Pharmaceutical composition for preventing or treating diseases or conditions associated with corneal endothelial disorders

Abstract

Provided is a pharmaceutical composition for preventing or treating diseases or conditions associated with corneal endothelial disorders.　It was discovered that, in diseases or conditions associated with corneal endothelial disorders, a compound having an estrogen activity inhibits the decrease in the density of corneal endothelial cells and inhibits the production of an extracellular matrix.

Description

Pharmaceutical compositions for the prevention or treatment of diseases or conditions related to corneal endothelial disorders.

[0001] This invention relates to a pharmaceutical composition for the prevention or treatment of diseases or conditions related to corneal endothelial disorders.

[0002] The cornea is the transparent tissue at the very front of the eyeball. It functions as a lens, taking in and refracting light and directing it into the eyeball, playing a crucial role in obtaining visual information. For good vision, a transparent cornea is essential.

[0003] The corneal endothelium plays a crucial role in maintaining corneal transparency by acting as a pump to regulate corneal moisture supply. While the cornea protects the surface of the eyeball, it is also susceptible to damage. Damage to the corneal endothelial cells located on the inner surface of the cornea can lead to corneal opacity and severe visual impairment due to bullous keratopathy.

[0004] In Fuchs endothelial corneal dystrophy (FECD), corneal endothelial cells undergo cell death, and the remaining corneal endothelial cells are unable to compensate for this, leading to a loss of corneal transparency, progressive opacity due to corneal edema, and ultimately bullous keratopathy. Furthermore, corneal endothelial cells in patients with Fuchs endothelial dystrophy produce excessive extracellular matrix, creating structures called guttaes on the thickened Descemet's membrane, which are primarily composed of extracellular matrix. Non-patent document 1 reports that in immortalized cell lines derived from patients with Fuchs endothelial dystrophy, corneal endothelial cells are more sensitive to oxidative stress than normal cells, resulting in increased DNA damage and apoptosis.

[0005] Fuchs' corneal endothelial dystrophy is a major cause of bullous keratopathy, but currently the only treatment available is corneal transplantation. The inventors have previously reported on culture techniques for human corneal endothelial cells for use in corneal transplantation, and methods for preserving corneal endothelial cells for use as therapeutic cells for corneal endothelial diseases, etc. (Non-Patent Document 2, Patent Document 1). Patent Document 2 also reports on methods for producing corneal endothelial cells derived from human embryonic stem cells and induced pluripotent stem cells as substitutes for donor corneas. Furthermore, the inventors have discovered that the deposition of extracellular matrix observed in Fuchs' corneal endothelial dystrophy, etc., can be suppressed by inhibiting the TGF-β pathway, and have developed a drug treatment for diseases related to extracellular matrix abnormalities in the corneal endothelium using a TGF-β signaling inhibitor (Patent Document 3). However, no pharmaceutical composition is known that suppresses both the deposition of extracellular matrix in the corneal endothelium and the decrease in cell density.

[0006] Patent No. 6664755 Special table 2022-513736 Patent No. 6403217

[0007] Investigative ophthalmology & visual science, 2011, 52.13: 9291-9297PloS one, 2013, 8.7: e69009

[0008] The object of this invention is to provide a pharmaceutical composition that can prevent or treat diseases or conditions related to corneal endothelial disorders.

[0009] As a result of diligent research to solve the above problems, the present inventors have discovered that compounds having estrogen activity suppress the decrease in corneal endothelial cell density and the production of extracellular matrix in diseases or conditions related to corneal endothelial damage, and have completed the present invention.

[0010] In other words, the present invention comprises the following: 1. A pharmaceutical composition for the prevention or treatment of a disease or condition related to corneal endothelial dysfunction, comprising an estrogen-active compound as an active ingredient. 2. The pharmaceutical composition according to item 1, wherein the estrogen-active compound is estradiol or a pharmaceutically acceptable salt thereof. 3. The pharmaceutical composition according to item 1 or 2, wherein the disease or condition related to corneal endothelial dysfunction is one or more diseases selected from the group consisting of Fuchs' corneal endothelial dystrophy, bullous keratopathy, corneal endothelial dysfunction after corneal transplantation, posterior polymorphic corneal dystrophy, congenital hereditary corneal endothelial dystrophy, iris-corneal endothelial syndrome, corneal edema, cytomegalovirus keratitis, herpes simplex virus keratitis, and exfoliation syndrome. 4. The pharmaceutical composition according to any one of items 1 to 3, wherein the disease or condition related to corneal endothelial dysfunction is a disease or condition characterized by a decrease in corneal endothelial cell density and accumulation of extracellular matrix. 5. A pharmaceutical composition according to any one of paragraphs 1 to 4 above, wherein the dosage form of the pharmaceutical composition is an eye drop or an eye ointment. 6. Use of estradiol or a pharmacopositically acceptable salt thereof for the manufacture of a pharmaceutical composition for the prevention or treatment of a disease or condition related to corneal endothelial disorder.

[0011] The pharmaceutical composition of the present invention showed excellent therapeutic effects in a Fuchs corneal endothelial dystrophy animal model in terms of corneal endothelial cell density. Furthermore, it showed excellent fibrosis inhibitory effects in cells derived from Fuchs corneal endothelial dystrophy patients. This confirms that the pharmaceutical composition of the present invention exhibits excellent effects against diseases or conditions related to corneal endothelial dysfunction.

