JP7542799B2 - Composition for inhibiting ocular tissue fibrosis - Google Patents

Description

The present invention relates to a composition for inhibiting fibrosis of ocular tissue.

When vital organs such as the lungs and liver are damaged and collagen fibers such as type I collagen accumulate during the repair process, the organ loses elasticity, hardens, and becomes dysfunctional, unable to express normal functions. Fibrosis is a disease that occurs widely in vital organs such as the lungs, heart, liver, kidneys, and skin. Fibrosis seen in solid organs (liver, heart, lungs, kidneys, digestive tract, etc.) is a serious chronic disease that can lead to death if left untreated. Although it is a disease that requires improvement in life prognosis, research has lagged significantly behind and there are no effective medicines. Research on organ fibrosis to date has focused mainly on the acquisition of immune function and collagen production ability, centering on the action of tissue growth factor β.

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, resulting in a pathological condition in which the tissue's natural functions fall below the threshold required to maintain physiological balance.

Collagen-producing cells such as myofibroblasts are thought to be involved in the pathology of fibrosis. However, it is presumed that the pathology is actually caused by the involvement of a wide variety of cells and complex cell-cell interactions. The molecular nature of the pathology has not yet been clarified, which is a major bottleneck in drug discovery. For example, one of the inventors reported in 2002 that macrophages (MPs), which gather at the affected area during the fibrosis stage, are involved in the onset of fibrosis, suggesting that drug development targeting Mps will enable the development of drugs for fibrosis, which has not had an effective treatment until now. The inventors have also been researching for many years a method to inhibit the function of Mps involved in the onset of fibrosis and a technology to inhibit chronic inflammation closely related to tissue fibrosis.

A condition in which inflammation persists and is marked by fibrosis (tissue remodeling) at the site of inflammation, angiogenesis, and the accumulation of specific immune cells is considered to be "chronic inflammation." Chronic inflammation has a variety of causes and pathologies, and internal and external environmental stress on an individual triggers inflammation as a biological defense mechanism mediated by the immune system and endocrine system. As inflammation persists or recurs, an asymptomatic pre-disease state is passed, and then an adaptive state of abnormal chronic inflammation accompanied by functional impairment of cells and tissues is established. As this chronic inflammatory state persists, tissue fibrosis occurs, and organ dysfunction becomes irreversible. When considering the process of disease formation at the tissue and organ level, tissue homeostasis is disrupted by the involvement of various factors, such as changes in the interactions between tissue-constituting cells and infiltrating immune cells, heterogeneous and diverse cells with different activation states, non-physiological metabolic responses, and disturbances in the extracellular matrix and humoral factor networks.

The eye is an immunologically special functional closed organ and is known as an "immune privileged site." Immune privilege is a self-defense mechanism that the body has. Immune privilege can be interpreted as a homeostatic mechanism that exists to protect the function of organs in which normal immune inflammatory reactions would cause severe tissue and functional damage. However, once inflammation exceeds a certain limit, the immune privilege mechanism is lost and ocular inflammation worsens. Damaged ocular tissues have difficulty recovering their function.

Immune responses are broadly classified into "acquired immunity," which is centered on T lymphocytes, and "innate immunity," which reacts 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.

Among the actual clinical pathologies of choroidal neovascular disease, such as age-related macular degeneration (AMD), many studies on choroidal neovascularization (CNV) have been conducted on its formation mechanism, but in actual clinical pathologies, the inflammatory response involved in the scar healing process in the macula that occurs after bleeding from CNV is important. In recent years, new treatments have been started, such as intravitreal administration of anti-VEGF antibodies such as bevacizumab to suppress the CNV formation process, and photodynamic therapy using verteporfin for already formed CNV, and a certain degree of therapeutic effect has been observed. However, the period when the new treatments are most effective is almost limited to the pre-onset and onset stages, making them insufficient in terms of practicality.

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, causing choroidal neovascularization (CNV). It is one of the serious visual impairment diseases in elderly people. Currently, the mainstream treatment for AMD is blood vessel ablation therapy using anti-VEGF antibodies that target the above-mentioned CNV.

The clinical pathology of choroidal neovascular disease is that "many patients only become aware of the disease when their vision deteriorates after macular hemorrhage, but from the perspective of visual recovery, the time has already come when recovery is difficult, and tissue scarring that has already formed will not recover" (Non-Patent Document 3). As a therapeutic target for choroidal neovascular disease, the process of macular dysfunction (scar healing) that occurs after bleeding and exudation from CNV is also considered to be important. In addition to the pathology of CNV formation, it is also important to suppress the pathology of retinal and choroidal scarring that is formed secondarily due to bleeding and exudation of blood components from CNV.

There are various problems with the currently widely used anti-VEGF therapy. (1) There is a high rate of non-responsive cases that resist the treatment, and resistant cases that lose their effectiveness over the course of the treatment. (2) Because anti-VEGF therapy aims to inhibit the progression of the disease, it is unlikely to improve visual function. (3) Multiple administrations over a long period of time are necessary, which carries the risk of complications and side effects (increased intraocular pressure, decreased vision, eye pain, retinal hemorrhage). (4) There are also cases where treatment is discontinued due to high medical costs. Due to these many problems, there is a strong demand in the clinical field for new AMD treatments that can replace anti-VEGF therapy.

As a cause of resistance to anti-VEGF antibodies, (i) a relationship with tissue fibrosis that occurs before CNV formation has been pointed out (Diego et al. 2013). In addition, as a cause of permanent vision loss, (ii) fibrous tissue that forms as a foundation for CNV development and remains as scar tissue even after blood vessel removal, and (iii) CNV itself is not necessarily formed only by VEGF, but many other angiogenic factors and angiogenic inhibitors are involved. Anti-VEGF therapy is completely ineffective against (i) to (iii).

Regarding retinal scar tissue, the inventors have reported the induction of local scarring 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 FIG. 38. The subretinal proliferative tissue of AMD patients is a mixture of proliferated and migrated RPE and Mps, and Mps and RPE are considered to be important in the pathogenesis. The present inventors reported that in a co-culture system of Mps and RPE, the production of proinflammatory cytokines is enhanced, the expression of complement activation genes such as C3 and CFB is enhanced, the expression of complement activation inhibitors such as CFH, CD59, and Clusterin is attenuated, the expression of VEGF is enhanced, and the expression of angiogenesis inhibitor PEDF is attenuated (Non-Patent Document 2). Intracellular αSMA increases in RPE co-cultured with Mps, and subretinal injection leads to the formation of subretinal scars. The present inventor has been searching for a substance that suppresses the production of inflammatory cytokines produced by Mps and its dendritic cell companions for many years, and has found a substance that maintains its effect even in the presence of fibrosis pathology aggravating factors related to tissue fibrosis, which became the starting point of the present invention.

Glaucoma, another important ocular tissue disease that is the subject of the present invention, is a disease characterized by characteristic changes such as damage to the optic nerve and narrowing of the visual field. When anti-glaucoma drugs are not sufficient to reduce intraocular pressure, trabeculectomy (TLE; surgery to remove the trabecular meshwork and drain aqueous humor out of the eye to reduce intraocular pressure) is performed to lower the intraocular pressure. Alternatively, trabeculotomy and insertion of implants for treating glaucoma are also performed for outflow reconstruction surgery. Improving the prognosis of glaucoma surgery is an important medical need.

In glaucoma surgery, for example trabeculectomy, aqueous humor is drained from the trabeculectomy site through under 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, adhesion, scarring, etc., intraocular pressure may rise again and glaucoma may worsen. Trabeculectomy has problems such as the risk of infection, the difficulty of forming and maintaining a filtering bleb, and the difficulty of controlling intraocular pressure over the long term.

Therefore, a glaucoma surgery that forms a filtering bleb over a wide area, maintains the vascular characteristics of the conjunctiva, and reduces intraocular pressure for a long period after surgery is desirable.
Mitomycin C (MMC) is applied during surgery to prevent postoperative inflammation, adhesion, and scarring. However, side effects of MMC include thinning of the conjunctiva, leakage of aqueous humor from the conjunctiva, and infection of the bleb. There are problems with the current trabeculectomy using MMC. Excessive wound healing causes thick connective tissue to form around the bleb, making it difficult to control intraocular pressure for a long period of time. Furthermore, it is widely known that the bleb becomes avascular and thin, increasing the risk of bleb infection through aqueous humor leakage.

