration of anti-VEGF antibodies such as bevacizumab, and for already formed CNV, photodynamic therapy using verteporfin. These treatments have demonstrated some therapeutic efficacy. However, the period in which these new treatments are effective is largely limited to the pre- and onset stages, making them insufficient from a practical standpoint.

[0009] Age-related macular degeneration (AMD) is a disease in which retinal pigment epithelial cells (RPE) in the macula degenerate due to aging and environmental factors such as oxidative stress, resulting in choroidal neovascularization (CNV). It is one of the most serious visual impairment diseases in elderly people. Currently, the mainstay of treatment for AMD is vascular ablation therapy using anti-VEGF antibodies that target the CNV.

[0010] The clinical pathology of choroidal neovascularization is that "many patients first become aware of the disease when vision deteriorates after macular hemorrhage, but from the perspective of visual recovery, recovery has already reached a difficult stage, and the tissue scarring that has already formed will not recover" (Non-Patent Document 3). The process of macular dysfunction (scar healing) that occurs after bleeding and exudation from CNV is also considered an important therapeutic target for choroidal neovascularization. In addition to the pathology of CNV formation, it is also important to inhibit the pathology of secondary choroidal scarring that forms due to bleeding and exudation of blood components from CNV.

[0011] Currently widely used anti-VEGF therapy has a number of problems. (1) There is a high incidence of non-responders who show resistance to treatment, and resistant cases in which the therapy loses effectiveness over time. (2) Because anti-VEGF therapy aims to suppress the progression of the disease, it is unlikely to improve visual function. (3) Long-term, multiple administrations are necessary, which carries the risk of complications and side effects (increased intraocular pressure, decreased vision, eye pain, retinal hemorrhage). (4) High medical costs have led to the discontinuation of treatment in some cases. Due to these many problems, there is a strong need in clinical settings for new AMD treatments that can replace anti-VEGF therapy.

[0012] Resistance to anti-VEGF antibodies has been linked to (i) tissue fibrosis that occurs before CNV formation (Diego et al. 2013). Permanent vision loss is also thought to be caused by (ii) fibrous tissue that forms the foundation for CNV development and remains as scar tissue even after devascularization. Furthermore, (iii) CNV itself is not necessarily formed solely by VEGF, but involves many other angiogenic and antiangiogenic factors. Anti-VEGF therapy is completely ineffective against these (i)-(iii).

[0013] Regarding retinal scar tissue, the inventors have reported that local scarring can be induced by injecting activated macrophages into the subretinal space of mice (Non-Patent Document 1). The clinical pathology of choroidal neovascular diseases such as AMD progresses as shown in Figure 38. The subretinal proliferative tissue of AMD patients contains a mixture of proliferated and migrated RPE and MPS, and MPS and RPE are thought to be important in the pathogenesis of the disease. The present inventors have reported that co-culture of MPS and RPE enhances the production of proinflammatory cytokines, enhances the expression of complement activation genes such as C3 and CFB, and attenuates the expression of complement activation inhibitors such as CFH, CD59, and Clusterin, enhances VEGF expression, and attenuates the expression of the antiangiogenic factor PEDF (Non-Patent Document 2). Intracellular α-SMA is elevated in RPE co-cultured with MPS, and subretinal injection leads to subretinal scar formation. The present inventors have long sought a substance that inhibits the production of inflammatory cytokines produced by MPS and their dendritic cell relatives. They discovered a substance that retains its effectiveness even in the presence of factors that exacerbate fibrotic pathology related to tissue fibrosis, which became the starting point for the present invention.

[0014] Glaucoma, another important ocular tissue disease targeted by the present invention, is a disease characterized by characteristic changes such as damage to the optic nerve and a narrowing of the visual field. When anti-glaucoma drugs are not sufficient to reduce intraocular pressure, trabeculectomy (TLE; a surgery in which the trabecular meshwork is removed to allow aqueous humor to drain out of the eye and reduce intraocular pressure) is performed to lower the intraocular pressure. Alternatively, outflow reconstruction surgery such as trabeculotomy and insertion of a glaucoma treatment implant is performed. Improving the prognosis of glaucoma surgery is an important medical need.

[0015] During glaucoma surgery, such as trabeculectomy, aqueous humor drains from the trabeculectomy site through the scleral flap and out of the eye, forming a filtering bleb under the conjunctiva. However, if the filtering bleb shrinks or disappears after surgery due to tissue inflammation, adhesions, scarring, etc., intraocular pressure may rise again, worsening glaucoma. Trabeculectomy has several issues, including the risk of infection, difficulty in forming and maintaining the filtering bleb, and difficulty in long-term intraocular pressure control.

[0016] Therefore, a glaucoma surgery that can form a filtering bleb over a wide area, maintain the vascularity of the conjunctiva, and reduce intraocular pressure for a long period after surgery is desired. Mitomycin C (MMC) is applied intraoperatively to prevent postoperative inflammation, adhesions, and scarring. However, MMC has side effects, such as thinning of the conjunctiva, leakage of aqueous humor from the conjunctiva, and infection of the filtering bleb. Current trabeculectomy using MMC has its own problems. Excessive wound healing leads to thick connective tissue around the filtering bleb, making long-term intraocular pressure control difficult. Furthermore, it is widely known that the avascularization and thinning of the filtering bleb increases the risk of bleb infection due to aqueous humor leakage.

[0017] Histone deacetylase (HDAC) inhibitors have been investigated in basic research to inhibit fibrosis, but their practical application remains hampered by significant bottlenecks and development has not been achieved. In the field of ophthalmology, the anti-fibrotic effect of the HDAC inhibitor SAHA on the conjunctiva has been reported in rabbit filtration surgery. However, the dose required is more than 1,000 times higher than that of the compound of the present invention, and the drug's ability to reach the affected area at effective concentrations is far from practical. Development has not been achieved because a compound with minimal side effects at practically effective concentrations has not been discovered, and the drug's properties do not meet the requirements for reaching the affected area at effective concentrations. In chronic tissue inflammation such as AMD, the pathological target is not clearly defined in time and space, and the appropriate timing and route of drug administration for local drug effects are unclear, resulting in a significant discrepancy with medical needs. Furthermore, practical effects that comprehensively inhibit fibrosis, angiogenesis, and tissue scarring cannot be expected. It has also been confirmed that the pharmacological effects of the small molecule compounds of the present invention cannot be achieved through HDAC inhibitory activity alone. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] Patent No. 3554707 [Non-patent literature]

[0019] [Non-Patent Document 1] Invest Ophthalmol Vis Sci 52 : 6089-6095 (2011) [Non-patent document 2] Invest Ophthalmol Vis Sci 57:5945-5953 (2016) [Non-patent document 3] Fukuoka Medical Journal. 99 (7), pp.137-143, 2008-07-25. Fukuoka Medical Society Summary of the Invention [Problem to be solved by the invention]

[0020] An object of the present invention is to provide a substance that has the effect of inhibiting fibrosis of ocular tissue. Another object of the present invention is to provide a method for treating ocular hypertension or glaucoma, more specifically, a method for treating which synergistically improves efficacy and reduces side effects. Another object of the present invention is to provide a method for treating AMD, which improves the aggravated pathology of AMD, and a method for treating patients who are expected to develop AMD.

[0021] The present inventors have conducted extensive research to solve the above-mentioned problems and have provided a glaucoma surgery technique that suppresses excessive fibrosis, promotes healthy conjunctival tissue repair, and maintains long-term intraocular pressure. Furthermore, in a laser-irradiated animal model, the inventors have discovered a small molecule compound that is effective at extremely low concentrations and dosages against choroidal tissue fibrosis, including RPE fibrosis, and that comprehensively inhibits multiple genes involved in tissue fibrosis. It also inhibits the expression of multiple genes involved in tissue fibrosis, angiogenesis, and scarring, including VEGF and PDGF, which are involved in angiogenesis, and LOX, which crosslinks collagen and is involved in scar formation. The inventors have successfully confirmed the compound's efficacy in an animal model, leading to the completion of the present invention. The inventors have continued their extensive research to develop a novel AMD treatment that combines a novel CNV suppression mechanism and the ability to inhibit scar tissue formation, based on their own unique molecular pathology theory.

[0022] The inventors have completed this invention, which addresses the two major unmet needs mentioned above, and the contents of this invention go beyond the examples shown in this specification and offer the possibility of application to other ocular tissue diseases and other organ diseases. [Means for solving the problem]

[0023] That is, the present invention is as follows. (1) A pharmaceutical composition comprising a substance that inhibits fibrosis of ocular tissue. (2) 2. The pharmaceutical composition according to 1, wherein the substance that suppresses fibrosis of ocular tissue is a substance that inhibits the expression of at least one gene that exacerbates pathology in each of the three stages of fibrosis, angiogenesis, and scar formation in ocular tissue in vivo. (3) 3. The pharmaceutical composition according to 2, wherein the pathology aggravating factor gene is selected from the group consisting of collagen 1A, collagen 3A1, collagen 4A1, TIMP 2, TIMP 3, TIMP 4, Thrombospondin 1, Thrombospondin 2, LOX, Loxl2, TGFb2, TGFb3, CTGF, VEGF, PDGF and Serpin. (4) 4. The pharmaceutical composition according to any one of 1 to 3, comprising a substance that suppresses fibrosis of ocular tissue at a dose of 100 pg / kg to 3000 pg / kg. (5) 4. The pharmaceutical composition according to any one of 1 to 3, comprising a substance that inhibits fibrosis of ocular tissue at a dose of 2 pg / eye to 9000 pg / eye.

[0024] (6) 6. The pharmaceutical composition according to any one of 1 to 5, wherein the substance that suppresses fibrosis of ocular tissue is a substance that suppresses fibrotic phase transition of cultured ocular tissue cells. (7) 7. The pharmaceutical composition according to any one of 1 to 6, comprising a substance that inhibits fibrotic phase transition in cultured ocular tissue cells at a concentration of 10 nM or less. (8) 8. The pharmaceutical composition according to any one of 1 to 7, comprising a substance having an inhibitory effect on HDAC activity in ocular tissue cells at a concentration of IC50=10 nM or less. (9) 9. The pharmaceutical composition according to any one of 1 to 8, wherein the substance that inhibits fibrosis of ocular tissue is a substance that has a filtering bleb-maintaining effect or an effect of improving the prognosis of glaucoma surgery. (10) 10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the substance that inhibits fibrosis of ocular tissue comprises a substance that has both an inhibitory effect on fibrosis and / or angiogenesis and an inhibitory effect on scar formation.

[0025] (11) Formula I: [ka] or the following formula II: [ka] (In the formula, R1 to R3 independently represent a hydrogen atom, a methyl group, or an ethyl group; R4 independently represent a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group; R5 to R8 independently represent a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 independently represent a hydrogen atom, a methyl group, or a protecting group; and R10 and R11 independently represent a hydrogen atom, a methyl group, or a protecting group.) 11. The pharmaceutical composition according to any one of 1 to 10, comprising a depsipeptide compound represented by the following formula: or a pharmaceutically acceptable salt thereof.