[0012] This graph shows the cell viability in immortalized corneal endothelial cell lines derived from Fuchs' corneal endothelial dystrophy patients (hereinafter sometimes simply referred to as "iFECD") in the presence of estradiol (hereinafter sometimes simply referred to as "E2"). The vertical axis shows the cell viability (%) at each E2 concentration (100 nM, 1 μM, 10 μM), with the control group (no E2 added) set to 100%. Error bars indicate the mean ± standard error. (Example 1) This shows the results of Western blots for Smad2, p-Smad2, Smad3, p-Smad3, and GAPDH in the evaluation of the effect of E2 addition on Smad proteins. From the left lane, the results are shown for the control group (no TGF-β2 added), the TGF-β2 added group, and the TGF-β + E2 (1 μM) added group. The left figure shows the results for iFECD cell lines derived from patients in whom the number of cytosine-guanine-thymine trinucleotide repeat sequences (hereinafter sometimes simply referred to as "CTG repeat sequences") in the TCF4 gene sequence is not 50 or more (hereinafter sometimes simply referred to as "RE-") (hereinafter sometimes simply referred to as "iFECD (RE-)"). The right figure shows the results for iFECD cell lines derived from patients in whom the number of CTG repeat sequences in the TCF4 gene sequence is 50 or more (hereinafter sometimes simply referred to as "RE+") (hereinafter sometimes simply referred to as "iFECD (RE+)"). (Example 2) The results of Western blots of Snail, ZEB1, and GAPDH in the evaluation of the effect of E2 addition on epithelial-mesenchymal transition are shown. From the left lane, the control group (no TGF-β2 addition), the TGF-β2 addition group, and the TGF-β + E2 (1 μM) addition group are shown. The left figure shows the results for iFECD (RE-), and the right figure shows the results for iFECD (RE+). (Example 2) Western blotting results for fibronectin, biglycan, and GAPDH in the evaluation of the effects of E2 addition on the extracellular matrix are shown. From the left lane, the control group (no TGF-β2 addition), the TGF-β2 addition group, and the TGF-β + E2 (1 μM) addition group are shown. The left figure shows the results for iFECD (RE-), and the right figure shows the results for iFECD (RE+). (Example 2) Immunostaining results for fibronectin in the evaluation of the effects of E2 addition on the accumulation of the extracellular matrix are shown.The left side shows the control group without TGF-β2, the center shows the group with TGF-β2, and the right side shows the group with TGF-β + E2 (1 μM). The top row shows the results for iFECD (RE-), and the bottom row shows the results for iFECD (RE+). (Example 3) Results of immunostaining of biglycan in evaluating the effect of E2 addition on the accumulation of extracellular matrix are shown. The left side shows the control group without TGF-β2, the center shows the group with TGF-β2, and the right side shows the group with TGF-β + E2 (1 μM). The top row shows the results for iFECD (RE-), and the bottom row shows the results for iFECD (RE+). (Example 3) Results of immunostaining of collagen I in evaluating the effect of E2 addition on the accumulation of extracellular matrix are shown. The left side shows the control group without TGF-β2, the center shows the group with TGF-β2, and the right side shows the group with TGF-β + E2 (1 μM). The top row shows the results for iFECD (RE-), and the bottom row shows the results for iFECD (RE+). (Example 3) The results of immunohistochemistry of aggresomes in evaluating the effect of E2 addition on the accumulation of denatured proteins are shown. The left side shows the control group without TGF-β2 addition, the center shows the group with TGF-β2 addition, and the right side shows the group with TGF-β + E2 (1 μM) addition. The top row shows the results for iFECD (RE-), and the bottom row shows the results for iFECD (RE+). (Example 3) The images of the central corneal endothelium of Col8a2Q455K / Q455K mice, an FECD model mouse, in evaluating the effect of E2 administration on the progression of FECD disease are shown. The left image shows the central corneal endothelium of an FECD model mouse that was orally administered a 0.1% ethanol solution from 8 weeks of age as a control, and the right image shows the central corneal endothelium of an FECD model mouse that was orally administered E2-containing drinking water from 8 weeks of age. (Example 4) Figure 9 shows graphs of corneal endothelial cell density for two groups in the evaluation of the effect of E2 administration on the progression of FECD disease. The left graph shows the data at 12 weeks of age, and the right graph shows the data at 16 weeks of age. In this graph, the vertical axis represents corneal endothelial cell density (cells / mm²). 2(Example 4) This shows panoramic and central images of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice, a late-onset FECD model mouse having a CTG (100 times) repeat sequence, used in the evaluation of the effects of E2 administration in Tcf4 CTG repeat sequence knock-in mice. The upper left image shows a panoramic image of the corneal endothelium of wild-type mice that ingested a 0.1% ethanol solution, the upper center image of Figure 11 shows a panoramic image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice that ingested a 0.1% ethanol solution, and the upper right image of Figure 11 shows a panoramic image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice that ingested E2-containing drinking water. The lower left image shows a representative central image of the corneal endothelium of wild-type mice that ingested a 0.1% ethanol solution, the lower center image of Figure 11 shows a representative central image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice that ingested a 0.1% ethanol solution, and the lower right image of Figure 11 shows a representative central image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice that ingested E2-containing drinking water. (Example 5) Evaluation of the effects of E2 administration in Tcf4 CTG repeat sequence knock-in mice: Guttae area percentage (%) and corneal endothelial cell density (cells / mm²) of the three groups shown in Figure 11. 2 The graphs below show the Guttae area percentage (%) and the corneal endothelial cell density (cells / mm²). The left graph shows the Guttae area percentage (%) and the right graph shows the corneal endothelial cell density (cells / mm²). 2 The graph shows the results. (Example 5)

[0013] The present invention relates to a pharmaceutical composition for the prevention or treatment of diseases or conditions related to corneal endothelial disorders (hereinafter sometimes simply referred to as "the pharmaceutical composition of the present invention"), comprising a compound having estrogenic activity as an active ingredient. The present invention also relates to the use of a compound having estrogenic activity for the manufacture of a pharmaceutical composition for the prevention or treatment of diseases or conditions related to corneal endothelial disorders, a compound having estrogenic activity for use in the prevention or treatment of diseases or conditions related to corneal endothelial disorders, and a method for the prevention or treatment of diseases or conditions related to corneal endothelial disorders comprising administering an effective amount of the compound having estrogenic activity to a target.

[0014] In this specification, "compounds having estrogenic activity" refers to compounds that exhibit all or part of the effects of estrogen. Estrogen, also known as follicular hormone or female hormone, is a well-known steroid hormone involved in the development and differentiation of human reproductive organs, pregnancy, and sexual behavior, but its effects are diverse. Estrogen deficiency is known to be a factor in various diseases, and estrogen replacement therapy is used for treatment. Examples of compounds having estrogenic activity include endogenous estrogens such as estradiol, estrone, estriol, estetrol, ecchiline, and ecchirenin; synthetic estrogens such as ethinylestradiol and mestranol; phytoestrogens; environmental estrogens; and solvates thereof (e.g., hydrates). Estradiol plays an important role in the human body, and its levels fluctuate with age and the menstrual cycle, decreasing significantly after menopause. Changes in its concentration affect various physiological and pathological processes. Estradiol exists as two isomers, α-estradiol and β-estradiol, depending on the coordination of the hydroxyl group at position 17. Both can be used in the present invention, but β-estradiol is preferred from the viewpoint of high estrogenic activity and the accumulation of knowledge regarding side effects.