In basic research to suppress fibrosis, histone deacetylase (HDAC) inhibitors have been investigated, but they are far from practical use and have a large bottleneck, so they have not been developed. In the field of ophthalmology, the anti-fibrotic effect of the conjunctiva has been reported in rabbit filtration surgery using the HDAC inhibitor SAHA, but the dosage required is more than 1000 times higher than that of the compound of the present invention, and in terms of the ability of the drug to reach the affected area at the effective concentration, it is far from practical use and has not been developed. This is because no compound with few side effects has been found at an effective concentration that reaches the practical range, and the drug characteristics do not satisfy the ability to reach the active concentration range at the active site. In chronic tissue inflammation such as AMD, the pathological target is not clearly defined in time and space, and the suitability of the drug application timing and route to exert a local drug effect is unclear, and there is a large gap with medical needs. In addition, practical effects that comprehensively suppress fibrosis, angiogenesis, and tissue scarring cannot be expected. It has also been confirmed that the pharmacological effects of the low molecular weight compounds of the present invention cannot be obtained solely through HDAC inhibitory activity.

Patent No. 3554707

Invest Ophthalmol Vis Sci 52:6089-6095 (2011) Invest Ophthalmol Vis Sci 57:5945-5953 (2016) Fukuoka medical magazine. 99(7), pp. 137-143, 2008-07-25. Fukuoka Medical Society

An object of the present invention is to provide a substance that has an inhibitory effect on 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 can synergistically improve effects and reduce side effects. Another object of the present invention is to provide a method for treating AMD to improve the aggravating pathology and a method for treating patients who are expected to develop AMD.

As a result of intensive research to solve the above problems, the inventor has provided a glaucoma surgery technique that suppresses excessive fibrosis, promotes healthy conjunctival tissue repair, and maintains a long-term low intraocular pressure effect. In addition, in a laser irradiation animal model, the inventor has found a low-molecular-weight compound that is effective against fibrosis of choroidal tissue including RPE at an extremely low concentration and dosage, and that exhibits a comprehensive inhibitory effect on multiple genes related to tissue fibrosis, and that exhibits an inhibitory effect not only on fibrosis but also on the expression of multiple genes related to tissue fibrosis, angiogenesis, and tissue scarring, such as VEGF and PDGF, which are involved in angiogenesis, and LOX, which crosslinks collagen and is involved in scar formation, and is expected to have a comprehensive and high therapeutic effect on the spatiotemporal multistage pathology of AMD patients, and has successfully confirmed its effectiveness in an animal model, thereby completing the present invention. Aiming to develop a new AMD treatment drug that combines a new CNV inhibitory mechanism based on a unique AMD molecular pathology theory and an inhibitory effect on scar tissue formation, the inventor has continued intensive research and completed one of the present inventions.

The inventors have completed this invention to address the two major Unmet needs mentioned above, but the content of this invention goes beyond the examples shown in this specification and offers the possibility of being applied to other ocular tissue diseases and other organ diseases.

That is, the present invention is as follows.
(1)
A pharmaceutical composition comprising a substance that inhibits fibrosis of ocular tissue.
(2)
The pharmaceutical composition according to 1, wherein the substance that suppresses fibrosis of ocular tissue is a substance that inhibits in vivo in ocular tissue the expression of at least one type of pathology aggravating factor gene associated with each of the three stages of fibrosis, angiogenesis, and scar formation.
(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 inhibits 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 suppresses fibrosis of ocular tissue at a dosage of 2 pg/eye to 9000 pg/eye.

(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 of 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.

又は次式ＩＩ：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩を含む、１～１０のいずれか１項に記載の医薬組成物。 (11)
The following formula I:

Or the following formula II:

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, or an ethyl group; R4 is 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 are independently a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 is a hydrogen atom, a methyl group, or a protecting group; and R10 and R11 are independently 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:

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩を含む、１１に記載の医薬組成物。 (12)
The following formula III:

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
12. The pharmaceutical composition according to claim 11, comprising a depsipeptide compound represented by the following formula: or a pharma- ceutically acceptable salt thereof.

(13)
13. The pharmaceutical composition according to claim 12, wherein R4 is an isopropyl group.
(14)
14. The pharmaceutical composition according to any one of claims 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 a filtering bleb tissue.

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

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 fibrosis of choroidal tissue at extremely low concentrations and dosages, and also has a comprehensive inhibitory effect on multiple genes involved in tissue fibrosis and precursor lesions. Many existing fibrosis inhibitor candidate substances are effective against TGFβ-induced fibrosis, but are ineffective against fibrosis caused by the combined action of TGFβ+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 the most important factors in maintaining the patient's vision.

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. This figure shows blebs on days 7 and 30 when 200 μl of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. FIG. 13 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. FIG. 1 shows Bleb on day 14 when OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. 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, and when it was administered on days 0, 1, 3, and 5 after surgery. 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, and on days 3, 5, and 7 after surgery. 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 was administered on days 0, 1, 3, 5, 7 and 9 after surgery, respectively. 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. This 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. 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. 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. FIG. 1 shows the inhibitory effect of OBP-801 on αSMA expression in HconF cells in which fibrosis was induced by TGF+TNF. FIG. 1 shows the inhibitory effect of OBP-801 on collagen and LOX expression in HconF cells in which fibrosis was induced by TGF+TNF. FIG. 1 shows the inhibitory effect on the expression of αSMA, col1, and col4 when OBP-801 was administered before and/or after fibrosis induction of HconF with TGF+TNF. FIG. 1 shows the inhibitory effect of αSMA expression when OBP-801 was treated at different concentrations and for different treatment times before fibrosis induction of HconF with TGF+TNF. FIG. 1 shows the time course of cell number following OBP-801 treatment. FIG. 1 shows HDAC and HAT activities when HconF induces fibrosis by TGF+TNF. FIG. 1 shows time-dependent changes in the amount of acetylated histones when HconF was treated with OBP-801. FIG. 1 shows genes whose expression levels were significantly changed two days after trabeculectomy and the inhibitory effect of OBP-801 on the expression of the genes. FIG. 1 shows genes whose expression levels were significantly changed 12 days after trabeculectomy and the inhibitory effect of OBP-801 on the expression of the genes. FIG. 1 shows genes whose expression levels were significantly changed 30 days after trabeculectomy and the inhibitory effect of OBP-801 on the expression of the genes. FIG. 1 shows the fibrosis-suppressing effect of HconF with OBP-801 and SAHA. FIG. 1 shows the cell proliferation inhibitory effect of HconF with OBP-801 and SAHA. FIG. 1 shows that OBP-801 inhibits myofibroblastic transformation of HTMCs. FIG. 1 shows the inhibitory effect of OBP-801 on collagen and LOX expression in HTMCs in which fibrosis was induced by TGF+TNF. FIG. 1 shows the CNV suppressive effect of OBP-801 administration. FIG. 1 shows the CNV suppressive effect of OBP-801 administration. FIG. 1 shows the CNV suppressive effect of OBP-801 administration. FIG. 1 shows the inhibitory effect of administration of OBP-801 on Collagen I expression. FIG. 1 shows the inhibitory effect of administration of OBP-801 on the expression of α-SMA. FIG. 1 shows the inhibitory effect of administration of OBP-801 on the expression of α-SMA. FIG. 1 shows the effect of suppressing CD31 expression by administration of OBP-801. FIG. 1 shows the inhibitory effect of OBP-801 on fibrosis in RPE cells. FIG. 1 shows the effect of OBP-801 on fibrosis-related gene expression. FIG. 1 shows the relationship with HDAC inhibitory activity of OBP-801. FIG. 1 shows the relationship with the HAT inhibitory activity of OBP-801. FIG. 1 shows the effect of OBP-801 on CD44 expression. FIG. 1 shows AMD pathology progression in drusen and laser-induced CNV models. FIG. 1 shows that OBP-801 suppressed collagen 1 expression 30 days after surgery. FIG. 1 shows that OBP-801 suppressed the expression of TGFβ2 and SERPINH1 30 days after surgery. FIG. 1 shows expression of ECM and ECM remodeling enzymes. FIG. 1 shows expression of inflammatory cytokines and chemokines. FIG. 1 shows expression of the TGFβ superfamily. FIG. 1 shows the expression of transcription factors. FIG. 1 shows the results of real-time RT-PCR. FIG. 1 shows the results of real-time RT-PCR. FIG. 1 shows the results of suppressing intraocular pressure by instillation of OBP-801. FIG. 1 shows the expression of a group of genes thought to be involved in maintaining intraocular pressure. FIG. 1 shows rabbit conjunctival tissue WB analysis. FIG. 13 shows the results of HE staining of rabbit filtering bleb tissue 30 days after surgery. FIG. 1 shows the results of α-SMA expression on 30 days after surgery by immunohistochemical staining of rabbit filtering bleb tissue. FIG. 1 shows the results of collagen I expression on day 30 after surgery by immunostaining of rabbit bleb tissue. FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue. FIG. 1 shows the results of an analysis of genes thought to be involved in maintaining intraocular pressure in human conjunctival tissue.