[0026] (12) Formula III: [ka] (wherein R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group). 12. The pharmaceutical composition according to 11, comprising a depsipeptide compound represented by the following formula (I): or a pharmaceutically acceptable salt thereof.

[0027] (13) 13. The pharmaceutical composition according to 12, wherein R4 is an isopropyl group. (14) 14. The pharmaceutical composition according to any one of items 1 to 13, wherein the ocular tissue is at least one selected from the group consisting of glaucoma-related tissue, conjunctiva-related tissue, and retina-related tissue. (15) 15. The pharmaceutical composition according to 14, wherein the glaucoma-related tissue is the trabecular meshwork or a tissue capable of controlling intraocular pressure. (16) 15. The pharmaceutical composition according to 14, wherein the retina-related tissue is a tissue involved in retinal pigment epithelium, choroidal neovascularization, or age-related macular degeneration. (17) 15. The pharmaceutical composition according to 14, wherein the conjunctiva-related tissue is filtering bleb tissue. [Effects of the Invention]

[0028] The present invention provides a compound that has the properties of suppressing excessive fibrosis, promoting healthy conjunctival tissue repair, and maintaining long-term intraocular pressure reduction. Furthermore, the present invention provides for the first time a compound that combines the effects of suppressing fibrosis and / or angiogenesis with the effect of suppressing scar formation, which are required but not yet provided in current AMD treatments, and will lead to the development of a useful means for innovative treatment in this field.

[0029] Such compounds, in part, promote the wound healing process after glaucoma surgery in trabecular meshwork cells and normalize aqueous humor dynamics. On the other hand, it is effective against choriocellular fibrosis at extremely low concentrations and dosages, and also exhibits comprehensive inhibitory effects on multiple genes involved in tissue fibrosis and precursor lesions. Many existing fibrosis-inhibiting candidate substances are effective against TGFβ-induced fibrosis, but are ineffective against fibrosis caused by the combined action of TGFβ and TNFα in chronic inflammatory tissue. The pharmaceutical composition of the present invention is also highly effective against this complex pathology, and is highly likely to directly inhibit damage (including fibrosis) to photoreceptors and retinal pigment epithelial cells, which are most important for maintaining a patient's vision. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 shows the results of measuring intraocular pressure up to day 30 when 200 μl of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 2] FIG. 1 shows blebs on days 7 and 30 after administration of 200 μl of 10 nM OBP-801 on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 3] FIG. 1 shows blebs (filtering blebs) on days 7 and 30 after administration of BSS as a control on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 4] FIG. 1 shows Blebs on day 14 when OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 5] FIG. 1 shows the results of measuring intraocular pressure up to day 30 when 200 μl of 10 nM OBP-801 was administered on postoperative days 0, 1, 3, 5, 7, and 9, and when it was administered on postoperative days 0, 1, 3, and 5. [Figure 6] FIG. 1 shows the results of measuring intraocular pressure up to day 30 when 200 μl of 10 nM OBP-801 was administered on postoperative days 0, 1, 3, 5, 7, and 9, and on postoperative days 3, 5, and 7. [Figure 7] FIG. 1 shows the results of measuring intraocular pressure up to day 15 when 200 μl of 10 nM, 1 μM, and 100 μM OBP-801 were administered on days 0, 1, 3, 5, 7, and 9 after surgery, respectively. [Figure 8] FIG. 1 shows the results of measuring intraocular pressure up to day 15 when 200 μl of 10 nM OBP-801 was administered on postoperative days 0, 1, 3, 5, 7, and 9, and when 200 μl of 100 μM OBP-801 was administered on postoperative day 0. [Figure 9] FIG. 1 shows the expression level of αSMA on day 14 when 200 μl of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 10] FIG. 1 shows the amount of collagen expression on day 14 when 200 μl of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 11]FIG. 1 shows the amount of collagen expression on day 30 when 200 μl of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. [Figure 12] FIG. 1 shows the inhibitory effect of OBP-801 on αSMA expression in HconF cells in which fibrosis was induced by TGF+TNF. [Figure 13] FIG. 1 shows the inhibitory effect of OBP-801 administration on collagen and LOX expression in HconF cells in which fibrosis was induced by TGF+TNF. [Figure 14] FIG. 1 shows the inhibitory effect of OBP-801 on the expression of αSMA, col1, and col4 when administered before and / or after fibrosis induction of HconF by TGF+TNF. [Figure 15] FIG. 1 shows the inhibitory effect of OBP-801 on αSMA expression when treated with OBP-801 at different concentrations and for different treatment times before induction of fibrosis in HconF with TGF+TNF. [Figure 16] FIG. 1 shows the time course of cell number following OBP-801 treatment. [Figure 17] FIG. 1 shows HDAC and HAT activity when HconF is induced to become fibrotic by TGF+TNF. [Figure 18] FIG. 1 shows the time course of changes in the amount of acetylated histones when HconF was treated with OBP-801. [Figure 19] FIG. 1 shows genes whose expression levels changed significantly two days after trabeculectomy and the inhibitory effect of OBP-801 on the expression of these genes. [Figure 20] FIG. 1 shows genes whose expression levels changed significantly 12 days after trabeculectomy and the inhibitory effect of OBP-801 on the expression of these genes. [Figure 21] FIG. 1 shows genes whose expression levels changed significantly 30 days after trabeculectomy and the inhibitory effect of OBP-801 on the expression of these genes. [Figure 22] FIG. 1 shows the inhibitory effects of OBP-801 and SAHA on HconF fibrosis. [Figure 23]FIG. 1 shows the cell proliferation inhibitory effects of OBP-801 and SAHA on HconF. [Figure 24] FIG. 1 shows that OBP-801 inhibits myofibroblastic transformation of HTMCs. [Figure 25] FIG. 1 shows the inhibitory effect of OBP-801 on collagen and LOX expression in HTMCs in which fibrosis was induced by TGF+TNF. [Figure 26] FIG. 1 shows the CNV suppression effect of OBP-801 administration. [Figure 27] FIG. 1 shows the CNV suppression effect of OBP-801 administration. [Figure 28] FIG. 1 shows the CNV suppression effect of OBP-801 administration. [Figure 29] FIG. 1 shows the inhibitory effect of OBP-801 administration on Collagen I expression. [Figure 30] FIG. 1 shows the inhibitory effect of OBP-801 administration on αSMA expression. [Figure 31] FIG. 1 shows the inhibitory effect of OBP-801 administration on αSMA expression. [Figure 32] FIG. 1 shows the suppressive effect of OBP-801 administration on CD31 expression. [Figure 33] FIG. 1 shows the inhibitory effect of OBP-801 on RPE cell fibrosis. [Figure 34] FIG. 1 shows the effect of OBP-801 on fibrosis-related gene expression. [Figure 35] FIG. 1 shows the relationship with the HDAC inhibitory activity of OBP-801. [Figure 36] FIG. 1 shows the relationship with the HAT inhibitory activity of OBP-801. [Figure 37] FIG. 1 shows the effect of OBP-801 on CD44 expression. [Figure 38] FIG. 1 shows the AMD pathological progression of drusen and laser-induced CNV models. [Figure 39] FIG. 1 shows that OBP-801 suppressed collagen 1 expression 30 days after surgery. [Figure 40] FIG. 1 shows that OBP-801 suppressed the expression of TGFβ2 and SERPINH1 30 days after surgery. [Figure 41] FIG. 1 shows the expression of ECM and ECM-remodeling enzymes. [Figure 42] FIG. 1 shows the expression of inflammatory cytokines and chemokines. [Figure 43] FIG. 1 shows the expression of the TGFβ superfamily. [Figure 44] FIG. 1 shows the expression of transcription factors. [Figure 45] FIG. 1 shows the results of real-time RT-PCR. [Figure 46] FIG. 1 shows the results of real-time RT-PCR. [Figure 47] FIG. 1 shows the results of suppressing intraocular pressure by instillation of OBP-801. [Figure 48] FIG. 1 shows the expression of a group of genes thought to be involved in maintaining intraocular pressure. [Figure 49] FIG. 1 shows WB analysis of rabbit conjunctival tissue. [Figure 50] FIG. 1 shows the results of HE staining of rabbit filtering bleb tissue 30 days after surgery. [Figure 51] FIG. 1 shows the results of immunostaining of rabbit filtering bleb tissue for αSMA expression 30 days after surgery. [Figure 52] FIG. 1 shows the results of immunostaining of rabbit filtering bleb tissue for collagen I expression 30 days after surgery. [Figure 53] FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. [Figure 54] FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. [Figure 55] FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. [Figure 56] FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. DETAILED DESCRIPTION OF THE INVENTION

[0031] 1. Overview The present invention relates to a pharmaceutical composition and method for restoring normal ocular tissue from ocular-related fibrotic tissue and for suppressing the loss of the original function of normal ocular tissue due to fibrosis. To solve the above-mentioned problems, the present inventors have conducted extensive research into (1) the spatiotemporal gene expression patterns associated with pathology in ocular tissue fibrosis models, and have actively sought substances that simultaneously suppress multiple gene groups induced not only by specific localized fibrotic stimuli but also by many other stimuli, including chronic inflammation associated with fibrosis. (3) Furthermore, the selection criteria set out a requirement that the substance be active even under conditions of cellular stress similar to that observed in chronic inflammatory tissues, where the effects of inflammatory cytokines are superimposed in the presence of factors well known to induce fibrosis. This approach was completely unknown in the past.

[0032] Furthermore, hydrophobic compounds that penetrate cells to suppress gene expression have extremely weak in vivo effects in terms of drug delivery unless the effective concentration is extremely low, and therefore cannot be expected to be effective as antifibrotic agents. In fact, as seen in the limitations of SAHA mentioned above, no practically usable antifibrotic agents have been discovered to date. The inventors have focused on this point and have diligently searched for compounds that meet the following strict selection criteria: (4) be effective at an effective concentration one thousandth that of the analogous compounds that have been studied, i.e., be expected to have an administration route that is practically usable in vivo.

[0033] The present invention was completed based on the discovery that compounds satisfying the above conditions (1) to (4) can reduce ECM such as collagen that has accumulated in ophthalmic tissues that are continuously exposed to various fibrotic stimuli in experimental animal models and human ocular tissue-related cells. This is a novel finding, as it has not been previously known that ECM involved in cell sclerosis can be reduced in ocular fibrotic tissues that are chronically exposed to multiple fibrotic stress stimuli.

[0034] Compounds that inhibit tissue fibrosis, which occurs before CNV formation, may enable early treatment before CNV formation and prevent vision loss. Furthermore, they may also be expected to be therapeutically effective against the aforementioned anti-VEGF resistance through their fibrosis-inhibiting effects. The present invention focuses on fibrosis, a functional phase transition triggered by the breakdown of the epigenetic regulatory mechanism of retinal pigment epithelial cells (RPE) during the progression of AMD pathology. The active ingredient of the pharmaceutical composition of the present invention has been found to be effective against choroidal tissue fibrosis, including RPE, at extremely low concentrations and dosages, and is the first compound to comprehensively inhibit multiple genes involved in tissue fibrosis. Furthermore, the active ingredient has been confirmed to inhibit the expression of multiple genes associated with each of the three key stages of pathogenesis, including fibrosis, such as VEGF and PDGF, which are involved in angiogenesis, and LOX, which cross-links collagen and contributes to scar formation. This suggests that the pharmaceutical composition of the present invention may be highly effective in treating a variety of AMD pathologies. Indeed, in CNV model mice, the pharmaceutical composition of the present invention has demonstrated significant inhibitory effects on both early fibrosis and neovascularization.