[0015] In this invention, we have discovered for the first time that a compound having estrogenic activity suppresses the decrease in corneal endothelial cell density. A pharmaceutical composition containing the compound having estrogenic activity of this invention as an active ingredient can suppress the production and accumulation of extracellular matrix in diseases or conditions related to corneal endothelial dysfunction, and can also suppress the decrease in corneal endothelial cell density.

[0016] The pharmaceutical composition containing the estrogenic compound of the present invention as an active ingredient may target diseases or conditions related to corneal endothelial dysfunction. The corneal endothelial cell layer maintains pump and barrier functions related to water transport, is involved in regulating the water content of the cornea, and is essential for maintaining corneal transparency. When corneal endothelial cells are damaged, they fall out, and the surrounding corneal endothelial cells compensate by expanding their area; cell proliferation does not occur. As the decrease in corneal endothelial cell density progresses, corneal endothelial function is impaired, eventually leading to bullous keratopathy due to corneal endothelial cell dysfunction. Specific examples of diseases or conditions related to corneal endothelial dysfunction include Fuchs' corneal endothelial dystrophy, bullous keratopathy, corneal endothelial dysfunction after corneal transplantation, posterior pleomorphic corneal dystrophy, congenital hereditary corneal endothelial dystrophy, iris-corneal endothelial syndrome, corneal edema, cytomegalovirus keratitis, herpes simplex virus keratitis, and exfoliation syndrome.

[0017] As described above, the pharmaceutical composition of the present invention can suppress the production and accumulation of extracellular matrix and suppress the decrease in corneal endothelial cell density. Therefore, it is presumed that the pharmaceutical composition of the present invention is particularly effective against diseases or conditions related to corneal endothelial disorders characterized by a decrease in corneal endothelial cell density and the accumulation of extracellular matrix. For example, Fuchs' corneal endothelial dystrophy is characterized by the formation of guttae due to the accumulation of extracellular matrix deposits in the early stages of the disease, and a decrease in corneal endothelial function due to a decrease in corneal endothelial cell density in the progressive stage, ultimately leading to bullous keratopathy. Bullous keratopathy is characterized by a severe decrease in corneal endothelial cells and the accumulation of extracellular matrix. In the examples described later, it was confirmed that compounds having estrogenic activity suppress the phosphorylation pathway of Smad, which transmits TGF-β signaling, in cells derived from Fuchs' corneal endothelial dystrophy patients. As a result, it was considered that one of the mechanisms of action of compounds having estrogenic activity is the suppression of TGF-β-induced extracellular matrix production. As mentioned above, estrogen is a well-known steroid hormone, and its effects are thought to be diverse. The effect of the estrogenic compound of the present invention on suppressing the production and accumulation of extracellular matrix in diseases or conditions related to corneal endothelial damage is not limited to a mechanism related to TGF-β inhibition, but is thought to be due to a wide range of effects. Furthermore, its effects are milder compared to drugs known as so-called TGF-β inhibitors, and the concern about side effects is considered low. Estrogen preparations can be applied with confidence because estrogen deficiency is known to be a factor in various diseases, and estrogen replacement therapy is used for treatment, and knowledge regarding side effects and other factors is accumulating. In addition, Fuchs' corneal endothelial dystrophy includes patients with "RE-" (repeat expansion negative), where the number of CTG repeat sequences in the TCF4 gene base sequence is less than 50, and patients with "RE+" (repeat expansion positive), where the number is 50 or more. However, it was confirmed in the examples described later that the pharmaceutical composition of the present invention can act effectively on both types of patients.In other words, the pharmaceutical composition of the present invention can be used for both patients with Fuchs' corneal endothelial dystrophy (RE+) and patients with Fuchs' corneal endothelial dystrophy (RE-).

[0018] In the present invention, "target" refers to a human or non-human mammal (e.g., monkey, cattle, horse, dog, cat, rat, etc.) that requires prevention or treatment, preferably a human, and more preferably a human that requires prevention, alleviation, improvement, or treatment of a disease or condition related to corneal endothelial dysfunction.

[0019] The pharmaceutical composition of the present invention comprises a compound having an effective amount of estrogen activity for therapeutic and / or prophylactic purposes. In this specification, “effective amount” means a sufficient amount of the active ingredient to achieve the intended use, including but not limited to therapeutic and prophylactic purposes. In this specification, “therapeutic effective amount” means an amount effective to achieve the desired therapeutic effect in the required dose and duration. In this specification, “prophylactic effective amount” means an amount effective to achieve the desired prophylactic outcome in the required dose and duration. The concentration of the active ingredient, the compound having estrogen activity, in the pharmaceutical composition of the present invention is not particularly limited as long as it is less than 10 μM, but is preferably 0.0001 to 9 μM, more preferably 0.0001 to 8 μM, more preferably 0.001 to 7 μM, more preferably 0.001 to 6 μM, more preferably 0.001 to 5 μM, more preferably 0.01 to 4 μM, more preferably 0.01 to 3 μM, more preferably 0.01 to 2 μM, more preferably 0.05 to 2 μM, more preferably 0.05 to 1 μM, and more preferably 0.1 to 1 μM.

[0020] In this specification, "active ingredient" means a substance capable of providing a preventive or therapeutic effect against one or more symptoms. The pharmaceutical composition of the present invention may contain one or more active ingredients.

[0021] In this specification, "pharmacologically acceptable salt" refers to a pharmacologically acceptable salt of a compound, which may form an acid addition salt or a salt with a base depending on the type of substituent. Examples include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; acid addition salts with organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyl tartaric acid, ditoluyl tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid, valeric acid, benzoic acid, cypionic acid, and propionic acid; salts with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts with organic bases such as methylamine, ethylamine, ethanolamine, lysine, and ornithine; salts with various amino acids and amino acid derivatives such as acetylleucine; ammonium salts; and solvates (e.g., hydrates) of these salts.

[0022] The pharmaceutical composition of the present invention is administered topically (for example, to the eyes, around the eyes, etc.), and topical administration to the eyes is preferred from the viewpoint of reducing side effects and ensuring stability of the drug's efficacy.