1. Overview The present invention relates to a pharmaceutical composition and method for restoring normal ocular tissue from ocular-related fibrotic tissue and for inhibiting the loss of the original function of normal ocular tissue due to fibrosis.
In order to solve the above problems, the present inventors have been conducting intensive research into (1) what kind of gene expression is expressed in a spatiotemporal manner in relation to pathology in a fibrosis model in ocular tissue, and have been searching for a substance that simultaneously suppresses not only a specific localized fibrosis stimulus but also (2) the multiple gene group induced by many stimuli including chronic inflammation related to fibrosis. (3) In addition, the selection criteria set forth that the substance acts under cellular stress similar to that of chronic inflammatory tissues where the action of inflammatory cytokines is superimposed in the presence of factors well known to induce fibrosis. Such an approach was completely unknown before.

In addition, hydrophobic compounds that penetrate into cells to suppress gene expression have extremely weak effects in vivo in terms of drug delivery unless the effective concentration is extremely low, and no effectiveness as an antifibrotic agent can be expected. In fact, as seen in the limitations of SAHA mentioned above, no antifibrotic agent that can be used in practice has been found to date. The inventors have focused on this point and have been actively searching for compounds that meet the strict selection criteria of (4) being effective at an effective concentration that is one thousandth of that of related compounds that have been studied, in other words, being able to be expected to have an administration route that can be used in vivo.

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

Compounds that have an inhibitory effect on tissue fibrosis that occurs before CNV formation can enable early treatment before CNV formation, and are expected to prevent vision loss. In addition, a therapeutic effect through the inhibitory effect on fibrosis formation can be expected against the above-mentioned anti-VEGF resistance. In the present invention, attention is focused on fibrosis, which is a functional phase transition triggered by the breakdown of the epigenetic regulatory mechanism of retinal pigment epithelial cells (RPE) in the progression of AMD pathology. It has been found that the active ingredient of the pharmaceutical composition of the present invention is effective against fibrosis of choroidal tissue including RPE at extremely low concentrations and dosages, and is the first compound to show a comprehensive inhibitory effect on multiple genes related to tissue fibrosis. Furthermore, it has been confirmed that the inhibitory effect is not only on fibrosis, but also on the expression of multiple genes related to each of the three important stages of pathogenesis, such as VEGF and PDGF involved in angiogenesis, and LOX, which crosslinks collagen and is involved in scar formation, and a high therapeutic effect is expected for the various pathologies of AMD patients. In fact, in CNV model mice, the pharmaceutical composition of the present invention has been shown to have a significant inhibitory effect on both early fibrosis and neovascularization.

Using a mouse model of retinal tissue fibrosis and angiogenesis, the pharmaceutical composition of the present invention has been shown to have a novel action profile that goes beyond the inhibition of collagen fiber crosslinking in the late stage and anti-angiogenesis effects to inhibit the transformation of fibroblasts into myofibroblasts early after laser irradiation, which correlates with anti-VEGF therapy resistance, and is superior to existing therapies. The drug properties of this compound are characterized by the simultaneous inhibition of the expression of multiple genes related to fibrosis, such as LOX, THBS1, Serpin, and MMP.

The first reason for using AMD as a model disease is that the limitations of anti-VEGF antibody therapy as an anti-angiogenic inhibitor are becoming apparent clinically. This antibody therapy only targets angiogenesis inhibition, which is only one of the phenotypes after onset and progression of the disease, and cannot directly inhibit damage (including fibrosis) to photoreceptors and retinal pigment epithelial cells (RPE), which are the most important factors in maintaining a patient's vision. Inhibition of fibrosis by the pharmaceutical composition of the present invention can maintain and restore normal function of retinal pigment epithelial cells and photoreceptor cells.

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

2. Fibrosis-inhibiting substance The present invention is a medicine containing 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 due to various stress stimuli on cells, such as oxidative stress, hypoxia, inflammation, and apoptosis. The pharmaceutical composition of the present invention can be used to inhibit stress on such tissues 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 caused by various stresses on such cells.

In addition, as a substance that suppresses fibrosis of ocular tissue, a substance that suppresses fibrotic phase transition of ocular tissue cells is also included in the present invention. "Fibrotic phase transition of ocular tissue cells" refers to a situation in which ocular tissues such as retina and conjunctival tissue undergo cellular function degeneration and tissue homeostasis is disrupted due to external or internal cellular stress (based on aging, etc.). Cell degeneration includes hardening of tissues due to functional changes in cells that constitute the tissue, such as transition to a fibroblast-like cell morphology, functional changes in cells called epithelial-mesenchymal transition (EMT), enhanced apoptosis, abnormal autophagy, enhanced production of extracellular matrix components, and abnormal cross-linking between proteins such as collagen and elastin that constitute the interstitium, and the substance of the present invention suppresses these.

Furthermore, as substances that suppress fibrosis of ocular tissue, substances that have a filtering bleb-maintaining effect or an effect of improving the prognosis of glaucoma surgery are also included in the present invention.
As pharmaceuticals for treating the above-mentioned pathological conditions, there are compounds that exhibit effects such as suppression of collagen and α-SMA production, TGF or TNF production inhibitors, TGF or TNF signal transduction inhibitors, and HDAC inhibitors. More preferred are compounds that (1) change the expression levels of related genes in a spatiotemporal manner in a fibrosis model of ocular tissue, (2) simultaneously suppress the multiple gene groups caused by many stimuli related to fibrosis, and (3) simultaneously act under cellular stress similar to that of chronic inflammatory tissues in which the action of inflammatory cytokines is superimposed in the presence of factors well known to induce fibrosis. A depsipeptide compound or a pharma- ceutical acceptable salt thereof, more preferably OBP-801 (details will be described later), is suitable as a compound that (4) has good penetration into cells in each tissue and exhibits antifibrotic and anti-scarring effects at a sufficiently low effective concentration.

3. Substance for Inhibiting Expression of a Pathological Condition Aggravating Factor Gene In another embodiment, the present invention relates to a medicine comprising a compound capable of regulating or controlling the expression of a pathological condition aggravating factor gene.
・Pathogenesis-aggravating factor genes expressed in conjunctival-related tissues, trabecular meshwork cells, retinal pigment epithelial cells, or choroidal neovascularization and choroidal tissue in mammals ・Pathogenesis-aggravating factor genes expressed in conjunctival-related tissues, trabecular meshwork cells, retinal pigment epithelial cells, or choroidal neovascularization and choroidal tissue in humans or rabbits ・Fibrosis-related genes in humans or rabbits ・Pathogenesis-aggravating factor genes whose expression changes due to glaucoma surgery in humans or rabbits

The pathology aggravating factor genes include fibrosis-inducing genes, angiogenesis-related genes, scarring-related genes, ECM-related genes, etc. By controlling the expression of the genes and controlling fibrosis and ECM accumulation in ocular tissue, it is possible to expect effects of maintaining filtration blebs, maintaining low intraocular pressure, inhibiting angiogenesis, and suppressing scar formation. For example, in ocular tissue in vivo, the expression of at least one pathology aggravating factor gene for each of the three stages of fibrosis, angiogenesis, and scar formation is inhibited. Examples of the pathology aggravating factor genes include 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, and the expression of these genes can be controlled by the pharmaceutical composition of the present invention.

The present invention also relates to a pharmaceutical composition that has an inhibitory effect on fibrosis and/or angiogenesis, and inhibits at least two of the stages of scar formation in ocular tissue cells in vivo, the pharmaceutical composition comprising a substance that inhibits the expression of at least one gene among the genes of exacerbation factors associated with each stage. The present invention also includes an activity regulator of a hub gene involved in the expression of a gene associated with a pathology exacerbation factor.

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 not only on fibrosis, but also on the expression of multiple genes, such as VEGF and PDGF, which are involved in angiogenesis, and LOX, which crosslinks collagen and is involved in scar formation, and is expected to have a high therapeutic effect on the diverse pathologies of AMD patients.

In order to inhibit the expression of the above-mentioned 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 the sequence information is available. The GenBank accession numbers of the genes are shown below.

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

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 fibrosis can be suppressed by administering antibodies to both genes.

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

4. Substance Having a Filtering Bleb-Maintaining Effect In another embodiment, the present invention is a medicine containing a compound having a filtering bleb-maintaining effect.
After glaucoma surgery, wound healing occurs in glaucoma-related tissues 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, fibrosis is stimulated by TGF+TNF in conjunctival fibroblasts, which hinders the maintenance of bleb. In the wound healing process of the subconjunctival tissue in the bleb after surgery, it undergoes a series of healing processes such as inflammation, proliferation, and scarring, resulting in morphological and functional changes.

In rabbit glaucoma surgery, morphological changes such as localization and avascularity in the filtration bleb are observed in the anterior segment. In the process of this change, the expression of fibrosis-related genes, scarring-related genes, and factors related to the increase in ECM is enhanced. The genes include 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.
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.