[0035] Using a mouse model of retinal fibrosis and angiogenesis, the pharmaceutical composition of the present invention has been shown to have novel action properties that go beyond the inhibition of collagen fiber crosslinking and anti-angiogenic effects in the late stage, and also inhibit the early stage of fibroblast-to-myofibroblast transformation after laser irradiation, which correlates with anti-VEGF therapy resistance, demonstrating its superiority over existing therapies. The pharmaceutical properties of this compound are characterized by the simultaneous inhibition of the expression of multiple fibrosis-related genes, such as LOX, THBS1, Serpin, and MMP.

[0036] The first reason for using AMD as a model disease is that the limitations of anti-VEGF antibody therapy as an anti-angiogenic inhibitor have become apparent clinically. This antibody therapy only targets angiogenesis inhibition, which is only one of the phenotypes after the onset or progression of the disease, and is unable to directly inhibit damage (including fibrosis) to photoreceptors and retinal pigment epithelial cells (RPE), which are most important for maintaining a patient's vision. Inhibition of fibrosis by the pharmaceutical composition of the present invention can maintain and restore normal RPE and photoreceptor function.

[0037] Based on the pioneering findings regarding retinal tissue described above, the inventors have further developed part of the present invention, which relates to the inhibition of fibrosis in ocular tissue, into ocular tissues related to glaucoma, and have discovered a technology that is widely applicable to ocular tissues and has excellent practicality.

[0038] 2. Fibrosis inhibitory substance The present invention relates to a pharmaceutical comprising a compound that inhibits fibrosis of multiple ocular tissues, such as retinal tissue and conjunctival tissue. Tissues attempt to repair themselves by accumulating extracellular matrix in response to various stresses on cells, such as oxidative stress, hypoxia, inflammation, and apoptosis. The pharmaceutical composition of the present invention can be used to inhibit such tissue stress and treat diseases caused by fibrosis of ocular tissues, such as retinal and conjunctival tissues. The pharmaceutical composition of the present invention can also be used to inhibit the production of extracellular matrix substances, such as collagen, that are produced by such various stresses on cells.

[0039] Furthermore, the present invention also encompasses substances that suppress fibrotic phase transition of ocular tissue cells as substances that suppress ocular tissue fibrosis. "Fibrotic phase transition of ocular tissue cells" refers to a state in which ocular tissues such as the retina and conjunctiva undergo cellular degeneration due to external or internal cellular stress (due to aging, etc.), resulting in disruption of tissue homeostasis. Cell degeneration includes hard tissue formation due to functional changes in the cells that make up the tissue, such as transition to a fibroblast-like cell morphology, a cellular functional change known as epithelial-mesenchymal transition (EMT), increased apoptosis, abnormal autophagy, increased production of extracellular matrix components, and abnormal cross-linking between proteins such as collagen and elastin that make up the interstitium, and the substances of the present invention suppress these changes.

[0040] Furthermore, as a substance that suppresses fibrosis of ocular tissue, a substance that has a filtering bleb-maintaining effect or an effect of improving the prognosis of glaucoma surgery is also included in the present invention. Pharmaceuticals for treating the above-mentioned pathologies include compounds that suppress collagen and α-SMA production, inhibit TGF or TNF production, inhibit TGF or TNF signaling, and inhibit HDAC. More preferred are compounds that, in an ocular tissue fibrosis model, (1) spatially and temporally alter the expression levels of related genes, (2) simultaneously suppress multiple gene groups in response to numerous fibrotic stimuli, and (3) act under cellular stress similar to that observed in chronic inflammatory tissues, where the effects of inflammatory cytokines are superimposed in the presence of factors well known to induce fibrosis. A depsipeptide compound or a pharmaceutically acceptable salt thereof, more preferably OBP-801 (details described below), is suitable as a compound that (4) has good intracellular penetration in various tissues and exhibits anti-fibrotic and anti-scarring effects at sufficiently low concentrations.

[0041] 3. Substances that suppress the expression of pathological factors In another aspect, the present invention is a pharmaceutical comprising a compound capable of regulating or controlling the expression of a pathology aggravating factor gene. Pathogenesis-aggravating factor genes expressed in mammalian conjunctival tissues, trabecular meshwork cells, retinal pigment epithelial cells, or choroidal neovascularization and choroidal tissue Pathogenesis-aggravating factor genes expressed in human or rabbit conjunctival tissues, trabecular meshwork cells, retinal pigment epithelial cells, or choroidal neovascularization choroidal tissues Fibrosis-related genes in humans or rabbits -Pathogenesis-aggravating genes whose expression changes after glaucoma surgery in humans or rabbits

[0042] The pathology-aggravating genes include fibrosis-inducing genes, angiogenesis-related genes, scarring-related genes, and ECM-related genes. Controlling the expression of these genes and suppressing fibrosis and ECM accumulation in ocular tissues can be expected to maintain filtration blebs, maintain low intraocular pressure, inhibit angiogenesis, and inhibit scar formation. For example, in ocular tissues, the expression of at least one pathology-aggravating gene associated with each of the three stages of fibrosis, angiogenesis, and scar formation can be inhibited in vivo. Examples of pathology-aggravating genes include collagen 1A, collagen 3A1, collagen 4A1, TIMP 2, TIMP 3, TIMP 4, thrombospondin 1, thrombospondin 2, LOX, Lox12, TGFb2, TGFb3, CTGF, VEGF, PDGF, and Serpin, and the expression of these genes can be controlled by the pharmaceutical composition of the present invention.

[0043] The present invention also relates to a pharmaceutical composition that inhibits the expression of at least one gene among the genes for exacerbating factors associated with each stage, and that has an inhibitory effect on fibrosis and / or angiogenesis, as well as inhibits at least two stages of scar formation in vivo in ocular tissue cells. The present invention also includes a substance that regulates the activity of a hub gene involved in the expression of a gene related to aggravating factors.

[0044] A more preferred form of such a substance is a depsipeptide compound, and even more preferably, OBP-801. OBP-801 has been found to be the first compound to demonstrate a comprehensive inhibitory effect on multiple genes involved in tissue fibrosis. Furthermore, OBP-801 has been confirmed to have an inhibitory effect on the expression of multiple genes, not only fibrosis but also angiogenesis-related genes such as VEGF and PDGF, as well as LOX, which cross-links collagen and is involved in scar formation. Therefore, it is expected to have a high therapeutic effect on the diverse pathologies of AMD patients.

[0045] To inhibit the expression of the above genes, inhibitory nucleic acids against these genes, such as antisense nucleic acids, decoy nucleic acids, microRNA, shRNA, or siRNA, can also be used. The nucleotide sequences of the genes to be inhibited are known, and sequence information for each gene is available. The GenBank accession numbers for each gene are listed below.

[0046] COL1A1: NM_000088 COL4A2: NM_001846 COL16A1: NM_001856 ITGA2: NM_002203 ITGA5: NM_002205 ITGB3: NM_000212 ITGAV: NM_002210 LAMA1: NM_005559 VCAN: NM_004385 TIMP1: NM_003254 CTGF: NM_001901

[0047] The pathological significance of inhibiting LOX expression is known to be antifibrotic chemotherapy. It has been reported that LOX and LOX2 gene expression levels increase during scar formation after CNV caused by laser irradiation surgery, and that the introduction of antibodies against these two genes can suppress fibrosis.

[0048] It has also been reported that administration of anti-LOX and anti-LOXL2 antibodies significantly reduces the transcription level of type I collagen A-1 (COL1A1), and that expression of LOX and LOX2 genes is enhanced in the Tenon's capsule and conjunctiva after glaucoma surgery.

[0049] 4. Substances that maintain filtering blebs In another aspect, the present invention is a medicament containing a compound having a filtering bleb-maintaining effect. After glaucoma surgery, wound healing occurs in glaucoma-related and conjunctival tissues due to postoperative inflammation, resulting in fibrosis or scarring of trabecular meshwork cells (HTMC) and conjunctival fibroblasts (HconF). It is known that after glaucoma surgery, conjunctival fibroblasts undergo fibrotic stimulation by TGF+TNF, which interferes with the maintenance of the filtering bleb. During the wound healing process in the subconjunctival tissue of the filtering bleb after surgery, a series of healing processes, including the inflammatory, proliferative, and scarring phases, are observed, resulting in morphological and functional changes.

[0050] In rabbits undergoing glaucoma surgery, morphological changes such as localized avascularity in the filtering bleb are observed in the anterior segment. During this process, increased expression of fibrosis-related genes, scarring-related genes, and factors related to increased ECM are observed. These genes include collagen 1A, collagen 3A1, collagen 4A1, TIMP 2, TIMP 3, TIMP 4, thrombospondin 1, thrombospondin 2, LOX, Lox12, TGFb2, TGFb3, CTGF, VEGF, PDGF, and Serpin. By controlling the expression of these genes, fibrosis of conjunctival fibroblasts is suppressed and the morphology and function of the filtering bleb are maintained.When the normal morphology and function of the filtering bleb are maintained, intraocular pressure can be maintained at a normal level.

[0051] 5. Substances that improve the prognosis of glaucoma surgery In another aspect, the present invention is a pharmaceutical comprising a compound having an effect of improving the prognosis of glaucoma surgery. Glaucoma is a disease characterized by functional and structural abnormalities, with characteristic changes in the optic nerve and visual field. Sufficient reduction of intraocular pressure usually improves or prevents optic nerve damage.

[0052] To treat this disease, the following surgical treatments are usually performed: Early surgery includes outflow reconstruction surgery such as trabeculotomy, filtration surgery such as trabeculectomy, and implant insertion (with or without a plate) for treating glaucoma, which are usually performed to restore the structure of the ocular tissue to a normal state.

[0053] Trabeculectomy is a method of reconstructing an artificial aqueous humor outflow pathway by absorbing aqueous humor from the conjunctiva and allowing it to evaporate from the surface. This procedure is highly invasive and counters the body's natural healing process, placing persistent stress on biological tissues, leading to delayed wound healing, delayed reduction in intraocular pressure, and often making intraocular pressure control difficult. The disadvantages of TLE are that the filtering bleb tends to become localized and avascular. In these situations, long-term intraocular pressure control, healthy bleb formation, and reduced risk of bleb infection are desired. TLE, which involves widespread bleb formation, maintains the vascularity of the conjunctiva, and provides long-term intraocular pressure reduction after surgery, are desired. Glaucoma surgery options include Ex-PRESS, INNFOCUS, Baerveldt Glaucoma Implant, Ahmed Glaucoma Valve, XEN Implant, and Hydrus Microstent.