[0023] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it can be used as a pharmaceutical product, and examples include eye drops, eye ointments, gels, sprays, creams, topical solutions, transdermal formulations, patches, injections, liquids, suspensions, etc. Preferably, it is an eye drop or eye ointment.

[0024] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives in addition to the above-mentioned active ingredients. Such additives include, for example, sugars, cooling agents, inorganic salts, organic acid salts, acids, bases, antioxidants, stabilizers, excipients, and preservatives, and can be used as needed. Examples of sugars include glucose, mannitol, sorbitol, xylitol, dextrin, honey, tragacanth, and trehalose. Examples of cooling agents include menthol and camphor. Examples of inorganic salts include sodium chloride and potassium chloride. Examples of organic acid salts include sodium citrate. Examples of acids include phosphoric acid, citric acid, sulfuric acid, and acetic acid. Examples of bases include trishydroxymethylaminomethane and monoethanolamine. Examples of antioxidants include tocopherol acetate and dibutylhydroxytoluene. Examples of stabilizers include sodium edetate and glycine. Examples of excipients include the above-mentioned sugars, alcohols, glycerin, starch, precipitated silica, gelatin, magnesium stearate, and mineral oil. Examples of preservatives include benzalkonium chloride, chlorhexidine gluconate, potassium sorbate, and polyhexanide hydrochloride. The pharmaceutical composition according to the present invention may also contain other active ingredients. The amount of each ingredient can be appropriately determined within a range acceptable for pharmaceutical use.

[0025] The method of administering the pharmaceutical composition of the present invention is not particularly limited and is determined appropriately on a case-by-case basis, taking into consideration the dosage form, symptoms, age, sex of the recipient, etc. For example, in the case of eye drops, 0.01 to 0.2 mL can be dropped into the eye (eyeball) 1 to 10 times, 1 to 8 times, 1 to 6 times, 1 to 4 times, or 1 to 3 times per day. The administration period is not particularly limited and can be set appropriately depending on the type and condition of the recipient, etc. For example, it may be 1 to 1000 days or 1 to 300 days, preferably 7 to 300 days, more preferably 14 to 150 days, more preferably 21 to 100 days, and more preferably 28 to 70 days.

[0026] The pharmaceutical compositions of the present invention can be manufactured according to conventional methods in the art. For example, in the case of eye drops, they can be manufactured by a general method for manufacturing eye drops, which includes mixing a compound having estrogenic activity, water, and optionally a pharmacochemically acceptable additive. The method may further include, if necessary, steps such as sterile filtration.

[0027] To aid in understanding the present invention, the present invention will be specifically described below with reference to examples, but it goes without saying that the present invention is not limited to these examples.

[0028] (Preparation Example) Preparation of immortalized corneal endothelial cell line (iFECD) derived from Fuchs' corneal endothelial dystrophy (FECD) patients. An immortalized corneal endothelial cell line was prepared from corneal endothelial cells derived from FECD patients.

[0029] (Method of acquisition) Corneal endothelial cells were obtained from three human patients who developed bullous keratopathy due to a clinical diagnosis of FECD and underwent corneal endothelial transplantation (Descemet's membrane endothelial corneal transplantation = DMEK), with written consent and approval from the Doshisha University Research Ethics Committee. During the DMEK procedure, pathological corneal endothelial cells were mechanically detached along with the basement membrane, Descemet's membrane, and immersed in Optisol-GS (Bausch + Lomb), a corneal preservation solution. Subsequently, collagenase treatment was performed to enzymatically recover corneal endothelial cells, which were then mixed with Opti-MEM I Reduced-Serum Medium, Liquid (INVITROGEN, 31985-070) and treated with 8% FBS (BIOWEST, S1820-500), 200 mg / ml CaCl2・2H2O (SIGMA, C7902-500G), 0.08% chondroitin sulfate (SIGMA, C9819-5G), 20 μg / ml ascorbic acid (SIGMA, A4544-25G), 50 μg / ml gentamicin (INVITROGEN, 15710-064), and 5 ng / ml Cells were cultured in a conditioned medium for 3T3 feeder cells supplemented with EGF (INVITROGEN, PHG0311), with the addition of SB431542 (1 μmol / l) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole <4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazole-5-yl]pyridine>) (1 μmol / l). Corneal endothelial cells derived from FECD patients were cultured, and the SV40 large T antigen and hTERT gene were amplified by PCR and introduced into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc). Subsequently, lentiviral vectors were used to infect 293T cells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) with three helper plasmids (pLP1, pLP2, pLP / VSVG; Life Technologies Inc.) using a transfection reagent (FugeneHD; Promega Corp., Madison, WI).After 48 hours of infection, the culture supernatant containing the virus was collected and added to the culture medium of corneal endothelial cells derived from FECD patients using 5 μg / ml polyblen to introduce the SV40 large T antigen and the hTERT gene, thereby creating an iFECD cell line. Genetic analysis confirmed that iFECD cells contained both RE+ and RE-. Phase-contrast microscopy images of iFECD cells confirmed that they had a single-layer polygonal morphology similar to normal corneal endothelial cells. iFECD cells were maintained in Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (P / S) (hereinafter sometimes referred to as "DMEM + 2% FBS + 1% P / S medium").

[0030] (Example 1) Measurement of cell viability in the presence of estradiol (E2) In this example, cell viability in the presence of E2 was measured using iFECD.

[0031] (Materials and Methods) iFECD was placed in a 96-well plate in 8 × 10⁶ units. 3 Individual seeds were seeded and cultured for 24 hours at 37°C under 5% CO2 conditions. The culture medium used was Dulbecco's modified Eagle medium (DMEM) (nacalai tesque, 08490-05) + 10% FBS (SIGMA, 172012) + 1% penicillin-streptomycin (nacalai tesque, 26252-94). Subsequently, the medium was removed and DMEM + 2% FBS + 1% P / S medium containing DMSO (Dimethyl Sulfoxide, Sterile-filtered) (nacalai tesque, 13408-64) and E2 (100 nM, 1 μM, 10 μM) (Sigma-Aldrich, E8875) was added, and cultured for 24 hours. After 24 hours, the same procedure was repeated. After that, CellTiter-Glo was performed according to the following procedure. TM Cell viability was analyzed using the Luminescent Cell Viability Assay. The culture medium was removed from each well to 50 μl, and CellTiter-Glo was used. TMThe Luminescent Cell Viability Assay solution (Promega, G756B) was added at 50 μl / well in a 1:1 ratio with the medium. The shaker was run at about 120 min -1 and mixed well for 2 minutes, then left standing for 10 minutes. After standing, 50 μl was transferred to an Assay plate (Corning, 3912, Assay plate 96 well, white polystyrene), and the absorbance was measured using a GloMax TM -Multi Detection System (Promega, E7051). The statistical significance in the comparison of multiple sample sets was analyzed using Dunnett's multiple comparison test.