5. Substance Having an Effect of Improving Prognosis of Glaucoma Surgery In another aspect, the present invention is a medicine containing a compound having an effect of improving the prognosis of glaucoma surgery.
Glaucoma is a disease characterized by characteristic changes in the optic nerve and visual field, as well as functional and structural abnormalities. In general, optic nerve damage can be improved or suppressed by sufficiently lowering intraocular pressure.

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 for treating glaucoma (with or without a plate) to restore the structure of the ocular tissue to a normal state.
Trabeculectomy is a method of reconstructing an artificial aqueous humor outflow route while absorbing aqueous humor from the conjunctiva and evaporating it from the surface. It involves strong invasion against the natural healing power of the body and continuous stress on biological tissues, which leads to delayed wound healing, delayed reduction in intraocular pressure, and often makes it difficult to control intraocular pressure. The disadvantage of TLE is that the filtration bleb is easily localized and avascular. In such a situation, it is desirable to control intraocular pressure for a long time, form a healthy filtration bleb, and reduce the risk of infection of the filtration bleb. It is desirable to have TLE in which the filtration bleb is formed over a wide area, the vascular properties of the conjunctiva are maintained, and the ocular pressure is reduced for a long time after surgery. Additionally, glaucoma surgery includes Ex-PRESS, INNFOCUS, Baerveldt Glaucoma Implant, Ahmed Glaucoma Valve, XEN Implant, Hydrus Microstent, and others.

6. Compound The present invention provides a pharmaceutical composition comprising a depsipeptide compound represented by the following formula I or II, or a pharma- ceutically acceptable salt thereof.

又は次式ＩＩ：

（式中、Ｒ１～Ｒ３は独立して水素原子、メチル基、エチル基、Ｒ４は水素原子、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｓｅｃ－ブチル基又はイソブチル基、Ｒ５～Ｒ８はそれぞれ独立して水素原子、メチル基、エチル基又はイソプロピル基、Ｒ８は水素原子、メチル基又は保護基、Ｒ１０及びＲ１１は、独立して水素原子、メチル基又は保護基を表す。） The following formula I:

Or the following formula II:

(In the formula, R1 to R3 are independently a hydrogen atom, a methyl group, or an ethyl group; R4 is 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 are independently a hydrogen atom, a methyl group, an ethyl group, or an isopropyl group; R8 is a hydrogen atom, a methyl group, or a protecting group; and R10 and R11 are independently a hydrogen atom, a methyl group, or a protecting group.)

（式中、Ｒ４はイソプロピル基、ｓｅｃ－ブチル基又はイソブチル基を表す。）
で示されるデプシペプチド化合物又はその製薬学的に許容可能な塩を含む、医薬組成物も提供する。
上記式ＩＩＩに示す化合物のうち、Ｒ４がイソプロピル基のものが好ましい。ＯＢＰ－８０１は、Ｒ４がイソプロピル基の化合物である。 The present invention also relates to a compound represented by the following formula III:

(In the formula, 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: or a pharma- ceutically acceptable salt thereof.
Of the compounds represented by formula III above, those in which R4 is an isopropyl group are preferred. OBP-801 is a compound in which R4 is an isopropyl group.

7. OBP-801
As the above-mentioned fibrosis-suppressing substance, the substance that suppresses the expression of pathology-aggravating factor genes, the substance that has an effect of maintaining filtration blebs, the medical treatment for suppressing the progression of pathology in AMD patients, the prevention of pathology that begins with neovascularization in the choroid and leads to blindness, and the substance that has an effect of improving the prognosis of glaucoma surgery, a medicine containing OBP-801 is preferred as an embodiment.

It has been found that OBP-801 is the first compound to show 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, including VEGF and PDGF, which are involved in angiogenesis, and LOX, which crosslinks collagen and is involved in scar formation, in addition to fibrosis, and is expected to have a high therapeutic effect on the various pathologies of AMD patients.
OBP-801 has a stronger antifibrotic effect than SAHA, and may be more effective in suppressing postoperative fibrosis. OBP-801 is also one of the compounds that penetrates into cells and exhibits an antifibrotic effect at a sufficiently low concentration to exert its effect. The present invention has revealed that OBP-801 is effective at a concentration 1/1000th that of SAHA. Since OBP-801 is effective at a lower concentration than SAHA, there is no risk of toxicity and it may be even 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-mentioned Patent Document.

8. Pharmaceutical Composition (1) Preparations 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.

Eye drops can be prepared using isotonic agents such as sodium chloride and concentrated glycerin; buffering agents such as sodium phosphate and sodium acetate; surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, and polyoxyethylene hydrogenated castor oil; stabilizers such as sodium citrate and sodium edetate; and preservatives such as benzalkonium chloride and parabens, as required. The pH should be within the range acceptable for ophthalmic preparations, but a range of 4 to 8 is usually preferred. Eye ointments can be prepared using commonly used bases such as white petrolatum and liquid paraffin.

In the present invention, the compound or its pharma- ceutically acceptable salt can be used in the form of gel, cream, and lotion. For example, it can be formulated for local or topical application to the eye, skin, and mucous membrane, and for application to the eye. The topical pharmaceutical composition is not limited in form, and can be, for example, 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 pharma-ceutically acceptable salts thereof may also be formulated as transdermal patches for transdermal administration.

In the present invention, topical pharmaceutical compositions containing the compounds or pharma- ceutically 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 oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, and the like.
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 may be used alone or as a mixture of one or more materials, are as follows:

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, iso-propyl isostearate, stearic acid, iso-butyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecane-2-ol, isocetyl alcohol, cetyl palmitate, dimethylpoly Siloxanes, di-n-butyl sebacate, iso-propyl myristate, iso-propyl palmitate, iso-propyl 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, e.g., ... propane, butane, iso-butane, dimethyl ether, carbon dioxide, and nitrous oxide; solvents such as ethyl alcohol, methylene chloride, iso-propanol, 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 smectites, trialkylarylammonium smectites, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymers, sodium carboxymethylcellulose, and ethylene glycol monostearate.

The sustained release agent or insert can be prepared by grinding and mixing the compound with a biodegradable polymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxyvinyl polymer, polyacrylic acid, etc., and compressing the powder. If necessary, excipients, binders, stabilizers, and pH adjusters can be used. Intraocular implant preparations can be prepared using biodegradable polymers, such as polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymers, hydroxypropyl cellulose, etc.

Injections can be prepared using agents selected as necessary 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 semisolid, such as a gel, eye ointment, etc.

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, etc. In addition, isotonicity agents such as sodium chloride, boric acid, and sodium citrate can be added to make the solution isotonic with tears, and buffers such as boric acid, buffer solutions, and phosphate buffer solutions can be added to maintain a constant pH of, for example, about 5.0 to 8.0. In addition, stabilizers such as sodium sulfite and propylene glycol, chelating agents such as sodium edetate, thickeners such as glycerin, carboxymethylcellulose, and carboxyvinyl polymer, and preservatives such as methylparaben and propylparaben can be added. These are sterilized, for example, by filtration through a bacteria-retaining filter or by heat sterilization.

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 polymer.

9. Dosage and Administration The dosage of the present compound may be appropriately varied depending on the dosage form, the severity of symptoms of the patient to be administered, the 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: 10 nM, 200 μl, subconjunctival injection, once a day (days 0, 1, 3, and 5)
For eye drops or inserts: 100 nM, 80 μl, instilled twice daily (days 0, 1, 2, 3, 4, 5, 6, 7)

In addition, 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 9,000 pg/eye, or 100 pg/kg to 3,000 pg/kg, depending on the form of conjunctival injection and eye drops.

In the case of suppression of conjunctival fibrosis (conjunctival injection), when the OBP-801 concentration is 10 nM and the liquid volume is 200 μl, the amount of OBP-801 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, administered by subconjunctival injection 30 minutes before surgery and 1, 3, and 5 days after surgery (a total of 4 administrations).
Alternatively, 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 1, 3, and 5 days after surgery (a total of 4 administrations).

- For 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, administered once, 15 times in the morning and evening, 30 minutes before surgery and on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days after surgery.

Alternatively, 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, should be administered once, 15 times in total, in the morning and evening, 30 minutes before surgery and on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days after surgery.

・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 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 administrations 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.
For ocular tissue culture cells:
The fibrotic phase transition of ocular tissue culture cells is suppressed at a concentration of 10 nM or less, and the inhibitory effect on HDAC activity in ocular tissue cells is at an IC50 of 10 nM or less.

10. Subject (1) Inhibition of ocular tissue fibrosis Definition:
Ocular tissues: conjunctival tissues, glaucoma-related tissues, retina-related tissues, corneal tissues, conjunctival tissues, scleral tissues, lens tissues, trabecular meshwork tissues, retinal choroidal tissues, optic nerve tissues, vitreous tissues Fibrosis: Cell proliferation of conjunctival fibroblasts (HconF) and trabecular meshwork cells (HTMC) progresses, and abnormal cross-linking of the extracellular matrix occurs due to overexpression of fibrosis-inducing genes such as TGF and TNF, and col1, col3, col4, col6, etc. As a result, the physiological functions of the fibrous cells and the parenchyma are reduced.