[0054] 6. Compound The present invention provides a pharmaceutical composition comprising a depsipeptide compound represented by the following formula I or II or a pharmaceutically acceptable salt thereof.

[0055] Formula I: [ka] or the following formula II: [ka] (In the formula, R1 to R3 independently represent a hydrogen atom, a methyl group, or an ethyl group; R4 independently represent a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group, or an isobutyl group; R5 to R8 independently represent a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 independently represent a hydrogen atom, a methyl group, or a protecting group; and R10 and R11 independently represent a hydrogen atom, a methyl group, or a protecting group.)

[0056] The present invention also provides a compound of formula III: [ka] (wherein R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group). Also provided is a pharmaceutical composition comprising a depsipeptide compound represented by the formula: or a pharmaceutically acceptable salt thereof. Among the compounds shown in the above formula III, those in which R4 is an isopropyl group are preferred. OBP-801 is a compound in which R4 is an isopropyl group.

[0057] 7.OBP-801 A preferred embodiment is a pharmaceutical containing OBP-801 as the above-mentioned substance that inhibits fibrosis, inhibits the expression of genes that exacerbate pathology, has a substance that has a filtering bleb-maintaining effect, is used in medical treatment to inhibit the progression of pathology in AMD patients, prevents pathology that begins with neovascularization in the choroid and leads to blindness, and has the effect of improving the prognosis of glaucoma surgery.

[0058] OBP-801 is the first compound to demonstrate comprehensive inhibitory effects on multiple genes involved in tissue fibrosis. Furthermore, OBP-801 has been confirmed to have inhibitory effects on the expression of multiple genes, including VEGF and PDGF, which are involved in angiogenesis, and LOX, which cross-links collagen and is involved in scar formation, in addition to fibrosis. This suggests that OBP-801 is expected to have a high therapeutic effect on the diverse pathologies of AMD patients.

[0059] OBP-801 has a stronger anti-fibrotic effect than SAHA and may be able to suppress postoperative fibrosis more effectively. Furthermore, OBP-801 is one of the compounds that penetrates into cells and exhibits anti-fibrotic effects at concentrations low enough to exert its effect. The present invention has revealed that OBP-801 is effective at a concentration 1 / 1000th that of SAHA. Because OBP-801 is effective at lower concentrations than SAHA, there is no risk of toxicity and it may be safer in terms of protecting the conjunctiva. The method for producing this compound is in accordance with a known method (Patent Document 1). The embodiments also include the above patent document.

[0060] 8. Pharmaceutical Compositions (1) Preparation The pharmaceutical composition of the present invention can be used in the form of eye drops, topical applications, sustained-release preparations, inserts, injections, ointments, and the like.

[0061] Eye drops can be prepared using an isotonic agent such as sodium chloride or concentrated glycerin; a buffering agent such as sodium phosphate or sodium acetate; a surfactant such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate or polyoxyethylene hydrogenated castor oil; a stabilizer such as sodium citrate or sodium edetate; or a preservative such as benzalkonium chloride or paraben, and the pH should be within the range acceptable for ophthalmic preparations, preferably in the range of 4 to 8. Eye ointments can be prepared using commonly used bases such as white petrolatum or liquid paraffin.

[0062] In the present invention, the compound or a pharmaceutically acceptable salt thereof can be used in the form of a gel, cream, or lotion. For example, it can be formulated for local or topical application to the eye, skin, and mucous membranes, or for application to the eye. The topical pharmaceutical composition can be in any form, including, but not limited to, a solution, cream, ointment, gel, lotion, emulsion, cleanser, moisturizer, spray, skin patch, etc. Solutions are formulated with appropriate salts as 0.01% to 10% isotonic solutions, pH 5 to 7. The compounds of the present invention or pharmaceutically acceptable salts thereof can also be formulated as transdermal patches for transdermal administration.

[0063] In the present invention, topical pharmaceutical compositions containing the compounds or pharmaceutically acceptable salts thereof can be mixed with a variety of carrier materials known in the art, such as, for example, water, alcohol, aloe vera gel, allantoin, glycerin, vitamin A and E oil, mineral oil, propylene glycol, PPG-2 myristyl propionate, etc. Other materials suitable for use in topical carriers include, for example, emollients, solvents, humectants, thickeners, and powders. Examples of each of these types of materials, which can be used alone or in combination with one or more materials, are as follows:

[0064] Representative topical or skin ointment agents include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecane-2-ol, isocetyl alcohol, cetyl palmitate, dimethylpoly Siloxanes, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, peanut oil, castor oil, acetylated lanolin alcohol, petroleum oil, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, and myristyl myristate; propellants, such as propellants propane, butane, isobutane, dimethyl ether, carbon dioxide, and nitrous oxide; solvents such as ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran; humectants such as glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, and gelatin; and powders such as chalk, talc, fuller's earth, kaolin, starch, gum, colloidal silicon dioxide, sodium polyacrylate, tetraalkylammonium smectite, trialkylarylammonium smectite, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethylcellulose, and ethylene glycol monostearate.

[0065] Sustained-release or intercalated formulations can be prepared by grinding and mixing the compound with a biodegradable polymer, such as hydroxypropyl cellulose, hydroxypropylmethylcellulose, carboxyvinyl polymer, or polyacrylic acid, and then compressing the resulting powder. If necessary, excipients, binders, stabilizers, and pH adjusters can be used. Intraocular implant formulations can be prepared using biodegradable polymers, such as polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, and hydroxypropyl cellulose.

[0066] Injectable preparations can be prepared using an agent selected as needed from among isotonic agents such as sodium chloride; buffering agents such as sodium phosphate; surfactants such as polyoxyethylene sorbitan monooleate; and thickeners such as methylcellulose. The compositions of the present invention may take the form of a liquid, such as a solution, emulsion or suspension, or a semi-solid, such as a gel, eye ointment, etc.

[0067] Diluents for aqueous solutions and suspensions include distilled water and physiological saline. Diluents for non-aqueous solutions and suspensions include vegetable oil, liquid paraffin, mineral oil, propylene glycol, p-octyldodecanol, and the like. To achieve isotonicity with tears, isotonicity agents such as sodium chloride, boric acid, and sodium citrate can be added, and buffers such as boric acid, buffer solutions, and phosphate buffer solutions can be added to maintain a constant pH, for example, between about 5.0 and 8.0. Furthermore, stabilizers such as sodium sulfite and propylene glycol, chelating agents such as sodium edetate, thickeners such as glycerin, carboxymethylcellulose, and carboxyvinyl polymers, and preservatives such as methylparaben and propylparaben can be added. These can be sterilized, for example, by filtration through a bacteria-retaining filter or by heat sterilization.

[0068] Eye ointments are based on petrolatum, selenium 50, plastibase, macrogol, etc., and surfactants can be added to enhance hydrophilicity. They may also contain gelling agents such as carboxymethylcellulose, methylcellulose, and carboxyvinyl polymers.

[0069] 9. Dosage and Administration The dosage of the present compound can be varied as appropriate depending on the dosage form, the severity of the symptoms of the patient to be administered, their age, body weight, the doctor's judgment, etc., but in the case of eye drops, the daily dosage for an adult is generally as follows: Injection: 10nM 200μl subconjunctival injection once daily (days 0, 1, 3, and 5) For eye drops or inserts: 100 nM, 80 μl, twice daily (days 0, 1, 2, 3, 4, 5, 6, and 7)

[0070] Furthermore, the pharmaceutical composition of the present invention can be used in the following dosage regimens, for example, in the case of OBP-801, at 2 pg / eye to 9000 pg / eye or 100 pg / kg to 3000 pg / kg, depending on the form of conjunctival injection or eye drops.

[0071] - In the case of suppressing conjunctival fibrosis (conjunctival injection) When the OBP-801 concentration is 10 nM and the solution volume is 200 μL, the amount of OBP-801 to be injected is 100 pg / kg to 3000 pg / kg, preferably 100 pg / kg to 500 pg / kg, more preferably 200 pg / kg to 400 pg / kg, and even more preferably 315 pg / kg, and is administered by subconjunctival injection 30 minutes before surgery and on days 1, 3, and 5 after surgery (a total of four administrations).

[0072] Alternatively, a single dose of 2 pg / eye to 9000 pg / eye, preferably 2 pg / eye to 1500 pg / eye, more preferably 4 pg / eye to 1200 pg / eye, and even more preferably 944 pg / eye, is administered by subconjunctival injection 30 minutes before surgery and on days 1, 3, and 5 after surgery (a total of four administrations).

[0073] - Inhibition of conjunctival fibrosis (eye drops) When the OBP-801 concentration is 100 nM and the liquid volume is 80 μl, the amount of OBP-801 injected is 100 pg / kg to 3000 pg / kg, preferably 2000 pg / kg to 3000 pg / kg, more preferably 2500 pg / kg to 3000 pg / kg, and even more preferably 2517 pg / kg, and is administered 15 times in total, in the morning and evening, 30 minutes before surgery and on days 1, 2, 3, 4, 5, 6, and 7 after surgery.

[0074] Alternatively, a single dose of 6 pg / eye to 27,000 pg / eye, preferably 5,000 pg / eye to 10,000 pg / eye, more preferably 7,000 pg / eye to 8,000 pg / eye, and even more preferably 7,552 pg / eye, is administered 15 times in total, in the morning and evening, 30 minutes before surgery and on days 1, 2, 3, 4, 5, 6, and 7 after surgery.

[0075] For CNV suppression (intravitreal injection): When the OBP-801 concentration is 10 nM and the liquid volume is 0.5 μl, the amount of OBP-801 to be injected is 100 pg / kg to 3000 pg / kg, preferably 100 pg / kg to 500 pg / kg, more preferably 100 pg / kg to 200 pg / kg, and even more preferably 118 pg / kg, administered once, for a total of 1 to 10 times per day. Alternatively, the dose is 2 pg / eye to 9000 pg / eye, preferably 2 pg / eye to 1500 pg / eye, more preferably 2 pg / eye to 10 pg / eye, and even more preferably 2.36 pg / eye, administered 1 to 10 times in total per day.

[0076] For ocular tissue culture cells: It inhibits the fibrotic phase transition of ocular tissue culture cells at concentrations of 10 nM or less, and its inhibitory effect on HDAC activity in ocular tissue cells is at IC50 = 10 nM or less.

[0077] 10. Target (1) Inhibition of ocular tissue fibrosis Definition: Ocular tissues: conjunctiva-related tissues, glaucoma-related tissues, retina-related tissues, corneal tissue, conjunctival tissue, scleral tissue, lens tissue, trabecular meshwork tissue, retinal choroid tissue, optic nerve tissue, vitreous tissue Fibrosis: Cell proliferation of conjunctival fibroblasts (HconF) and trabecular meshwork cells (HTMC) progresses, and excessive expression of fibrosis-inducing genes such as TGF and TNF, as well as col1, col3, col4, and col16, causes abnormal cross-linking of the extracellular matrix. As a result, the physiological functions of the cells and the stroma are impaired.