[0032] (Results) The results are shown in Figure 1. CellTiter-Glo TM As a result of measuring cell viability by the Luminescent Cell Viability Assay, when E2 was added at concentrations of 100 nM and 1 μM, no cytotoxicity was observed. On the other hand, when E2 was added at a concentration of 10 μM, the cell number decreased significantly (* indicates P < 0.05 in Figure 1. n = 8). Therefore, it was suggested that E2 has high toxicity to cells at concentrations of 10 μM or higher.

[0033] (Example 2) Evaluation of the effects of E2 addition on Smad proteins, epithelial-mesenchymal transition, and extracellular matrix. In this example, the effects of adding E2 on Smad proteins, epithelial-mesenchymal transition, and extracellular matrix were examined.

[0034] (Materials and methods) iFECD was seeded in a 12-well plate at 9×10 4Seeds were seeded individually and cultured for 24 hours at 37°C under 5% CO2 conditions. The culture medium used was Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (P / S). Subsequently, the medium was removed, and DMEM+2%FBS+1%P / S medium containing DMSO and E2 (1 μM) was added, and cultured for 24 hours as a pretreatment. After 24 hours, the medium was removed, and DMEM+2%FBS+1%P / S medium containing DMSO, 10 ng / ml recombinant human TGF-β2 (Wako, 200-19911), and E2 (1 μM) was added, and cultured for 24 hours. After 24 hours, Western blotting of proteins was performed according to the following procedure to evaluate the effects on Smad protein, epithelial-mesenchymal transition, and extracellular matrix.

[0035] 1) To recover the protein, suspension, and dead cells, the culture medium was collected on ice, and the solution obtained by washing the cells twice with 1×PBS(-) was also collected. The mixture was centrifuged at 4°C at 800 g for 10 minutes, the supernatant was discarded, and a precipitate was obtained. The washed cells were then extracted for protein by adding protein extraction buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) on ice. Subsequently, the precipitate obtained after centrifugation of the suspension and dead cells was also suspended and extracted. The collected solution was ground three times in cold water for 30 seconds using an ultrasonic device (BIORUPTOR, TOSHO DENKI), then centrifuged at 4°C at 15000 rpm for 10 minutes, and the protein supernatant was collected.

[0036] 2) Western blotting method The extracted protein was separated by SDS-PAGE and transferred to a FluoroTrans W PVDF Transfer Membrane (PALL, BSP0161). The transferred membrane was blocked with 3% Nonfat Dry Milk (Cell signaling, 9999S) at room temperature for 1 hour. Then, rabbit anti-Smad2 antibody (Cell Signaling, 5339) (1:1000), rabbit anti-p-Smad2 antibody (Cell Signaling, 3108) (1:1000), rabbit anti-Smad3 antibody (Cell Signaling, 9523) (1:1000), rabbit anti-p-Smad3 antibody (Cell Signaling, 9520) (1:1000), rabbit anti-Snail antibody (Cell Signaling, 3879) (1:1000), rabbit anti-ZEB1 antibody (CellSignaling, 3396) (1:1000), mouse anti-Fibronectin antibody (BD Biosciences, M171-3) (1:15000), rabbit anti-Biglycan antibody (Sigma Aldrich, A117664) (1:1000), and mouse anti-GAPDH antibody (MBL, M171-3) (1:3000) were diluted in 3% Nonfat Dry Milk and subjected to an antibody reaction at 4°C for 18 hours. After the antibody reaction, peroxidase-labeled anti-rabbit antibody (GE healthcare biosciences, NA934V) (1:5000) and anti-mouse antibody (GE healthcare biosciences, NA931V) (1:5000) were diluted in 3% Nonfat Dry Milk as secondary antibodies and subjected to an antibody reaction at room temperature for 1 hour. Chemi Lumi ONE Ultra (nacalai tesque, 11644-40) was used for detection, and detection and analysis were performed using an Image Quant LAS 4000mini (Fujifilm, 622471).

[0037] (Results) The results are shown in Figures 2-4. Figure 2 shows the results of Western blotting of Smad proteins in iFECD (RE-) and iFECD (RE+). When iFECD was stimulated with TGF-β2 in the absence of E2, the expression levels of p-Smad2 and p-Smad3 increased in both cell lines compared to the TGF-β2-unstimulated control, and phosphorylation activity of Smad2 and Smad3 was observed. On the other hand, the addition of E2 (1 μM) suppressed the increase in p-Smad2 and p-Smad3 expression in both cell lines, confirming that the phosphorylation activity of Smad2 and Smad3 induced by TGF-β2 was suppressed. Therefore, it was suggested that E2 suppresses the phosphorylation pathway of Smad that transmits TGF-β signaling. Figure 3 shows the results of Western blotting of proteins related to epithelial-mesenchymal transition in iFECD (RE-) and iFECD (RE+). In the absence of E2, stimulation of iFECD with TGF-β2 resulted in increased expression of Snail and ZEB1 in both cell lines. Conversely, the addition of E2 (1 μM) suppressed the TGF-β2-induced increase in Snail and ZEB1 expression in both cell lines. Therefore, E2 is suggested to suppress epithelial-mesenchymal transition, which is involved in the production of the extracellular matrix. Figure 4 shows the results of Western blot analysis of extracellular matrix constituent proteins in iFECD (RE-) and iFECD (RE+). In the absence of E2, stimulation of iFECD with TGF-β2 resulted in increased expression of fibronectin and biglycan in both cell lines. Conversely, the addition of E2 (1 μM) suppressed the TGF-β2-induced increase in fibronectin and biglycan expression in both cell lines. Therefore, E2 is suggested to suppress the production of the extracellular matrix.

[0038] (Example 3) Evaluation of the effect of E2 addition on the accumulation of extracellular matrix and denatured proteins In this example, the effect of using E2 on the accumulation of extracellular matrix and denatured proteins was investigated.