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), dominant negative p38 MAPK gene by adenovirus, 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, cell proliferation inhibitor gene p21, Bevacizumab, Ranibizumab, MMC, steroids, NSAIDS, 5FU, Anti-VEGF antibody, Anti-LOXL1 antibody, Anti-LOX antibody

(2) Glaucoma-Related Tissues Glaucoma-related tissues include tissues that are composed of trabecular meshwork and trabecular meshwork cells (HTMCs) and that 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

(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 about 0.3 mL, and the aqueous humor is exchanged every 1-2 hours, providing nutrients to avascular tissues, transporting waste products, and maintaining the homeostasis of intraocular pressure.

(2-2) Tissues that can control 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 with this 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 front of the eyeball. Aqueous humor is produced in the ciliary body and flows out of the space just below the cornea (anterior chamber) through the gap between the iris and the lens (posterior chamber). It then passes through the trabecular meshwork in the part called the angle at the base of the cornea and iris, and is discharged by the Schlemm's canal. If this flow is blocked and the amount of aqueous humor increases, intraocular pressure rises. When intraocular pressure is 21 mmHg or higher, it is determined to be high intraocular pressure. However, it is known that in Japanese people, the incidence of normal tension glaucoma, in which the optic disc is damaged even though the intraocular pressure is lower than that, is high.

(3) Conjunctival-related tissues Conjunctival-related tissues are composed of bleb tissue, connective tissue around the bleb, and aqueous humor tissue. Their functions are to absorb aqueous humor from the conjunctiva, evaporate water from the surface, and at the same time, form an artificial aqueous humor outflow route to control the outflow of aqueous humor from within the eye and maintain normal intraocular pressure.
Types of conjunctival-associated tissues include the conjunctival epithelium, lamina propria, Tenon's capsule, episclera, and sclera.

(4) Retina-related tissues The retina is located inside the choroid, and more than 100 million photoreceptor cells form a thin membrane of 0.2 to 0.5 mm. It is considered the most important part for sensing light and color and seeing things. The macula is the part of the retina where the light entering through the pupil hits the front of the fundus and looks slightly yellower than the surrounding retina. The macula also has a point in the center that is slightly thinner than the surrounding retina called the fovea centralis, which is a point where the eyesight is most sensitive, with no blood vessels except for a high concentration of cone cells. The optic disc is located on the fundus a little inside (towards the nose) of the macula, and is where the nerve fibers connected to the photoreceptor cells on the retina are gathered. The light information received by the retina leaves the eyeball from here and is sent to the brain to become an image. The optic disc is also the gathering point of blood vessels in the retina, and from here the retinal arteries and retinal veins spread throughout the retina.

(4-1) Retinal Pigment Epithelium The retinal pigment epithelium is located in the outermost layer of the retina's ten layers, and is a single layer of epithelial cells. The tip, called the outer segment, is constantly phagocytosed by the retinal pigment epithelium and replaced with a new one. The retinal pigment epithelium has the ability to phagocytose photoreceptors and regenerate visual substances (retinal, etc.), and constitutes the blood-retinal barrier. It is also the main focus of age-related macular degeneration. Photoreceptors are one of the cells that make up the retina. The retinal pigment epithelium is called a photoreceptor and converts light energy into electrical energy. Fibrosis of the retinal pigment epithelium tissue is a serious pathological condition common to intraocular proliferative diseases, and a common treatment method called fibrosis inhibition can be used to treat multiple diseases. Age-related macular degeneration (AMD), proliferative vitreous band retinopathy, and proliferative diabetic retinopathy (PDR) are all age-related diseases, and are considered to be disease pathologies caused by acquired epigenetic gene changes. One of these is the fibrotic pathology of the retinal pigment epithelium cell tissue (RPE). When fibrosis of the posterior ocular stroma leads to poor visual prognosis, the pathology becomes more malignant and undergoes a functional phase transition of cells. Subsequently, as cellular senescence of the retinal pigment epithelium (RPE) advances, epithelial-mesenchymal transition leads to fibrosis, leading to age-related macular degeneration.

(4-2) Choroidal neovascularization Light that enters the eye passes through the cornea, lens, and vitreous body 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 that control vision are concentrated, and it distinguishes most of the information from light, such as the shape, size, color, solidity, and distance of objects.
Behind the macula, new pathological blood vessels (choroidal neovascularization) that are prone to rupture are formed, and each one leaks blood and exudates into the fundus. As a result, the macula degenerates and becomes damaged, impairing central vision and causing a decline in vision. Vascular endothelial growth factor (VEGF) is closely involved in the development and growth of pathological choroidal neovascularization.

(4-3) Tissues that progress to age-related macular degeneration Tissues that develop age-related macular degeneration include retina-related tissues, the retina that constitutes the blood-retinal barrier, retinal pigment epithelium, and photoreceptor cells that constitute the retina. In addition, the macula is present in the center of the retina, and the macula is a part of the retina where important cells that control vision are concentrated. Behind the macula, pathological blood vessels that are prone to rupture (choroidal neovascularization) are newly formed, and blood leaks from these vessels and overflows into the fundus. As a result, the macula develops the above-mentioned disease due to degeneration or injury, and central vision is impaired, resulting in symptoms such as decreased vision.

(5) Target Diseases Since the pharmaceutical composition of the present invention targets the above-mentioned tissues, the pharmaceutical composition of the present invention can be administered to any of diseases associated with fibrosis, inflammation, and angiogenesis.
Typically, for example, 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 intraocular inflammatory diseases such as ocular inflammatory diseases selected from retinopathies, particularly diabetic retinopathy, arterial hypertension-induced hypertensive retinopathy, radiation-induced retinopathy, sunlight-induced solar retinopathy, trauma-induced retinopathy, for example Purcher's retinopathy, retinopathy of prematurity (ROP) and hyperviscosity-related 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, vitreous-retinal surgery, eye muscle surgery, ocular plastic surgery, eye tumor surgery, conjunctival surgery including pterygium, and/or surgery involving the lacrimal apparatus, in particular after complicated eye surgery, surgery after trauma, and/or non-complicated eye surgery. In particular, diseases of the ocular tissue 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 corneal ulcer, superficial keratitis, macular keratitis, filamentous keratitis, snow blindness, punctate keratitis, e.g. dry keratitis, for: 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; for iridocyclitis in general, in particular acute iridocyclitis, subacute iridocyclitis and chronic iridocyclitis, primary iridocyclitis, recurrent iridocyclitis and secondary iridocyclitis, phacogenic iridocyclitis, Fuchs heterochromic cyclitis, Vogt-Koyanagi syndrome; for iritis; for chorioretinal inflammation in general, in particular focal and disseminated chorioretinal inflammation, chorioretinitis, choroiditis, retinitis, retinochoroiditis, posterior cyclitis, Harada's disease, infectious and parasitic diseases. post-operative 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, vitreous-retinal surgery, eye muscle surgery, ocular plastic surgery, eye tumor surgery, conjunctival surgery including pterygium, and surgery involving the lacrimal apparatus, preferably intraocular inflammation, in particular post-operative intraocular inflammation, preferably post-operative intraocular inflammation 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 Eales' disease and retinal perivasculitis; retinopathies in general, in particular diabetic retinopathy, (arterial hypertension induced ) inflammatory and non-inflammatory diseases of the eye selected from hypertensive retinopathy, exudative retinopathy, radiation-induced retinopathy, sun-induced photoretinopathy, trauma-induced retinopathies, such as Purcher's retinopathy, retinopathy of prematurity (ROP) and/or hyperviscosity-related retinopathy, non-diabetic proliferative retinopathy, and/or proliferative vitreous retinopathy; blebitis; endophthalmitis; sympathetic ophthalmia; styes; chalazions; blepharitis; eyelid dermatitis and other inflammations; dacryoadenitis; dacryoductitis, particularly acute and chronic canaliculitis; dacryocystitis; orbital inflammation, particularly orbital cellulitis, orbital periostitis, orbital Tenon's capsuleitis, orbital granuloma and orbital myositis; suppurative endophthalmitis and parasitic endophthalmitis.

The present invention will be described in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.
[Example 1]
(In vivo study on the effect of maintaining bleb after trabeculectomy)

1. Methods: Rabbit model of glaucoma filtration surgery using a cannula, administration of OBP-801, and observation. OBP-801 was diluted with ocular irrigation fluid (BSS: Balanced Salt Solution) to prepare a pharmaceutical composition for administration to the treatment group.