[0078] Types of inhibitors: anti-TGFb2 antibodies, siRNA (anti-TGFb), siRNA (anti-TGFb2 receptor), tranilast, genistein, suramin, angiotensin-converting enzyme inhibitors, chymase inhibitors, Smad7 gene transfer, ROCK (Rho-associated kinase) inhibitors, decorin, ribozymes, Aptamaers (ARC 126 and ARC 127), adenovirus-mediated dominant negative p38MAPK gene, Simvastatin, HMG-CoA reductase inhibitor, Lovastatin, Follistatin, MMC-containing hydrogel, 5FU sustained-release agent, Paclitaxel, Bleomycin, Thiotepa (alkylating agent), Retinoic acid and its derivatives (vitamin A), IFN-α, Lectin, Saporin, Cytostatic gene p21, Bevacizumab, Ranibizumab, MMC, Steroids, NSAIDS, 5FU, Anti-VEGF antibody, Anti-LOXL1 antibody, Anti-LOX antibody

[0079] (2) Glaucoma-related organizations Glaucoma-related tissues include tissues composed of the trabecular meshwork and trabecular meshwork cells (HTMC), which are capable of controlling intraocular pressure by regulating the flow of aqueous humor. Types of glaucoma-related tissues: trabecular meshwork, Schlemm's canal, collecting duct, episcleral vein

[0080] (2-1) Trabecular Meshwork The trabecular meshwork is located in the outflow path of aqueous humor that accumulates in the anterior chamber of the eye, and plays a role in filtering it. To maintain normal aqueous humor dynamics, the total volume of aqueous humor is approximately 0.3 mL, and the aqueous humor is exchanged every 1-2 hours, providing nutrients to avascular tissue, transporting waste products, and maintaining homeostasis of intraocular pressure.

[0081] (2-2) Tissue capable of controlling intraocular pressure Intraocular pressure refers to the pressure of the intraocular fluid that fills the eyeball. It is slightly higher than atmospheric pressure, and the difference between this and atmospheric pressure is expressed as the intraocular pressure value. It is expressed in mmHg. Intraocular pressure is controlled by the amount of aqueous humor circulating in the front of the eyeball. Aqueous humor is produced by the ciliary body and flows through the gap between the iris and lens (the posterior chamber) into the space just below the cornea (the anterior chamber). It then passes through the trabecular meshwork in the angle at the base of the cornea and iris, before being drained by the Schlemm's canal. If this flow is blocked and the amount of aqueous humor increases, intraocular pressure rises. Intraocular pressure of 21 mmHg or higher is considered high. However, it is known that Japanese people have a high incidence of normal-tension glaucoma, in which the optic disc is damaged even when the intraocular pressure is lower than this.

[0082] (3) Conjunctival-related tissues The conjunctival tissue is composed of the filtering bleb tissue, the connective tissue surrounding the filtering bleb, and aqueous humor tissue. Its function is to absorb aqueous humor from the conjunctiva and evaporate it from the surface, while at the same time forming an artificial aqueous humor outflow pathway, thereby controlling the outflow of water within the eye and maintaining normal intraocular pressure. Types of conjunctival-related tissues include the conjunctival epithelium, the lamina propria mucosa, Tenon's capsule, the episclera, and the sclera.

[0083] (4) Retina-related tissues The retina, located inside the choroid, is a thin membrane of over 100 million photoreceptor cells, measuring 0.2–0.5 mm. It is considered the most important part of the retina for perceiving light and color and for seeing. The macula is the area of ​​the retina where light entering through the pupil strikes the front of the fundus, appearing slightly yellower than the surrounding retina. The macula also has a central area called the fovea, which is slightly thinner than the surrounding retina. It is densely packed with cone cells and lacks blood vessels, making it the most sensitive point of vision. The optic disc, located slightly medial to the macula (toward the nose), is the area where nerve fibers connecting to the retinal photoreceptors converge. Light information received by the retina leaves the eyeball and is sent to the brain, where it becomes an image. The optic disc is also the hub of retinal blood vessels, from which retinal arteries and veins extend throughout the retina.

[0084] (4-1) Retinal pigment epithelium The retinal pigment epithelium (RPE) is a single-layer epithelial cell located in the outermost layer of the retina (the 10 layers). Its distal end, called the outer segment, is constantly phagocytosed by the RPE and replaced with new ones. The RPE is capable of phagocytosing photoreceptors and regenerating visual pigments (such as retinal), forming the blood-retinal barrier. It is also the primary lesion in age-related macular degeneration (AMD). Photoreceptors are one of the cells that make up the retina. The RPE is a photoreceptor that converts light energy into electrical energy. Fibrosis of the RPE tissue is a serious condition common to intraocular proliferative diseases, and a common treatment, namely, fibrosis prevention, can be used to target multiple diseases. Age-related macular degeneration (AMD), proliferative vitreous band retinopathy, and proliferative diabetic retinopathy (PDR) are all age-related diseases and are thought to be pathological conditions resulting from acquired epigenetic changes. One of these is fibrosis of the retinal pigment epithelium (RPE). Posterior stromal fibrosis leads to poor visual prognosis, and the condition progresses to malignancy, leading to a functional phase transition of cells. Subsequently, as cellular senescence of the retinal pigment epithelium (RPE) progresses, epithelial-mesenchymal transition leads to fibrosis, eventually resulting in age-related macular degeneration.

[0085] (4-2) Choroidal neovascularization Light entering the eye passes through the cornea, lens, and vitreous humor to form an image on the retina at the back of the eye. The macula is located in the center of the retina. The macula is the part of the retina where important cells responsible for vision are concentrated, and it is where most of the information from light, such as the shape, size, color, three-dimensionality, and distance of objects, is identified.

[0086] New, pathological, fragile blood vessels (choroidal neovascularization) form behind the macula, leaking blood and exudates into the fundus. As a result, the macula degenerates and becomes damaged, impairing central vision and resulting in a decrease in visual acuity. Vascular endothelial growth factor (VEGF) is closely involved in the development and growth of these pathological choroidal neovascularization.

[0087] (4-3) Tissues that progress to age-related macular degeneration Tissues that develop age-related macular degeneration include retinal-related tissues, the retina that forms the blood-retinal barrier, the retinal pigment epithelium, and photoreceptors, which are cells that make up the retina. The macula is located in the center of the retina, where important cells that control vision are concentrated. Behind the macula, pathologically fragile blood vessels (choroidal neovascularization) are newly formed, from which blood leaks and overflows into the fundus. As a result, the macula develops the disease due to degeneration or damage, impairing central vision and resulting in symptoms such as decreased vision.

[0088] (5) Target disease Since the pharmaceutical composition of the present invention targets the above-mentioned tissues, the diseases to which the pharmaceutical composition of the present invention is administered can be any of diseases associated with fibrosis, diseases associated with inflammation, and diseases associated with angiogenesis.

[0089] Typical examples include intraocular inflammatory diseases such as glaucoma, diabetic macular edema (DME), age-related macular degeneration (AMD) (particularly exudative or non-exudative age-related macular degeneration), cataract, infectious or non-infectious uveitis, scleritis, corneal surgery, non-infectious keratitis, iritis, chorioretinal inflammation, inflammatory diseases that damage the retina of the eye, and retinopathies, particularly diabetic retinopathy, arterial hypertension-induced hypertensive retinopathy, radiation-induced retinopathy, sunlight-induced solar retinopathy, trauma-induced retinopathies, e.g., Purcher's retinopathy, retinopathy of prematurity (ROP), and hyperviscosity-associated retinopathy. Further examples of target diseases include intraocular inflammation after anterior / posterior eye surgery, such as cataract surgery, laser eye surgery, glaucoma surgery, refractive surgery, corneal surgery, vitreoretinal surgery, ocular muscle surgery, ocular plastic surgery, ocular tumor surgery, conjunctival surgery including pterygium, and / or surgery involving the lacrimal apparatus, particularly after complicated eye surgery, surgery after trauma, and / or uncomplicated eye surgery. Diseases of the ocular tissue in particular include uveitis, in particular anterior, intermediate and / or posterior uveitis, sympathetic uveitis and / or panuveitis; general scleritis, in particular anterior scleritis, marginal scleritis, posterior scleritis and scleritis with corneal disorders; general episcleritis, in particular transient periodic episcleritis and nodular episcleritis; retinitis; corneal surgery; mucopurulent conjunctivitis, atopic conjunctivitis, toxic conjunctivitis, pseudomembranous conjunctivitis, serous conjunctivitis, chronic conjunctivitis, giant papillary conjunctivitis, follicular conjunctivitis, vernal conjunctivitis, blepharoconjunctivitis and / or pingueculitis; general non-infectious keratitis, in particular keratoconjunctivitis; Membrane ulcers, superficial keratitis, macular keratitis, filamentous keratitis, snow blindness, punctate keratitis, e.g., dry eye syndrome (keratoconjunctivitis sicca), neurotrophic keratoconjunctivitis, nodular ophthalmia, phlyctenular keratoconjunctivitis, vernal keratoconjunctivitis and other keratoconjunctivitis, interstitial conjunctivitis and deep keratitis, sclerosing keratitis, corneal neovascularization and other keratitis; general iridocyclitis, in particular acute iridocyclitis, subacute iridocyclitis and chronic iridocyclitis, primary iridocyclitis, recurrent iridocyclitis and secondary iridocyclitis, phacogenic iridocyclitis, Fuchs heterochromic cyclitis, Vogt-Koyanagi syndrome; iritis;Chorioretinal inflammation in general, in particular focal chorioretinal inflammation and disseminated chorioretinal inflammation, chorioretinitis, choroiditis, retinitis, retinochoroiditis, posterior cyclitis, Harada's disease, chorioretinal inflammation in infectious and parasitic diseases; postoperative inflammation of the eye after anterior and / or posterior surgery, for example after cataract surgery, laser eye surgery (e.g. laser in situ keratotomy (LASIK)), glaucoma surgery, refractive surgery, corneal surgery, vitreoretinal surgery, ocular muscle surgery, ocular plastic surgery, ocular tumor surgery, conjunctival surgery including pterygium, and surgery involving the lacrimal apparatus, preferably intraocular inflammation, in particular postoperative intraocular inflammation, preferably after complex and / or non-complicated eye surgery, for example inflammation of the bleb after a procedure; inflammatory diseases damaging the retina of the eye; retinal vasculitis, in particular eel retinal perivascular inflammation; general retinopathies, in particular diabetic retinopathy, (arterial hypertension-induced) hypertensive retinopathy, exudative retinopathy, radiation-induced retinopathy, sunlight-induced solar retinopathy, trauma-induced retinopathies, e.g., Purcher's retinopathy, retinopathy of prematurity (ROP) and / or hyperviscosity-associated retinopathy, non-diabetic proliferative retinopathy, and / or proliferative vitreoretinopathy; blebitis; endophthalmitis; sympathetic These include, but are not limited to, ophthalmia; hordeolum; chalazion; blepharitis; eyelid dermatitis and other inflammations; dacryoadenitis; lacrimal ductitis, especially acute and chronic canaliculitis; dacryocystitis; orbital inflammation, especially orbital cellulitis, orbital periostitis, orbital Tenon's capsuleitis, orbital granuloma and orbital myositis; inflammatory and non-inflammatory eye diseases selected from suppurative endophthalmitis and parasitic endophthalmitis;

[0090] Example The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0091] [Example 1] (In vivo study on the effect of maintaining filtering blebs after trabeculectomy) 1. Method Cannula-based glaucoma filtration surgery rabbit model, OBP-801 administration, observation OBP-801 was diluted with ocular irrigation fluid (BSS: Balanced Salt Solution) to prepare a pharmaceutical composition for administration to the treatment group.