[0039] (Materials and Methods) iFECD was placed in a round cover glass (Matsunami Glass, C013001) in a 24-well plate with 8 × 10 3 Individual seeds were seeded and cultured for 24 hours at 37°C under 5% CO2 conditions. The culture medium used was Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin (P / S). Subsequently, the medium was removed, and DMEM+2%FBS+1%P / S medium containing DMSO and E2 (1 μM) was added and cultured for 24 hours as a pretreatment. After 24 hours, the medium was removed, and DMEM+2%FBS+1%P / S medium containing DMSO, 10 ng / ml recombinant human TGF-β2, and E2 (1 μM) was added, and cultured for 24 hours. After 24 hours, immunohistochemistry and aggresome staining were performed to evaluate the effects on the accumulation of extracellular matrix and denatured proteins. For observation with a fluorescence microscope, the medium was removed from the iFECD on a round coverslip and washed twice with 1×PBS(-). After adding 4% paraformaldehyde (PFA) (Nacalai Tesque, 26126-25) and fixing at room temperature for 10 minutes, 0.5% Triton X100 (Nacalai Tesque, 28229-25) was added and permeabilized at room temperature for 5 minutes. The samples were incubated with 2% bovine serum albumin (BSA) (Nacalai Tesque, 01863-77) at 37°C, 25 rpm, for 45 minutes. Mouse anti-Fibronectin antibody (BD Biosciences, M171-3) (1:1000), rabbit anti-Biglycan antibody (Sigma Aldrich, A117664) (1:1000), and rabbit anti-Collagen I antibody (RKL Rockland Immunochemicals, 600-401-103S) (1:200), diluted in 2% BSA, were added, and antibody reactions were carried out at 37°C, 25 rpm, for 45 minutes. After the antibody reaction, Alexa Fluor stains fibronectin, biglycan, denatured protein aggregates, and cell nuclei. TM 488 goat anti-Mouse IgG (H+L) (invitrogen, A11001) (1:1000), Alexa Fluor TM488 donkeyanti-Mouse IgG (H+L) (invitrogen, A21206) (1:1000), Cellstain DAPI Solution (4',6-Diamidino-2-phenylindole Dihydrochloride Solution) (DOJINDO, VB073) (1:1000), and Aggresome Detection Reagent (Enzo, ENZ-51035-K100) (1:1000), diluted in 2% BSA, were added, and the antibody reaction was carried out at 37°C, 25 rpm, for 45 minutes. The samples were observed using a confocal microscope (Leica, DMI4000B), and stained images were obtained.

[0040] (Results) The results are shown in Figures 5-8. Figure 5 shows the results of immunostaining of fibronectin in iFECD (RE-) and iFECD (RE+). When iFECD was stimulated with TGF-β2 in the absence of E2, fibronectin expression increased in both cell lines. On the other hand, it was confirmed that the increase in fibronectin expression induced by TGF-β2 was suppressed in both cell lines by the addition of E2 (1 μM). Figure 6 shows the results of immunostaining of biglycan in iFECD (RE-) and iFECD (RE+). When iFECD was stimulated with TGF-β2 in the absence of E2, biglycan expression increased in both cell lines. On the other hand, it was confirmed that the increase in biglycan expression induced by TGF-β2 was suppressed in both cell lines by the addition of E2 (1 μM). Figure 7 shows the results of immunostaining of collagen I in iFECD (RE-) and iFECD (RE+). In the absence of E2, stimulation of iFECD with TGF-β2 increased collagen I expression in both cell lines. On the other hand, the addition of E2 (1 μM) suppressed the TGF-β2-induced increase in collagen I expression in both cell lines. Figure 8 shows the staining results of aggresomes in iFECD (RE-) and iFECD (RE+). Aggresomes are aggregates of denatured proteins. Corneal endothelial cells from FECD patients exhibit excessive production of extracellular matrix, a component of guttae, and the associated accumulation of denatured proteins. In the absence of E2, stimulation of iFECD with TGF-β2 enhanced the accumulation of denatured proteins in both cell lines. On the other hand, the addition of E2 (1 μM) suppressed the TGF-β2-induced accumulation of denatured proteins in both cell lines. From these results, it was confirmed that E2 can suppress guttae formation in FECD by suppressing the production and accumulation of extracellular matrix.

[0041] (Example 4) Evaluation of the effect of E2 administration on the progression of FECD disease In this example, the effect of E2 on the progression of FECD disease was investigated using FECD model mice (Col8a2Q455K / Q455K mice, provided by Johns Hopkins University) as a disease model. Homozygous mutant knock-in mice (Col8a2Q455K / Q455K) with a point mutation causing a glutamine-to-lysine substitution (Q455K) at the 455th amino acid position of the alpha2 collagen 8 (COL8A2) gene cause early onset of symptoms similar to those of human FECD.

[0042] (Methods and Materials) E2 (Sigma-Aldrich, E8875) was dissolved in 95% ethanol to prepare an E2 solution with a concentration of 5 mg / ml. The prepared E2 solution was mixed with drinking water to adjust the final concentration of E2 to 4 μg / ml and the final concentration of ethanol to 0.1%, thereby preparing E2-containing drinking water. The prepared E2-containing drinking water was placed in a water bottle and administered orally to 12 Col8a2Q455K / Q455K mice, an early-onset FECD model mouse, from 8 weeks to 28 weeks of age. These 12 mice were used as the Estradiol group. As a control, 11 Col8a2Q455K / Q455K mice were given a 0.1% ethanol solution in the same manner and for the same period as ad libitum.

[0043] (Method of observing the corneal endothelium) The corneal endothelium was observed using a contact specular microscope (KONAN MEDICAL, Cell Check C). For observation, mice were placed in an induction anesthesia box (Natsume Seisakusho, KN-1010), and sevoflurane inhalation anesthetic solution (Nikko Pharmaceutical, 1313700Q2070) was administered at an induction concentration of 5% and a maintenance concentration of 2.5-4% using a simple inhalation anesthesia device for small animal experiments such as mice and rats (Natsume Seisakusho, KN-1071-S) to induce general anesthesia. Under general anesthesia, benoxil (Santen Pharmaceutical, 126718), a local anesthetic eye drop, was instilled into the right eye of the mouse to dislocate the eyeball. Then, scopizole® ophthalmic solution (Senju Pharmaceutical, 131980AQ1038) was applied to reduce friction, and the corneal endothelium was observed and recorded on video.