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

After the cannula is sutured and fixed to the sclera, the conjunctiva is sutured, atropine eye drops and Rinderon A ointment are injected, and the surgery is completed. After surgery, antibiotics and steroids are administered locally. OBP-801 is administered subconjunctivally on the 1st, 3rd, and 5th days after surgery. From the 1st to the 30th day, the degree of ocular inflammation, anterior chamber depth, and properties of the conjunctival filtration bleb are observed once every 2-3 days in accordance with the postoperative examination, and the size of the filtration bleb and intraocular pressure are measured. Furthermore, the animal is euthanized by overdosing on anesthetics, etc., to prevent pain, and the eyeball is removed and the effect of intraocular perfusate, MMC, and OBP-801 on maintaining the filtration bleb is examined histologically. If any abnormality such as sudden weight loss is observed, the animal is euthanized by overdosing on pentobarbital, and this is the humane endpoint. The above one-month experiment constituted one course.

Intraocular pressure was measured three times in both eyes using a Tonovet, and the median value was recorded as the measured value.
◆ Western blotting Tissues frozen with liquid nitrogen were crushed in a mortar, and 30 μl of RIPA buffer (plus protease inhibitor) was added per 10 mg of tissue. Then, the tissue was vortexed, ultrasonically crushed (5 min, 30 sec intervals), and dissolved by rotation at 4°C (several hours), and the remaining lysate was precipitated and removed at 10,000 g at 4°C for 20 min, and the supernatant was collected. To quantify the protein in this sample (BCA kit), electrophoresis was performed on SDS-page: 30 μg/lane [iBlot 4-12% Bis-Tris Plus Gel], and transferred to a PVDF membrane [iBlot PVDF Transfer Stack Regular]. Blocking and antibody reaction (iBind Western System) were detected by ECL chemiluminescence (NOVEX ECL CHEMI SUBSTRATE) (LAS3000 (Fuji film)).

◆ Tissue staining Cryostat sections (10-μm thick) were coated with 2% silane (3-aminopropyltriethoxysilane) and collected on slide glass. The sections were fixed for 15 min using cold methanol (-30°C) and then air-dried. Next, blocking was performed by reacting the sections with a reaction solution containing 1% bovine albumin at room temperature for about 30 to 60 min.
After primary antibody reaction: overnight at 4°C, and secondary antibody reaction: at room temperature for 60 minutes, the specimen was mounted with a mounting medium containing DAPI (VECTASHELS with DAPI). After mounting with nail polish, the specimen was stained with picrosirius red and observed under a fluorescent microscope.

2. Results [Pharmacological effects of OBP-801]
(1) Effect of OBP-801 Administration on Maintaining Low Intraocular Pressure A decrease in intraocular pressure was observed immediately after surgery in both the OBP-801 and BSS groups.
In the OBP-801 administration group, intraocular pressure remained lower 30 days after surgery compared to pre-surgery levels (Figures 1 and 2).
On the other hand, in the BSS administration group, intraocular pressure increased from 15 days after surgery onwards, and compared with the OBP-801 administration group, intraocular pressure could not be maintained low for a long period of time (FIGS. 1 and 3).

(2) Suppression of conjunctival congestion at the filtering bleb On the 14th day after surgery, severe congestion was observed in the conjunctiva at the filtering bleb in the BSS administration group, whereas conjunctival congestion at the filtering bleb was mild in the OBP-801 administration group (Figure 4).
(3) Number of OBP-801 Administrations The number of OBP-801 administrations was reduced from six times (just before surgery, and 1, 3, 5, 7, and 9 days after surgery) to four times (just before surgery, and 1, 3, and 5 days after surgery), and the same level of low intraocular pressure was maintained up to 30 days after surgery (Figure 5).

(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). By administering OBP-801 only during the efficacy phase (3, 5, and 7 days after surgery) and also during the perioperative period (just before surgery and 1 day after surgery), lower intraocular pressure could be maintained (Figure 6).
(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).

(6) Administration Method It was shown that a lower intraocular pressure was maintained when an appropriate dose (200 μl of 10 nM OBP-801 subconjunctival injection) was administered multiple times compared to when a single dose was increased (100 μl of 100 μM OBP-801 subconjunctival injection) ( FIG. 8 ).

[Optical properties of OBP-801]
(7) Relationship between intraocular pressure reduction and αSMA expression level In three rabbits in which intraocular pressure reduction was observed 14 days after surgery, the expression level of αSMA in the filtration bleb tissue was examined by immunohistochemistry. The expression level of αSMA varied from individual to individual, suggesting that there is little correlation between the intraocular pressure reduction effect and the expression level of αSMA ( FIG. 9 ).
(8) Relationship between decrease in intraocular pressure and collagen expression level 14 days after surgery The expression levels of type I collagen and type III collagen fibers were analyzed by picrosirius red staining. The same level of collagen was expressed in rabbits in which a decrease in intraocular pressure was observed 14 days after surgery and in rabbits in which a decrease in intraocular pressure was not observed, so the relationship between the decrease in intraocular pressure and the collagen expression level 14 days after surgery is unclear ( FIG. 10 ).

(9) Relationship between decrease in intraocular pressure and collagen expression level 30 days after surgery The expression levels of type I collagen and type III collagen fibers were analyzed by picrosirius red staining. In rabbits in which a decrease in intraocular pressure was observed on the 30th day and rabbits in which a decrease in intraocular pressure was not observed, the expression level of collagen in the filtration bleb was low in the rabbits in which a decrease in intraocular pressure was observed, whereas the expression level of collagen was high in the rabbits in which a decrease in intraocular pressure was not observed, suggesting a correlation between intraocular pressure and collagen expression level ( FIG. 11 ).
[Example 2]
(In vitro study on the effect of maintaining bleb after trabeculectomy)

1. Methods Cell culture, OBP-801 addition (including pulse), and observation Conjunctival fibroblasts (HconF) (P1) were purchased from ScienCell (#6570).
◆Culture (Culture according to the instructions):
Culture medium: Fibroblast Medium (#2301) was supplemented with the accompanying medium additives, antibiotics, and FBS.
The cells were seeded at 5×10 ³ cells/cm ² on a culture vessel coated with poly-l-lysine.
All experiments were performed at passage number (P) 3.

Drug 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 culture medium to prepare a pharmaceutical composition to be administered to the treatment group.
When the cells reached 80-90% confluence, the medium was replaced with one lacking 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.
◆Observation Live cells were observed using a phase contrast microscope.
◆Immunostaining After removing the medium, the cells were washed with PBS 3 times for 5 min, and then fixed with cold methanol (-30°C) for 15 min. The cells were air-dried, and blocked with a reaction solution containing 1% bovine albumin for about 30 to 60 minutes at room temperature. The primary antibody reaction was performed at 4°C O/N with PBS 3 times for 5 min, and the secondary antibody reaction was performed at room temperature for 60 minutes. Nuclear staining was performed with 5 μg/ml DAPI at room temperature for 15 minutes, and the cells were washed with PBS 3 times for 5 min, and then observed with a fluorescent microscope with PBS in the cells.

Western blotting Extraction reagent: SDS-HBS; 50 μl of 1% SDS, 150 mM NaCl in 10 mM Hepes (pH 7.4) was used, and the mixture was boiled for 3 minutes and sonicated for 5 minutes (15 sec-10 sec pause) in a 35 mm dish. Protein quantification (BCA kit) was performed by SDS-page electrophoresis: 30 μg/lane [iBlot 4-12% Bis-Tris Plus Gel], followed by transfer to a PVDF membrane [iBlot PVDF Transfer Stack Regular], blocking and antibody reaction [iBind Western System], and detection [LAS3000 (Fuji film)] by ECL chemiluminescence [NOVEX ECL CHEMI SUBSTRATE].
Measurement of HDAC activity Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002).
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 non-deacetylated substrate was detected with an anti-acetylated histone antibody.

Measurement of HAT activity Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002).
EpiQuik HAT Activity/Inhibition Direct Assay Kit (Epigenetic #P-4003) was used.
After reacting the sample with the deacetylated substrate coated on the plate, the acetylated substrate was detected with an anti-acetylated antibody.
* PCR Array The following operations were carried out according to the kit protocol.
RNA extraction: RNeasy mini kit (QIAGEN #74104)
Reverse transcription reaction: 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)

2. Results Drug properties of OBP-801 (1) Inhibition of muscle fibrosis induction by HconF by OBP-801 It was shown that the expression of αSMA and Type IV Collagen (col4) was induced in conjunctival fibroblasts (HconF) by stimulation with TGF + TNF (Figures 12 and 13). It is believed that the HconF culture system can be used as a model for fibrotic tissue formation in filtering blebs.
We confirmed that OBP-801 has an inhibitory effect on the expression of αSMA and Type IV Collagen LOX 12 induced by TGF+TNF stimulation (FIGS. 12 and 13), suggesting that OBP-801 may be effective in maintaining the filtering bleb during glaucoma surgery.
(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 (Figure 14).