[0092] After topical anesthesia, domestic rabbits (Japanese white, female, weighing 2.5-2.99 kg) were given intraocular irrigation solution, MMC, or OBP-801 subconjunctival injection 30 minutes later. Under general anesthesia (intramuscular injection of ketamine (50 mg / kg) and xylazine (10 mg / kg)), 6-0 nylon was sutured to the cornea at 12 o'clock and pulled to expose the surgical field. A fornix-based conjunctival flap was created, and the subconjunctival tissue and sclera were bluntly dissected 15 mm posterior to the limbus to expose the sclera. A semi-thickness scleral tunnel was created 4 mm posterior to the limbus using an MVR Lance (20 g) until the corneal stroma in the anterior chamber was visible. A venous cannula was inserted into the scleral tunnel and inserted into the anterior chamber.

[0093] After suturing and securing the cannula to the sclera, the conjunctiva was sutured, and atropine and Rinderon A ointment were injected to complete the surgery. Postoperatively, topical antibiotics and steroids were administered. Subconjunctival administration of OBP-801 was performed on days 1, 3, and 5 after surgery. From days 1 to 30, the animals were examined every 2–3 days, according to postoperative examinations, to assess the degree of ocular inflammation, anterior chamber depth, and conjunctival bleb characteristics, as well as to measure bleb size and intraocular pressure. Furthermore, the animals were euthanized without pain by administering an overdose of anesthetics, and the eyeballs were then enucleated and the effects of intraocular perfusate, MMC, and OBP-801 on bleb maintenance were examined histologically. If any abnormalities, such as rapid weight loss, were observed, the animals were euthanized by administering an overdose of pentobarbital, marking the humane endpoint. The above-described one-month experiment constituted one cycle.

[0094] ◆Intraocular pressure measurement method Both eyes were measured three times using the Tonovet, and the median value was used as the measurement value. Western blotting Tissue samples frozen in liquid nitrogen were ground in a mortar and pulverized in RIPA buffer (plus protease inhibitor) at a ratio of 30 μl per 10 mg of tissue. Subsequently, the tissue was lysed by vortexing and sonication (5 min, 30-second intervals) with rotation at 4°C for several hours. The remaining tissue was precipitated at 10,000 xg for 20 min at 4°C, and the supernatant was collected. For protein quantification (BCA kit), the samples were electrophoresed on SDS-page gel at 30 μg / lane using iBlot 4-12% Bis-Tris Plus Gel and transferred to a PVDF membrane using iBlot PVDF Transfer Stack Regular. Blocking and antibody reactions using the iBind Western System were detected using ECL chemiluminescence (NOVEX ECL CHEMISUBSTRATE) on a Fujifilm LAS3000.

[0095] ◆Tissue staining Cryostat sections (10 μm thick) were coated with 2% silane (3-aminopropyltriethoxysilane) and then mounted on glass slides. They were fixed in cold methanol (-30°C) for 15 minutes and then air-dried. Blocking was then performed using a reaction solution containing 1% bovine albumin at room temperature for approximately 30–60 minutes. After incubation with the primary antibody overnight at 4°C and 60 minutes at room temperature for the secondary antibody, the sections were mounted in VECTASHELS with DAPI. After sealing with nail polish, the sections were stained with picrosirius red and observed under a fluorescence microscope.

[0096] 2.Results [Pharmacological effects of OBP-801] (1) Maintenance of low intraocular pressure by OBP-801 administration In both the OBP-801 and BSS groups, a decrease in intraocular pressure was observed immediately after surgery. In the OBP-801 group, intraocular pressure remained lower than before surgery 30 days after surgery (Figures 1 and 2). On the other hand, in the BSS group, intraocular pressure increased from 15 days after surgery, and intraocular pressure could not be maintained low for a long period of time compared to the OBP-801 group (Figures 1 and 3).

[0097] (2) Suppression of conjunctival congestion in filtering blebs On the 14th day after surgery, severe conjunctival congestion was observed in the area of ​​the filtering bleb in the BSS-treated group, whereas conjunctival congestion in the filtering bleb was mild in the OBP-801-treated group (Figure 4).

[0098] (3) Number of OBP-801 doses Even when the number of OBP-801 administrations was reduced from six (just before surgery, and 1, 3, 5, 7, and 9 days after surgery) to four (just before surgery, and 1, 3, and 5 days after surgery), the same level of low intraocular pressure was maintained up to 30 days after surgery (Figure 5).

[0099] (4) Timing of OBP-801 administration OBP-801 was able to maintain low intraocular pressure up to 30 days after surgery even when administered only during the efficacy phase (3, 5, and 7 days after surgery) (Figure 6). Even lower intraocular pressure could be maintained by administering the drug perioperatively (just before surgery and 1 day after surgery) in addition to administering it only during the efficacy phase (3, 5, and 7 days after surgery) (Figure 6).

[0100] (5) Dose of OBP-801 Subconjunctival injection of 200 μl of 10 nM OBP-801 maintained a lower intraocular pressure than administration of the same amount of 1 μM or 100 μM OBP-801 (FIG. 7).

[0101] (6) Administration method It was shown that a lower intraocular pressure was maintained when an appropriate dose (200 μl of 10 nM OBP-801 administered subconjunctivally) was administered multiple times compared to when a single dose was increased (100 μl of 100 μM OBP-801 administered subconjunctivally) (Figure 8).

[0102] [OBP-801 action characteristics] (7) Relationship between intraocular pressure reduction and αSMA expression In three rabbits in which a decrease in intraocular pressure was observed 14 days after surgery, the expression level of αSMA in the filtering bleb tissue was examined using immunohistochemistry. The expression level of αSMA varied from individual to individual, suggesting that there was little correlation between the intraocular pressure-lowering effect and the expression level of αSMA (Figure 9).

[0103] (8) Relationship between intraocular pressure reduction and collagen expression 14 days after surgery The expression levels of type I collagen and type III collagen fibers were analyzed using picrosirius red staining. The rabbits in which a decrease in intraocular pressure was observed 14 days after surgery and those in which a decrease was not observed showed similar levels of collagen expression, so the relationship between a decrease in intraocular pressure and the amount of collagen expression 14 days after surgery is unclear (Figure 10).

[0104] (9) Relationship between intraocular pressure reduction and collagen expression 30 days after surgery The expression levels of type I collagen and type III collagen fibers were analyzed using picrosirius red staining. At 30 days, rabbits with and without a decrease in intraocular pressure showed low collagen expression levels in the filtering bleb, while those without a decrease in intraocular pressure showed high collagen expression levels, suggesting a correlation between intraocular pressure and collagen expression levels (Figure 11).

[0105] [Example 2] (In vitro study of the effect of maintaining filtering blebs after trabeculectomy) 1. Method ◆Cell culture, OBP-801 addition (including pulse), observation Conjunctival fibroblasts (HconF) (P1) were purchased from ScienCell (#6570). ◆Cultivation (Cultivated according to the procedure): Culture medium: Fibroblast Medium (#2301) supplemented with the accompanying medium additives, antibiotics, and FBS. 5x10 cells in a poly-l-lysine coated culture vessel 3 cells / cm 2 The seeds were sown in. All experiments were performed at passage number (P) 3.

[0106] ◆ Chemical treatment OBP-801 was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 μM to prepare a stock solution, which was then diluted with the medium to prepare a pharmaceutical composition to be administered to the treatment groups. When the cells reached 80-90% confluence, the medium was changed to one without FBS and OBP-801 (0-5 nM) was added. After 0-24 hours, TGFβ (20 ng / ml) and TNFα (10 ng / ml) were added and the cells were exposed for 24 hours.

[0107] Observation Live cells were observed under a phase-contrast microscope. ◆Immunostaining After removing the medium, the sections were washed with PBS for 5 minutes three times and then fixed in cold methanol (-30°C) for 15 minutes. They were then air-dried and blocked with a reaction solution containing 1% bovine albumin for approximately 30-60 minutes at room temperature. Primary antibody reactions were performed overnight at 4°C with PBS, followed by 5-minute washes three times, and secondary antibody reactions were performed at room temperature for 60 minutes. Nuclei were stained with 5 μg / ml DAPI at room temperature for 15 minutes, followed by 5-minute washes three times with PBS, and then observed under a fluorescence microscope with PBS in place.

[0108] Western blotting Extraction reagent: 50 μl of SDS-HBS (1% SDS, 150 mM NaCl in 10 mM Hepes (pH 7.4)) was used. The mixture was boiled for 3 minutes in a 35-mm dish and sonicated for 5 minutes (15-second, 10-second pause). Protein quantification (BCA kit) was performed using SDS-page electrophoresis (30 μg / lane) with iBlot 4-12% Bis-Tris Plus Gel. Protein was then transferred to a PVDF membrane (iBlot PVDF Transfer Stack Regular). After blocking and antibody reaction (iBind Western System), the protein was detected by ECL chemiluminescence (NOVEX ECL CHEMI SUBSTRATE) on a Fujifilm LAS3000.

[0109] ◆HDAC activity measurement Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002). The EpiQuik HDAC Activity / Inhibition Direct Assay Kit (Epigenetic #P-4034) was used. After reacting the sample with the acetylated histone substrate coated on the plate, the undeacetylated substrate was detected with an anti-acetylated histone antibody.

[0110] ◆HAT activity measurement Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002). The EpiQuik HAT Activity / Inhibition Direct Assay Kit (Epigenetic #P-4003) was used. After the sample was reacted with the deacetylated substrate coated on the plate, the acetylated substrate was detected with an anti-acetylated antibody.

[0111] ◆PCR array The following procedures were carried out in accordance with the kit protocol. RNA extraction: RNeasy mini kit (QIAGEN #74104) Reverse transcription: RT2 First Strand Kit (QIAGEN #330401) PCR reaction: RT 2 SYBR Green ROX qPCR Mastermix (QIAGEN #330522) RT 2 Profiler TM PCR Array Rabbit Fibrosis] (QIAGEN #PANZ-120ZC)

[0112] 2.Results Drug properties of OBP-801 (1) Inhibition of HconF-induced myofibrillation by OBP-801 In conjunctival fibroblasts (HconF), stimulation with TGF+TNF was shown to induce the expression of αSMA and Type IV Collagen (col4) (Figures 12 and 13). The HconF culture system may be useful as a model for fibrotic tissue formation in filtering blebs. We confirmed that OBP-801 inhibited the expression of αSMA and Type IV Collagen LOX 12 induced by TGF+TNF stimulation (Fig. 12, Fig. 13), suggesting that OBP-801 may be effective in maintaining the filtering bleb during glaucoma surgery.