[0044] (Analysis of Corneal Endothelial Cell Density) Using a program developed by the inventors, in-focus images were extracted from video footage acquired by a contact specular microscope. The area of ​​the out-of-focus region was quantified using a normalized surface roughness coefficient, and 400 images with the best focus were extracted at approximately 0.2-second intervals every (video playback time) / 400 seconds, in 480 x 720 pixel grayscale. Furthermore, 100 images were automatically selected from the 400 images using Laplacian transformation. From the extracted 100 images, the image in which all cells were covered and in focus was designated as the corneal endothelial central image. One representative image with the best focus was blindly extracted from the corneal endothelial central images for each individual, and 100 corneal endothelial cells in the image were manually and continuously filled in using the paint tool ProCreate (Savage Interactive Pty Ltd.) to calculate the corneal endothelial cell density.

[0045] (Statistical Analysis) The statistical significance (P-value) of the mean difference between the two samples was determined using Student's t-test.

[0046] (Results) Figure 9 shows central corneal endothelial images of Col8a2Q455K / Q455K mice, which are FECD model mice. The left image in Figure 9 shows central corneal endothelial images of FECD model mice that were orally administered a 0.1% ethanol solution from 8 weeks of age as a control, and the right image in Figure 9 shows central corneal endothelial images of FECD model mice that were orally administered E2-containing beverages from 8 weeks of age. Both images were taken at 16 weeks of age, 8 weeks after the start of administration, and it was confirmed that the decrease in corneal endothelial cell density was suppressed in FECD model mice administered E2-containing beverages compared to the control.

[0047] Figure 10 shows the corneal endothelial cell density (cells / mm²) of the two groups shown in Figure 9. 2 The graphs shown are as follows: Figure 10 left shows the graph at 12 weeks of age, and Figure 10 right shows the graph at 16 weeks of age. Compared to the control, corneal endothelial cell density was significantly higher in FECD model mice administered E2-containing drinking water. More specifically, at 12 weeks of age, the control had an average of 2757 cells / mm².2 Mice administered E2-containing drinking water produced an average of 3063 cells / mm³. 2 At 16 weeks of age, the control group averaged 2456 cells / mm². 2 Mice administered E2-containing drinking water produced an average of 2837 cells / mm³. 2 Therefore, the inhibitory effect of E2 on the decrease in corneal endothelial cell density in FECD model mice administered long-term was maintained throughout the administration period and lasted for at least 4 weeks. From these results, it was revealed that E2 can suppress the decrease in corneal endothelial cell density in FECD.

[0048] (Example 5) Evaluation of the effects of E2 administration in Tcf4 CTG repeat sequence knock-in mice In this example, we used Tcf4 CTG repeat sequence knock-in mice (Tcf4(CTG)100 / (CTG)100 mice), which were created as previously reported (Invest Ophthalmol Vis Sci. 2025 Jun 2;66(6):18) as an FECD model mouse in which a trinucleotide repeat sequence in TCF4, the most frequently observed gene mutation in FECD patients (25-80%), was inserted 100 times, and investigated the effect of E2 on the progression of FECD disease. All experimental procedures using animals were carried out in accordance with protocols approved by the Animal Experiment Committee of Doshisha University and in compliance with the ARVO (Association for Research in Vision and Ophthalmology) statement on the use of animals in ophthalmological and vision research.

[0049] (Methods and Materials) E2 (Sigma-Aldrich, E8875) was dissolved in 95% ethanol to prepare an E2 solution with a concentration of 5 mg / ml. The prepared E2 solution was mixed with drinking water to adjust the final concentration of E2 to 4 μg / ml and the final concentration of ethanol to 0.1%, thereby preparing E2-containing drinking water. The prepared E2-containing drinking water was placed in a water bottle and administered orally to 10 Tcf4(CTG)100 / (CTG)100 mice, a late-onset FECD model mouse with a CTG (100 repetitions) repeat sequence, from 8 weeks to 28 weeks of age. In addition, 6 wild-type mice and 10 Tcf4(CTG)100 / (CTG)100 mice that were given a 0.1% ethanol solution in the same manner and for the same period were used as controls.

[0050] (Transmission electron microscopy analysis) Eyeballs of Tcf4(CTG)100 / (CTG)100 mice and wild-type mice were fixed overnight at 4°C in 0.1 M phosphate buffer (pH 7.4) containing 2% paraformaldehyde and 2% glutaraldehyde. The corneas were excised, washed with 0.1 M phosphate buffer (4°C, 30 minutes), and then post-fixed with 2% osmium tetroxide (4°C, 3 hours). After dehydration, the samples were embedded in epoxy resin, and 70 nm ultrathin sections were collected on a copper grid. The sections were counterstained with uranyl acetate aqueous solution and tungstic acid phosphate (1 hour each), and then stained with Reynolds lead citrate (20 minutes). Further staining was performed using 2% uranyl acetate (15 minutes) and lead staining solution (3 minutes). The images were acquired using a JEM-1400Plus transmission electron microscope (JEOL) operating at 100 kV and equipped with a CCD camera.

[0051] (Analysis of Guttae area ratio and corneal endothelial cell density) Corneal endothelium observation and statistical analysis were performed in the same manner as in Example 4 above. 100 images selected in the same manner as in Example 4 were combined using AutoStitch64 software developed at the University of British Columbia to generate a comprehensive panoramic image, which was used to calculate the Guttae area ratio and corneal endothelial cell density. Guttae and cell boundaries were identified using the program used in Example 4. This program is a validated U-Net-based deep learning model developed by the inventors using manually annotated corneal endothelial images obtained from Col8a2L450W / L450W knock-in mice as training data (Cornea. 2022;41:901-907).