(3) Concentration and Treatment Time of OBP-801 When cells were treated with 5 nM OBP-801 for 5 hours prior to the induction of fibrosis, a strong inhibitory effect on the expression of αSMA was observed (FIG. 15).
(4) Changes in Cell Number When cells were treated with 5 nM OBP-801 for 5 hours prior to the induction of fibrosis, no decrease in cell number was observed (FIG. 16).

(5) Relationship with HDAC inhibitory activity No changes were observed in HDAC activity or HAT activity when comparing HconF before and after fibrosis induction by TGF + TNF stimulation. Therefore, it was suggested that the pharmacological effect of OBP-801 is not dependent on HDAC inhibitory activity (Figure 17).
After treatment of HconF with OBP-801, the amount of acetylated histones increased for up to 2 days, but was reduced by half by day 7. As mentioned above, the pharmacological effect of OBP-801 persisted up to 30 days after surgery, suggesting that the pharmacological effect of OBP-801 is not dependent on HDAC inhibitory activity (Figure 18).

(6) Comprehensive analysis of changes in expression of fibrosis-related genes after trabeculectomy in rabbits The peak expression of fibrosis-related genes after trabeculectomy differed depending on the gene. They were roughly classified into three groups based on the time of expression, and are shown in Figures 19 to 21. In the figures, red plots indicate the OBP-801 administration group, and blue plots indicate 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 to 21). In addition, OBP-801 suppressed multiple genes regardless of the expression stage of the genes (Figs. 19 to 21).

(7) Comparison with HDAC inhibitor SAHA OBP-801 at 1 nM has the same level of fibrosis suppression effect as SAHA at 1 μM ( FIG. 22 ).
It was shown that OBP-801 had a cell proliferation inhibitory effect on HconF cells in which muscle fibrosis was induced by administration of TGFβ and TNFα (FIG. 23).
OBP-801 at 0.25 nM has the same cell proliferation inhibitory effect as SAHA at 0.25 μM. Combined with its Col16 expression inhibitory effect, it is expected to have a low intraocular pressure maintenance effect by inhibiting excessive fibrotic scar formation (FIG. 23).
[Example 3]
(In vitro test on glaucoma suppression 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 group.
(1) OBP-801 suppressed the expression of αSMA, collagen, and LOX genes in HTMCs whose fibrosis was induced by TGF+TNF, and was shown to inhibit the myofibroblastic transformation of HTMCs (FIGS. 24 and 25).
[Example 4]
(In vivo test on the effect of inhibiting age-related macular degeneration)

1. Method <Laser-irradiated CNV-induced mouse model, OBP-801 administration, observation>
(iii) Laser irradiation Mice were anesthetized with 0.15 ml of Ketalar 9 mg/ml + Seractal 1 mg/ml i.p.
Mydriasis: One drop of Mydrin P eye drops was instilled into the eye, and about 5 minutes later, the mouse was ready for treatment. PBS was then applied at appropriate times to prevent drying, and laser irradiation was then performed.
Laser irradiation: Scopisol eye drops were applied, a cover glass was attached, and the irradiation was performed under the following conditions.
Irradiation: Red, 200 mW, 100 ms, 50 μm. Irradiation was performed on one eye only, at a site away from the optic nerve, in the 3, 6, 9, and 12 o'clock directions.

■ OBP-801 administration Vitreous injection was performed on the control group (1 ml PBS + 1 μl DMSO) and the experimental group (1 ml PBS + 1 μl 10 μM OBP-801), and the sclera near the ciliary body was incised using a 22.5° slit knife (the incision width was approximately the diameter of a 30 G needle). A 32 G was inserted into the incision with the bevel up, and 1 μl of solution was injected. The crystalline lens was avoided, and taking into consideration the amount of liquid and leakage at the bevel up insertion site, the injection was performed at a position where the inserted needle was slightly visible, but did not reach the retina, with a little more than the 0.5 μl mark.

◆Isolectin B4 staining After euthanasia, the eyeballs were removed and kept in 4% PFA/PBS at room temperature for about 1 hour, and the anterior segment and sclera were excised to prepare retinal flatmounts. After fixation for 10 minutes using -20°C methanol, the retina was washed with 4% PFA/PBS (room temperature for 10 minutes) and then blocked (1% fetal calf serum, 0.1% Triton X-100 in PBS RT, shaking for 1 hour).
Alx594-conjugated isolectin B4 (1:100) (Invitrogen #I21413) was shaken overnight at 4°C and fixed with 4% PFA/PBS at room temperature for 10 minutes. After processing with a DAPI-containing mounting medium (VECTASHELD), observation and photography were performed using a fluorescent microscope.

◆Choroidal Flat-mount
After euthanasia, the mouse was excised and kept in 1% PFA/PBS at room temperature for 2 hours. After removing the anterior segment and the lens, four incisions were made radially and the retina was removed.
Immunostaining (choroidal flat-mount):
The tissue was shaken in PBS buffer at room temperature for 30 min (500 μl/well, 48-well plate), and then shaken in blocking (5% BSA/PBS) at room temperature for 1 hour (200 μl/well, 48-well plate). The antibody was reacted with the plate at 4° C. overnight with shaking (100 μl/well, 48-well plate), and the plate was washed three times with 0.1% TX100/PBS. Next, the second antibody was incubated at room temperature for 1 hour (100 μl The cells were incubated in a 48-well plate (1 well) and washed three times with 0.1% TX100/PBS, then mounted (VECTASHELD with DAPI) and observed and photographed using a confocal laser microscope.

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 body, neovascularization was confirmed in the retina on the choroid side, demonstrating that the mouse could be used as a CNV model ( FIG. 26 , control). When 10 nM OBP-801 was administered at 1 μl/eye immediately after laser irradiation, no neovascularization was confirmed and CNV was suppressed ( FIG. 26 , OBP-801 treatment group).

(2) Inhibitory effect of OBP-801 administration on CNV (fluorescein fundus angiography)
Nine days after laser irradiation of the mouse vitreous, leakage of the dye that had been administered into 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).
(3) Inhibitory effect on collagen I expression In control mice, collagen I signals were observed around all laser-irradiated areas, but collagen I signals were reduced in OBP-801-treated mice (Figure 29).

(4) Inhibitory effect on αSMA expression In the control mice, αSMA signals were observed around all laser irradiated areas, whereas the αSMA signals were reduced in the OBP-801-treated mice (FIGS. 30 and 31).
(5) Inhibitory Effect on CD31 Expression In the control mice, CD31 signals were observed around all laser-irradiated areas, whereas the CD31 signals were reduced in the OBP-801-treated mice (FIG. 32).
[Example 5]
(In vitro test on the effect of inhibiting 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)
Culture medium: DMEM/F12 (Invitrogen: 11330-032) supplemented with 10% FBS and antibiotics.
All experiments were performed at passages P23-26.
◆ Human retinal pigment epithelial cells (H-RPE: 00194987 LONZA) (P2) were purchased and subjected to the following treatment.
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.

Drug 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 culture medium to prepare a pharmaceutical composition to be administered to the treatment group.
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 using a phase contrast microscope.
◆Immunostaining The medium was removed, and the cells were washed three times with PBS for 5 min each. The cells were fixed in cold methanol (-30°C) for 15 min and air-dried. Blocking: The cells were reacted in 1% bovine albumin solution at room temperature for approximately 30-60 minutes. The cells were reacted with the primary antibody overnight at 4°C, and then washed three times with PBS for 5 minutes each. The cells were reacted with the secondary antibody for 60 minutes at room temperature. Nuclear staining was performed using 5 μg/ml DAPI for 15 minutes at room temperature. The cells were washed three times with PBS for 5 minutes each. The cells were observed under a fluorescent microscope with PBS in the cells.

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 min, followed by sonication for 5 min (15 sec-10 sec pause). A BCA kit was used for protein quantification. 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 [iBind Western System], ECL chemiluminescence [NOVEX ECL CHEMISTRY] was used. The mixture was subjected to SUBSTRATE and detected using an LAS3000 (Fuji film).

Measurement of HDAC activity Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002).
Using EpiQuik HDAC Activity/Inhibition Direct Assay Kit (Epigenetic #P-4034), the sample was reacted with an acetylated histone substrate coated on a plate, and the non-deacetylated substrate was detected with an anti-acetylated histone antibody.
Measurement of HAT activity Nuclear extraction was performed using EpiQuik Nuclear Extraction Kit I (Epigenetic #OP-0002).
Using EpiQuik HAT Activity/Inhibition Direct Assay Kit (Epigenetic #P-4003), the sample was reacted with the deacetylated substrate coated on the plate, and then the acetylated substrate was detected with an anti-acetylated antibody.

◆ PCR array RNA extraction was performed using RNeasy mini kit (QIAGEN #74104), followed by reverse transcription using RT2 First Strand Kit (QIAGEN #330401), and PCR using ^RT2 SYBR Green ROX qPCR Mastermix (QIAGEN #330522) and ^{RT2 ProfilerTM} PCR Array Human Fibrosis (QIAGEN #PAHS-120ZC).