[0113] (2) Order of fibrosis induction and OBP-801 administration Administration of OBP-801 before fibrosis induction showed a stronger inhibitory effect on αSMA expression than administration of OBP-801 after fibrosis induction (FIG. 14).

[0114] (3) Concentration and treatment time of OBP-801 Treatment with 5 nM OBP-801 for 5 hours before fibrosis induction showed a strong inhibitory effect on αSMA expression (FIG. 15).

[0115] (4) Changes in cell number When cells were treated with 5 nM OBP-801 for 5 hours before fibrosis induction, no decrease in cell number was observed (FIG. 16).

[0116] (5) Relationship with HDAC inhibitory activity No changes in HDAC or HAT activity were observed in HconF cells before and after fibrosis induction by TGF+TNF stimulation, suggesting that the pharmacological effects of OBP-801 are not dependent on HDAC inhibitory activity (Figure 17). After treatment of HconF with OBP-801, the amount of acetylated histones increased by day 2, but was halved by day 7. As mentioned above, the pharmacological effect of OBP-801 persisted up to day 30 after surgery, suggesting that the pharmacological effect of OBP-801 is not dependent on HDAC inhibitory activity (Figure 18).

[0117] (6) Comprehensive analysis of changes in fibrosis-related gene expression after trabeculectomy in rabbits The peak expression of fibrosis-related genes after trabeculectomy varied depending on the gene. They were roughly classified into three groups based on the time of expression, as shown in Figures 19 to 21. In the figures, red plots represent the OBP-801 administration group, and blue plots represent the control group. OBP-801 was shown to suppress multiple genes involved in cell-cell interactions, cell-extracellular matrix interactions, fibrogenesis, cell proliferation, and wound healing (Figs. 19-21). Furthermore, it suppressed multiple genes regardless of the timing of gene expression (Figs. 19-21).

[0118] (7) Comparison with HDAC inhibitor SAHA At 1 nM, OBP-801 has the same level of fibrosis-inhibiting effect as SAHA at 1 μM (FIG. 22). It was shown that OBP-801 has a cell proliferation inhibitory effect on HconF cells that had been administered with TGFβ and TNFα and induced myofibrosis (Figure 23). At 0.25 nM, OBP-801 has the same level of cell proliferation inhibitory effect as SAHA at 0.25 μM. Combined with its inhibitory effect on Col16 expression, it is expected to have the effect of maintaining low intraocular pressure by suppressing excessive fibrotic scar formation (Figure 23).

[0119] [Example 3] (In vitro test for glaucoma prevention effect) OBP-801 was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 μM to prepare a stock solution, which was then diluted with the medium to prepare a pharmaceutical composition to be administered to the treatment groups.

[0120] (1) OBP-801 suppressed the expression of αSMA, collagen, and LOX genes in HTMCs induced by TGF+TNF, indicating that it inhibited the myofibroblastic transformation of HTMCs (Figures 24 and 25).

[0121] [Example 4] (In vivo test for the inhibitory effect on age-related macular degeneration) 1. Method <Laser-irradiated CNV-induced mouse model, OBP-801 administration, observation> ■ Laser irradiation Mice were anesthetized with 0.15 ml ip of Ketalar 9 mg / ml + Selactal 1 mg / ml. Mydriasis: One drop of Mydrin P eye drops was instilled into the eye, and after about 5 minutes, the mouse was ready for treatment. PBS was then added at the appropriate time to prevent drying, and laser irradiation was performed. Laser irradiation: Scopisol eye drops were applied, a cover glass was attached, and the irradiation was carried out under the following conditions. Irradiation: Red, 200mW, 100ms, 50μm. Irradiation was performed on only one eye, away from the optic nerve, at 3, 6, 9, and 12 o'clock.

[0122] OBP-801 administration Intravitreal injections were performed on the control group (1 ml PBS + 1 μl DMSO) and the experimental group (1 ml PBS + 1 μl 10 μM OBP-801). A 22.5° slit knife was used to incise the sclera near the ciliary body (the incision width was approximately the diameter of a 30G needle). A 32G needle was inserted into the incision with the bevel up, and 1 μl of solution was injected. Avoiding the lens, and taking into consideration the volume of fluid and leakage at the bevel up insertion site, the needle was injected slightly more than the 0.5 μl mark, at a point where it was slightly visible but did not reach the retina.

[0123] ◆Isolectin B4 staining After euthanasia, the eyes were enucleated and stored in 4% PFA / PBS at room temperature for approximately 1 hour. The anterior segment and sclera were then removed and retinal flatmounts were prepared. The retinal flatmounts were fixed in -20°C methanol for 10 minutes, then washed with 4% PFA / PBS (room temperature for 10 minutes), followed by blocking (1% fetal calf serum, 0.1% Triton X-100 in PBS at room temperature for 1 hour with shaking). Alx594-conjugated isolectin B4 (1:100) (Invitrogen #I21413) was shaken overnight at 4°C, and then fixed in 4% PFA / PBS for 10 min at room temperature. After mounting with DAPI-containing mounting medium (VECTASHELD), the sections were observed and photographed under a fluorescence microscope.

[0124] ◆Choroidal Flat-mount After euthanasia, the mice were euthanized, and their eyeballs were enucleated and placed in 1% PFA / PBS at room temperature for 2 hours. After removing the anterior segment and lens, four radial incisions were made in the eye, and the retina was removed. Immunostaining (choroidal flat-mount):

[0125] The tissues were incubated in PBS buffer for 30 min with shaking at room temperature (500 μl / well, 48-well plate). Then, they were blocked with 5% BSA / PBS for 1 h with shaking at room temperature (200 μl / well, 48-well plate). Then, the first antibody was incubated overnight at 4°C with shaking (100 μl / well, 48-well plate). The plate was then washed three times with 0.1% TX100 / PBS. The second antibody was incubated for 1 h at room temperature (100 μl / well, 48-well plate). The plate was then washed three times with 0.1% TX100 / PBS. The plates were then mounted in VECTASHELD with DAPI and observed and photographed using a confocal laser microscope.

[0126] 2.Results [Pharmacological effects of OBP-801] (1) Suppression of CNV by OBP-801 administration (Isolectin B4 staining) Thirty-five days after laser irradiation of the mouse vitreous, neovascularization was confirmed in the retina on the choroid side, demonstrating that the mouse could be used as a CNV model (Figure 26, control). When 10 nM OBP-801 was administered at 1 μl / eye immediately after laser irradiation, no new blood vessels were observed and CNV was suppressed (FIG. 26, OBP-801-treated group).

[0127] (2) Suppression of CNV by OBP-801 administration (fluorescein angiography) Nine days after laser irradiation of the mouse vitreous, leakage of the dye that had been administered to the blood vessels 5 minutes prior to the observation was observed (FIG. 27). On the other hand, when OBP-801 was administered to the rabbit vitreous after laser irradiation, leakage of the dye administered to the blood vessels 5 minutes before observation was suppressed (FIG. 28).

[0128] (3) Collagen I expression inhibitory effect In control mice, collagen I signals were observed around all laser-irradiated areas, but in OBP-801-treated mice, collagen I signals were reduced (Figure 29).

[0129] (4) Inhibitory effect on αSMA expression In the control mice, αSMA signals were observed around all laser-irradiated areas, but in the OBP-801-treated mice, the αSMA signals were reduced (FIGS. 30 and 31).

[0130] (5) Inhibitory effect on CD31 expression In the control mice, CD31 signals were observed around all laser-irradiated areas, but in the OBP-801-treated mice, the CD31 signals were reduced (FIG. 32).

[0131] [Example 5] (In vitro test for the inhibitory effect on age-related macular degeneration) 1. Method <Cell culture, OBP-801 addition, observation> The human retinal pigment epithelial cell line, ARPE-19 (ATCC CRL-2302 (registered trademark), Lot. 60279299) (P19), was purchased. Culture (culture according to the procedure manual) Culture medium: DMEM / F12 (Invitrogen: 11330-032) supplemented with 10% FBS and antibiotics. All experiments were performed at passage numbers P23-26.

[0132] ◆Human retinal pigment epithelial cells (H-RPE: 00194987 LONZA) (P2) were purchased and treated as follows. Culture: The media used were Growth medium: RtEGM (200 ml), and Plating medium: Growth medium + 2% FBS. All experiments were performed at passages P3-5.

[0133] ◆ Chemical treatment OBP-801 was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 μM to prepare a stock solution, which was then diluted with the medium to prepare a pharmaceutical composition to be administered to the treatment groups. When the cells reached 80-90% confluence, the medium was replaced with one lacking FBS, and OBP-801 (0-1 nM) was added. After 24 hours, TGFβ (20 ng / ml) and TNFα (10 ng / ml) were added and the cells were exposed for 48 hours. Observation Live cells were observed under a phase-contrast microscope.

[0134] ◆Immunostaining The medium was removed, and the sections were washed three times with PBS for 5 minutes each. The sections were fixed in cold methanol (-30°C) for 15 minutes and air-dried. Blocking: The sections were incubated in 1% bovine albumin solution at room temperature for approximately 30-60 minutes. The primary antibody was incubated overnight at 4°C, followed by three 5-minute washes with PBS. The secondary antibody was incubated for 60 minutes at room temperature. Nuclei were stained using 5 μg / ml DAPI for 15 minutes at room temperature. The sections were washed three times with PBS for 5 minutes each. The sections were observed under a fluorescence microscope with PBS in place.

[0135] Western blotting Protein extraction reagent (SDS-HBS; 1% SDS, 150 mM NaCl in 10 mM Hepes (pH 7.4)) was prepared and boiled for 3 minutes, followed by 5 minutes of sonication (15 seconds, 10 seconds pause). Protein was quantified using a BCA kit. SDS-page electrophoresis (30 μg / lane [iBlot 4-12% Bis-Tris Plus Gel]) was performed, and the protein was transferred to a PVDF membrane [iBlot PVDF Transfer Stack Regular]. After blocking and antibody reaction [iBind Western System], ECL chemiluminescence [NOVEX ECL CHEMI SUBSTRATE] was detected [LAS3000 (Fuji film)].

[0136] ◆HDAC activity measurement Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002). Using the EpiQuik HDAC Activity / Inhibition Direct Assay Kit (Epigenetic #P-4034), samples were reacted with acetylated histone substrate coated on a plate, and the undeacetylated substrate was detected with an anti-acetylated histone antibody.

[0137] ◆HAT activity measurement Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002). Using the EpiQuik HAT Activity / Inhibition Direct Assay Kit (Epigenetic #P-4003), the sample was reacted with the deacetylated substrate coated on the plate, and the acetylated substrate was then detected with an anti-acetylated antibody.