[0052] (Results) The upper part of Figure 11 shows panoramic images of the corneal endothelium. More specifically, the upper left image of Figure 11 is a panoramic image of the corneal endothelium of wild-type mice that ingested a 0.1% ethanol solution, the upper center image of Figure 11 is a panoramic image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice that ingested a 0.1% ethanol solution, and the upper right image of Figure 11 is a panoramic image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice that ingested E2-containing drinking water. The lower part of Figure 11 shows central images of the corneal endothelium. More specifically, the lower left image in Figure 11 shows a representative central image of the corneal endothelium of wild-type mice ingested with a 0.1% ethanol solution, the lower center image in Figure 11 shows a representative central image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice ingested with a 0.1% ethanol solution, and the lower right image in Figure 11 shows a representative central image of the corneal endothelium of Tcf4(CTG)100 / (CTG)100 mice ingested with E2-containing drinking water. All images were taken at 28 weeks of age, 20 weeks after the start of administration. As shown in each image in Figure 11, Tcf4(CTG)100 / (CTG)100 mice ingested with a 0.1% ethanol solution showed significantly more guttae formation in the corneal endothelial basement membrane, a characteristic of FECD, compared to wild-type mice. On the other hand, in Tcf4(CTG)100 / (CTG)100 mice administered with E2-containing drinking water, it was confirmed that guttae formation in the corneal endothelial basement membrane was suppressed. Furthermore, Tcf4(CTG)100 / (CTG)100 mice ingested a 0.1% ethanol solution showed a significant decrease in corneal endothelial cell density compared to wild-type mice. However, administration of E2-containing drinking water maintained a high corneal endothelial cell density in Tcf4(CTG)100 / (CTG)100 mice, and it was confirmed that the decrease in cell density was suppressed. Figure 12 (left) shows a graph of the Guttae area percentage (%) for the three groups shown in Figure 11. Tcf4(CTG)100 / (CTG)100 mice ingested a 0.1% ethanol solution had a significantly higher Guttae area percentage compared to wild-type mice, and administration of E2-containing drinking water significantly suppressed the Guttae area percentage (in Figure 12 (left), ***p < 0.001. The specific p values ​​from left to right are p = 4.02 × 10⁻⁶). -8 and p = 3.49 × 10 -4 (This is the case.) The right panel of Figure 12 shows the corneal endothelial cell density (cells / mm²) of the three groups shown in Figure 11. 2The graph shows the results. Tcf4(CTG)100 / (CTG)100 mice that ingested a 0.1% ethanol solution showed a significant decrease in corneal endothelial cell density compared to wild-type mice, and the decrease in cell density was significantly suppressed by administration of E2-containing drinking water (Figure 12, right panel, **p < 0.01. Note that the specific p values ​​from left to right are p = 3.64 × 10⁻⁶). -3 and p = 7.15 × 10 -3 (The above results indicate that E2 suppresses guttae formation in the corneal endothelial basement membrane, maintains a high density of corneal endothelial cells, and suppresses cell damage.)

[0053] (Reference Example) Creation of Tcf4 CTG Repeat Sequence Knock-In Mice Tcf4 CTG repeat sequence knock-in mice were created using genome editing with CRISPR / Cas9 as previously reported (Investigative Ophthalmology & Visual Science, 2025, 66.6: 18-18). More specifically, based on the location of CTG repeat sequences in FECD patients, the CTG100 repeat sequence was designed to be inserted into the intron 2-3 region of the mouse Tcf4 gene. Prior to the creation of the knock-in mice, sequence analysis was performed and a high degree of conservation was observed between human TCF4 and mouse Tcf4, with sequence identity of 66% in the intron 2-3 region, 92% in the open reading frame, and 98% at the protein level confirmed. The wild-type mouse Tcf4 genome does not contain CTG repeat sequences in this region. A plasmid co-expressing a single guide RNA (sgRNA) targeting Cas9 and Tcf4 exon 2 was constructed using the pX459 vector (No. 48139, Addgene). A homology repair template was prepared by incorporating 1.6 kb and 2.0 kb homology arms flanking the target site and a CTG100 repeat sequence into a pBluescript II SK(+) vector. The guide RNA (gRNA) was designed using CRISPRdirect, and a gRNA exhibiting high on-target activity while minimizing predicted off-target effects based on a reporter assay was selected. The Cas9 / sgRNA plasmid and repair template were co-introduced into EGR-G01 embryonic stem (ES) cells. Successfully edited ES cell clones were injected into 8-cell stage Institute of Cancer Research (ICR) embryos, and the resulting chimeric blastocysts were transplanted into pseudopregnant female mice. The founder of the chimera was crossed with C57BL / 6J mice to establish the heterozygous Tcf4+ / (CTG)100 mouse. Subsequently, homozygous Tcf4(CTG)100 / (CTG)100 mice were produced by crossing the heterozygous mice. Mice were obtained from CLEA Japan, SLC Japan, and Shimizu Laboratory Animals, and were reared in a standard laboratory environment with a 12-hour light-dark cycle.All recombinant DNA manipulations were performed in accordance with the guidelines approved by the Doshisha University Recombinant DNA Experiment Committee.

[0054] The present invention can provide a pharmaceutical composition for the prevention or treatment of diseases or conditions related to corneal endothelial dysfunction. A pharmaceutical composition containing the estrogenic compound of the present invention as an active ingredient can be widely applied to diseases or conditions related to corneal endothelial dysfunction. Although the estrogenic compound of the present invention has a milder effect compared to so-called TGF-β inhibitors, estrogen preparations are already used as pharmaceuticals, so knowledge regarding side effects and other factors has been accumulated, and they can be applied with confidence.

Claims

1. A pharmaceutical composition for the prevention or treatment of diseases or conditions related to corneal endothelial disorders, comprising a compound having estrogen activity as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the compound having estrogenic activity is estradiol or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the disease or condition related to corneal endothelial dysfunction is one or more diseases selected from the group consisting of Fuchs' corneal endothelial dystrophy, bullous keratopathy, corneal endothelial dysfunction after corneal transplantation, posterior polymorphic corneal dystrophy, congenital hereditary corneal endothelial dystrophy, iris-corneal endothelial syndrome, corneal edema, cytomegalovirus keratitis, herpes simplex virus keratitis, and exfoliation syndrome.

4. The pharmaceutical composition according to claim 1 or 2, wherein the disease or condition related to corneal endothelial dysfunction is a disease or condition characterized by a decrease in corneal endothelial cell density and an accumulation of extracellular matrix.

5. The pharmaceutical composition according to claim 1 or 2, wherein the dosage form of the pharmaceutical composition is an eye drop or an eye ointment.

6. Use of estradiol or a pharmacopositically acceptable salt thereof for the manufacture of pharmaceutical compositions for the prevention or treatment of diseases or conditions related to corneal endothelial disorders.

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