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, whose expression was increased by treating RPE cells with TGFβ alone, TNFα alone, or TGFβ + TNFα, demonstrating that OBP-801 inhibits RPE cell fibrosis (Figure 33).
(2) Effect of OBP-801 on fibrosis-related gene expression OBP-801 suppressed the expression of MMP9, CD44, and αSMA, whose expression was increased by the treatment of RPE cells with TGFβ alone, TNFα alone, or TGFβ + TNFα. In particular, the inhibitory effect of OBP-801 on the expression of MMP9 and CD44 was higher than that of TSA (Figure 34).

(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. This suggests that the pharmacological effect of OBP-801 is not dependent on HDAC inhibitory activity (Figure 35).
When RPE cells were induced to undergo fibrosis, HAT activity was decreased. OBP-801 did not inhibit HAT activity either before or after fibrosis induction. This suggests that the pharmacological effect of OBP-801 is not dependent on HDAC inhibitory activity (Figure 36).
Furthermore, an investigation of the effect on CD44 expression revealed that OBP-801 had a suppressive effect (Figure 37).
[Example 6]
Intraocular pressure suppression test and gene expression test using OBP-801

1. Methods: Rabbit model of glaucoma filtration surgery using a cannula, administration of mitomycin and OBP-801, and observation. Mitomycin C (MMC) was dissolved in water for injection and administered at 0.02% (w/v) in a volume of 100 μl 30 minutes before surgery. Glaucoma surgery, administration of OBP-801, and administration of intraocular irrigation solution 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.

* PCR Array The following operations were carried out according to the kit protocol.
RNeasy mini kit (QIAGEN #74104) was used for RNA extraction.
For the reverse transcription reaction, RT2 First Strand Kit (QIAGEN #330401) was used.
RT ² SYBR Green ROX qPCR Mastermix (QIAGEN #330522) was used for the PCR reaction.
RT ² Profiler® PCR Array Rabbit Fibrosis (QIAGEN #PANZ-120ZC) was used.

◆Real-Time PCR
The following operations were carried out in accordance with the kit protocol.
RNeasy mini kit (QIAGEN #74104) was used for RNA extraction.
PrimeScript® RT Master Mix (TAKARA #RR036A) was used for the reverse transcription reaction.
TB Green® Premix Ex Taq® II (TAKARA #RR820B) was used for PCR reaction (intercalator). Primers provided PDGFB, LOX, LOXL2, PDGFRA, and PDGFRB (designed and purchased by TAKARA). TaqMan Fast Advanced Master Mix (Invitrogen #4444963) was used for PCR reactions (fluorescently labeled probes). Primers used were Oc03398424_m1 (TGFB2), Oc03399251_m1 (ACTA2), Oc03396112_m1 (COL1A2), and Oc03395687_g1 (CTGF) (Applied Biosystems #4453320).

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 an ocular irrigation solution (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 (with a 30-second interval between each dose) per course, with one course being administered 30 minutes before surgery and then the morning and evening on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th days after surgery, for a total of two courses. From 1st to 30th days after surgery, the degree of ocular inflammation, anterior chamber depth, and properties of the conjunctival filtration bleb were observed in accordance with the postoperative examination, and the size of the filtration bleb and intraocular pressure were measured once every 2-3 days.

2. Results (1) Comparison with Mitomycin C The effect of mitomycin C (MMC), which is currently used in glaucoma surgery, on gene expression after trabeculectomy was compared with that of OBP-801 (FIGS. 39 to 46).
The expression of type I collagen (COL1A), which is 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 not suitable for long-term (30 days or more) intraocular pressure control (Figure 39). In addition, OBP-801 inhibited the expression of all of the genes thought to be involved in COL1A expression (TGFB2, SERPINH1, αSMA, CTGF, PDGFB) at 30 days, indicating the superiority of OBP-801 in long-term intraocular pressure control (Figures 40, 45, and 46).

Furthermore, the effects of OBP-801 and MMC on the expression of fibrosis-related genes after trabeculectomy were comprehensively analyzed and compared. They were roughly classified into six categories according to function, and the results are shown in Figures 41 to 44.
It has also been reported that LOX and LOXL2 are involved in scar tissue formation and have an inhibitory effect on the maintenance of blebs. In the BSS group (Control), increased expression was observed on the second day after surgery, but both OBP-801 and MMC showed inhibition of 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).

(2) Effect 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).
In addition, the effect of OBP-801 eye drop on fibrosis-related gene expression was comprehensively analyzed and compared with the results of OBP-801 subconjunctival injection. 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 filtration blebs, were suppressed by both subconjunctival injection and eye drop (Figure 48).
[Example 7]
Inhibitory effect of OBP-801 on gene expression in rabbit conjunctiva and bleb tissues

1. Methods ◆ Western blotting Conjunctival tissue and bleb tissue were collected from rabbit eyeballs, and protein was extracted using RIPA buffer. The tissue was dissolved by sonication for 5 min (15 sec-10 sec pause) and O/N rotation at 4°C, and the remaining tissue was removed by centrifugation (10,000 g for 10 min). A BCA kit was used for protein quantification. SDS-page electrophoresis (30 μg/lane [iBlot 4-12% Bis-Tris Plus Gell]) was performed, and the tissue was transferred to a PVDF membrane [iBlot PVDF Transfer Stack Regular]. After blocking and antibody reaction (iBind Western System), the antibodies were subjected to ECL chemiluminescence (NOVEX ECL CHEMI SUBSTRATE) and detected (LAS3000 (Fuji film)).

◆Tissue immunostaining Conjunctival tissue and bleb tissue were collected from rabbit eyeballs, embedded in compound (SurgiPath FSC 22), and frozen in liquid nitrogen. Sections (10-μm thick) prepared using a cryostat (CM3050S: Leica) were collected on slides coated with 2% silane (3-aminopropyltriethoxysilane). After fixation for 15 min using cold methanol (-30°C), the tissues were air-dried. Subsequently, blocking was performed using a reaction solution containing 1% bovine albumin, and the tissues were reacted at room temperature for about 30 to 60 minutes. Primary antibody reaction: 4°C O/N, secondary antibody reaction: reacted at room temperature for 60 minutes, and then mounted with DAPI mounting medium (VECTASHELS with DAPI). After sealing the surrounding area with nail polish, the specimen was observed using a fluorescence microscope.

2. Results (1) Western blotting analysis of rabbit conjunctival tissue (Figure 49)
In the untreated (BSS) group 30 days after surgery, when intraocular pressure was seen to rise again, increased expression of type I collagen and aSMA was observed. The expression of these was suppressed by OBP-801. This is consistent with the results of RT-PCR (RNA expression).
(2) Immunostaining of rabbit filtering bleb tissue (Figs. 50 to 52)
Thirty days after surgery, when intraocular pressure was increasing again, the expression of collagen I and α-SMA in the bleb was increased in untreated (BSS) tissues. The expression of these proteins was suppressed in OBP-801-treated tissues. Furthermore, the expression of these proteins was significantly increased in MMC-treated tissues.
[Example 8]
Human conjunctival tissue genetic analysis results

1. Methods 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 by PCR array.
The following operations were carried out in accordance with the kit protocol.
RNeasy mini kit (QIAGEN #74104) was used for RNA extraction.
For the reverse transcription reaction, RT2 First Strand Kit (QIAGEN #330401) was used.
RT2 SYBR Green ROX qPCR Mastermix (QIAGEN #330522) was used for the PCR reaction.
RT2 Profiler® PCR Array Human Fibrosis (QIAGEN #PAHS-120ZC) was used.

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

Claims (8)

The following formula III:

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
or a pharma- ceutical acceptable salt thereof for inhibiting fibrosis of a trabecular meshwork, a filtration bleb tissue, a conjunctiva, a subconjunctival tissue, a Schlemm's canal, or a sclera. The pharmaceutical composition of claim 1 , wherein R4 is an isopropyl group. The following formula III:

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
or a pharma- ceutical composition for inhibiting choroidal neovascularization, comprising a depsipeptide compound represented by the formula: The following formula III:

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
or a pharma- ceutical composition for inhibiting choroidal neovascularization, comprising a depsipeptide compound represented by the formula: The following formula III:

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
or a pharmaceutical acceptable salt thereof, wherein the depsipeptide compound or a pharmaceutical acceptable salt thereof is administered at a dose of 100 pg/kg to 500 pg/kg. The following formula III:

(In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
or a pharmaceutical acceptable salt thereof, wherein the depsipeptide compound or a pharmaceutical acceptable salt thereof is administered at a dose of 2 pg/eye to 10 pg/eye. 7. The pharmaceutical composition according to claim 3, wherein R4 is an isopropyl group. A drug for treating age-related macular degeneration, comprising the pharmaceutical composition according to any one of claims 3 to 7.

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