[0138] ◆PCR array RNA was extracted using the RNeasy mini kit (QIAGEN #74104), followed by reverse transcription using the RT2 First Strand Kit (QIAGEN #330401). PCR was performed using the RT2 First Strand Kit (QIAGEN #330401). 2 SYBR Green ROX qPCR Mastermix (QIAGEN #330522) was used for RT. 2 Profiler TM PCR was performed using PCR Array Human Fibrosis (QIAGEN #PAHS-120ZC).

[0139] 2.Results [Drug properties of OBP-801] (1) Inhibitory effect of OBP-801 on RPE cell fibrosis 1 nM OBP-801 suppressed the expression of ZO-1 and αSMA, which were increased by treating RPE cells with TGFβ alone, TNFα alone, or TGFβ + TNFα, demonstrating that OBP-801 inhibits RPE cell fibrosis (Figure 33).

[0140] (2) Effect of OBP-801 on fibrosis-related gene expression OBP-801 suppressed the expression of MMP9, CD44, and αSMA, which were increased by treatment of RPE cells with TGFβ alone, TNFα alone, or TGFβ + TNFα. In particular, OBP-801 had a stronger inhibitory effect on MMP9 and CD44 than TSA (Figure 34).

[0141] (3) Relationship with HDAC inhibitory activity HDAC activity was not affected by fibrosis induction in RPE cells. OBP-801 inhibited HDAC activity to the same extent both before and after fibrosis induction. Therefore, it was suggested that the pharmacological effects of OBP-801 are not dependent on HDAC inhibitory activity (Figure 35). When RPE cells were induced to undergo fibrosis, HAT activity was reduced. OBP-801 did not inhibit HAT activity either before or after fibrosis induction. Therefore, it was suggested that the pharmacological effects of OBP-801 are not dependent on HDAC inhibitory activity (Figure 36). Furthermore, the effect on CD44 expression was examined, and a suppressive effect by OBP-801 was observed (Figure 37).

[0142] [Example 6] Intraocular pressure suppression test and gene expression test using OBP-801 1. Method Cannula-based glaucoma filtration surgery rabbit model, mitomycin administration, OBP-801 administration, observation Mitomycin C (MMC) was dissolved in water for injection and administered at 100 μl at 0.02% (w / v) 30 minutes before surgery. Glaucoma surgery, OBP-801, and intraocular irrigation solution administration were performed as described above. For the purpose of collecting RNA, the bulbar conjunctival epithelium of the filtering bleb, the lamina propria mucosa, and Tenon's capsule were collected 2, 5, 12, and 30 days after surgery.

[0143] ◆PCR array The following procedures were carried out in accordance with the kit protocol. The RNeasy mini kit (QIAGEN #74104) was used for RNA extraction. The RT2 First Strand Kit (QIAGEN #330401) was used for the reverse transcription reaction. RT for PCR reactions 2 SYBR Green ROX qPCR Mastermix (QIAGEN #330522) was used. RT 2 The Profiler® PCR Array Rabbit Fibrosis (QIAGEN #PANZ-120ZC) was used.

[0144] ◆Real-Time PCR The following procedures were carried out in accordance with the kit protocol. The RNeasy mini kit (QIAGEN #74104) was used for RNA extraction. PrimeScript® RT Master Mix (TAKARA #RR036A) was used for the reverse transcription reaction.

[0145] For PCR reactions (intercalator), TB Green® Premix Ex Taq® II (TAKARA #RR820B) was used. Primers used were PDGFB, LOX, LOXL2, PDGFRA, and PDGFRB (designed and purchased from TAKARA). For PCR reactions (fluorescently labeled probes), TaqMan Fast Advanced Master Mix (Invitrogen #4444963) was used. Primers used were Oc03398424_m1 (TGFB2), Oc03399251_m1 (ACTA2), Oc03396112_m1 (COL1A2), and Oc03395687_g1 (CTGF) (Applied Biosystems #4453320).

[0146] ◆Eye drops OBP-801 was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 μM to prepare a stock solution, which was then diluted with ocular irrigation fluid (BSS: Balanced Salt Solution) to prepare a pharmaceutical composition for administration to the treatment group. The amount of OBP-801 was 100 nM, 20 μl x 4 times (30 seconds between each dose) for one course, with one course administered 30 minutes before surgery and two courses administered in the morning and evening on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days after surgery. From 1st to 30th days after surgery, the degree of ocular inflammation, anterior chamber depth, and properties of the conjunctival filtering bleb were observed once every 2-3 days, in accordance with the postoperative examination, and the size of the filtering bleb and intraocular pressure were measured.

[0147] 2.Results (1) Comparison with mitomycin C The effects of mitomycin C (MMC), which is currently used in glaucoma surgery, on gene expression after trabeculectomy were compared with those of OBP-801 (FIGS. 39 to 46).

[0148] The expression of type I collagen (COL1A), which has been suggested to be involved in the re-elevation of intraocular pressure, was suppressed in the OBP-801 group 30 days after surgery, but was significantly increased in the MMC group. This suggests that MMC is unsuitable for long-term (30 days or longer) intraocular pressure control (Figure 39). Furthermore, OBP-801 inhibited the expression of all genes thought to be involved in COL1A expression (TGFB2, SERPINH1, αSMA, CTGF, PDGFB) at 30 days, demonstrating the superiority of OBP-801 in long-term intraocular pressure control (Figures 40, 45, 46).

[0149] Furthermore, the effects of OBP-801 and MMC on the expression of fibrosis-related genes after trabeculectomy were comprehensively analyzed and compared. The results were roughly classified into six categories based on function, and are shown in Figures 41 to 44. LOX and LOXL2 have also been reported to be involved in scar tissue formation and to have an inhibitory effect on the maintenance of filtering blebs. In the BSS group (control), increased expression was observed on postoperative day 2, but both OBP-801 and MMC inhibited expression. However, 30 days after surgery, only the OBP-801 group showed an inhibitory effect on both LOX and LOXL2 expression (Figures 45 and 46).

[0150] (2) Effects of eye drops In the OBP-801 eye drop group, low intraocular pressure was maintained until 30 days after surgery, but in the BSS subconjunctival injection group, a gradual increase in intraocular pressure was observed from 15 days after surgery. Even eye drops at a concentration 10 times that of the subconjunctival injection were effective (Figure 47). We also comprehensively analyzed the effects of OBP-801 eye drops on fibrosis-related gene expression and compared the results with those of subconjunctival injection of OBP-801. Both subconjunctival injection and eye drops showed inhibitory effects on the expression of COL1A, which is involved in maintaining low intraocular pressure; TGFB3 and SERPINH1, which are involved in COL1A expression; and LOX, which acts to suppress the maintenance of filtering blebs (Figure 48).

[0151] [Example 7] Gene expression suppression effect of OBP-801 in rabbit conjunctiva and filtering bleb tissue 1. Method Western blotting Conjunctival and bleb tissue samples were collected from rabbit eyes and subjected to protein extraction using RIPA buffer. The tissue was lysed by sonication for 5 min (15 sec, 10 sec pause) and overnight rotation at 4°C. Residual tissue was removed by centrifugation (10,000g for 10 min). Protein was quantified using a BCA kit. SDS-page electrophoresis (30 μg / lane [iBlot 4-12% Bis-Tris Plus Gel]) was performed, followed by transfer to a PVDF membrane [iBlot PVDF Transfer Stack Regular]. After blocking and antibody reaction using the iBind Western System, ECL chemiluminescence (NOVEX ECL CHEMI SUBSTRATE) was detected using an LAS3000 (Fuji film).

[0152] ◆Tissue immunostaining Conjunctival and bleb tissues were collected from rabbit eyes, embedded in compound (SurgiPath FSC 22), and frozen in liquid nitrogen. Sections (10 μm thick) were prepared using a cryostat (CM3050S: Leica) and mounted on 2% silane (3-aminopropyltriethoxysilane)-coated slides. The sections were fixed in cold methanol (-30°C) for 15 minutes and then air-dried. Blocking was then performed at room temperature for approximately 30–60 minutes using a reaction solution containing 1% bovine albumin. Primary antibody incubation was performed overnight at 4°C, and secondary antibody incubation was performed at room temperature for 60 minutes. The sections were then mounted with DAPI-containing mounting medium (VECTASHELS with DAPI). The surrounding area was covered with nail polish and observed under a fluorescence microscope.

[0153] 2.Results (1) Western blotting analysis of rabbit conjunctival tissue (Figure 49) In the untreated (BSS) group, where intraocular pressure re-elevated 30 days after surgery, increased expression of type I collagen and aSMA was observed. These expressions were suppressed by OBP-801, which is consistent with the results of RT-PCR (RNA expression).

[0154] (2) Immunostaining of rabbit filtering bleb tissue (Figures 50-52) Thirty days after surgery, when intraocular pressure re-elevated, increased expression of collagen I and α-SMA was observed in the filtering bleb in untreated (BSS) tissue. These expressions were suppressed in OBP-801-treated tissue. Furthermore, MMC-treated tissue significantly increased these expressions.

[0155] [Example 8] Genetic analysis results of human conjunctival tissue 1. Method Total RNA was extracted from normal human subconjunctival tissue and human filtering bleb tissue (at the time of reoperation for glaucoma), and gene expression analysis was performed using PCR array. The following procedures were carried out in accordance with the kit protocol.

[0156] The RNeasy mini kit (QIAGEN #74104) was used for RNA extraction. The RT2 First Strand Kit (QIAGEN #330401) was used for the reverse transcription reaction. RT2 SYBR Green ROX qPCR Mastermix (QIAGEN #330522) was used for the PCR reaction. The RT2 Profiler® PCR Array Human Fibrosis (QIAGEN #PAHS-120ZC) was used.

[0157] 2.Results Increased levels of α-SMA and collagen were observed in human filtering bleb tissue at the time of reoperation (Figure 53). When intraocular pressure rose again in rabbits (day 30), increased expression of TGFb2, 3, TGFR, CTGF, PDGFA, SERPINH1, etc. was observed in bleb tissue compared to untreated tissue (day 0). In contrast, increased expression of TGFb1, TGFb3, CTGF, and SERPINH1 was observed in human bleb tissue after reoperation (Figure 54). A significant increase in the expression of TNF was observed. IL1A was also significantly increased, suggesting that inflammation may have occurred in the human bleb tissue at the time of reoperation (Figs. 55 and 56).

Claims

1. The following formula III: 【Chemistry 1】 (wherein R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.) or a pharmaceutically acceptable salt thereof, wherein the depsipeptide compound or a pharmaceutically acceptable salt thereof is administered at a dose of 2 pg / eye to 1500 pg / eye.

2. The following formula III: 【Chemistry 2】 (wherein R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.) or a pharmaceutically acceptable salt thereof, wherein the depsipeptide compound or a pharmaceutically acceptable salt thereof is administered at a dose of 2 pg / eye to 1500 pg / eye.

3. A pharmaceutical composition according to claim 1 or 2, wherein R4 is an isopropyl group.

4. A drug for treating age-related macular degeneration, comprising the pharmaceutical composition described in any one of claims 1 to 3.

Images