Agent for promoting corneal endothelial cell adhesion

Abstract

A means for efficiently proliferating corneal endothelial cells and a means for the stable supply of a corneal endothelial preparation. A corneal endothelial cell adhesion promoter comprising a Rho kinase inhibitor, a corneal endothelial cell culture medium comprising a Rho kinase inhibitor, a corneal preservation solution comprising a Rho kinase inhibitor, and a method for manufacturing a corneal endothelial preparation comprising the step of culturing corneal endothelial cells using the culture medium.

Description

Technology Field

[0001] The present invention relates to a corneal endothelial cell adhesion promoter, which is used for the adhesion, maintenance, or preservation of corneal endothelial cells. Background Technology

[0002] Visual information is perceived when light introduced through the cornea, the transparent tissue on the front surface of the eye, reaches the retina, excites retinal nerve cells, and the resulting electrical signals are transmitted via the optic nerve to the visual field of the cerebrum. To achieve good vision, the cornea must be transparent. The transparency of the cornea is maintained by keeping its water content constant through the pump and barrier functions of corneal endothelial cells.

[0003] Human corneal endothelial cells exist at a density of approximately 3,000 per square millimeter at birth, but they lack the ability to regenerate once damaged. In bullous keratosis, which occurs due to corneal endothelial degeneration or dysfunction of the corneal endothelium caused by various factors, the cornea undergoes edema and opacity, leading to a significant decline in vision. Currently, for bullous keratosis, full-thickness corneal transplantation, which involves transplanting all three layers of the corneal structure—epithelial, stroma, and endothelium—is performed. However, corneal donations in Japan are insufficient; for approximately 5,500 patients on the waiting list for corneal transplants, the number of corneal transplants performed domestically annually is only about 2,700.

[0004] Recently, the concept of "part transplantation," which involves transplanting only the damaged tissue to reduce the risk of rejection or postoperative complications and to obtain better visual function, has been gaining attention. Among corneal transplants, epithelial transplantation, in which only the corneal epithelium is transplanted; cultured oral mucosal epithelial transplantation, in which oral mucosa is transplanted instead of corneal epithelium; and deep superficial corneal transplantation, in which stromal tissue is transplanted, have been performed. Methods for transplanting only the corneal endothelium are also being considered. For the transplantation of the corneal endothelium, a corneal endothelium-shaped sheet consisting of a corneal endothelium layer cultured on a collagen layer is known (see Patent Documents 1 and 2). However, regarding corneal endothelial cells, particularly human-derived corneal endothelial cells, corneal donors are limited, and in vitro culture is difficult; furthermore, obtaining the number of cultured cells required for transplantation requires time and cost.

[0005] Human embryonic stem (ES) cells possess high self-replicating and pluripotency, attracting attention for their medical applications; however, they have practical issues, such as a significant decrease in cell count, as cell death is easily induced by dispersing the cells during the culture process. Recently, it was discovered that cell death occurring when human ES cells are cultured is caused by the activation of Rho kinase (ROCK), and that cell death is significantly suppressed by the inhibition of ROCK. It was reported that mass culture of human ES cells and the generation of cerebral cells using ROCK inhibitors such as Y-27632 became possible (Non-patent Literature 1).

[0006] It is known that Rho kinase (ROCK) inhibitors have various functions. For example, Patent Document 3 discloses that a Rho kinase inhibitor promotes corneal nerve process formation and that a Rho kinase inhibitor is used as a corneal sensation recovery agent. Additionally, Patent Document 4 discloses that a Rho kinase inhibitor has an action promoting axonal extension of retinal ganglion cells and is used in the treatment of visual dysfunction. Prior art literature

[0007] Patent Document 1: Japanese Patent Publication No. 2004-24852 Patent Document 2: Japanese Patent Publication No. 2005-229869 Patent Document 3: WO2005 / 118582 Pamphlet Patent Document 4: WO2002 / 083175 Pamphlet

[0008] Non-patent literature 1: Nat Biotechnol. 2007, 25, 681 The problem to be solved

[0009] The object of the present invention is to provide a means capable of efficiently adhering to corneal endothelial cells and a means for the stable supply of corneal endothelial preparations. means of solving the problem

[0010] Taking the above-mentioned problem into account, the inventors carefully examined the matter and discovered that culturing corneal endothelial cells in the presence of a Rho kinase inhibitor dramatically promotes the adhesion of the cells to the culture substrate, thereby completing the present invention. That is, the present invention is as described below.

[0011] [1] A corneal endothelial cell adhesion promoter comprising a Rho kinase inhibitor.

[0012] [2] The promoter described in [1], wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0013] [3] The promoter described in [1] above, in which the corneal endothelial cells are of human origin.

[0014] [4] An accelerator described in any one of [1] to [3] above, which is a preparation for the prevention or treatment of corneal endothelial damage.

[0015] [5] The accelerator described in [4] above in the form of an anterior chamber injection solution or ocular irrigation solution.

[0016] [6] Culture medium of corneal endothelial cells containing a Rho kinase inhibitor.

[0017] [7] A culture medium described in [6] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0018] [8] A culture medium described in [6] above in which corneal endothelial cells are of human origin.

[0019] [9] Corneal preservation solution containing a Rho kinase inhibitor.

[0020]

[10] A preservative solution described in [9] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0021]

[11] A preservative solution described in [9] above, in which the cornea is of human origin.

[0022]

[12] A method for preparing a corneal endothelial preparation, comprising the step of culturing corneal endothelial cells using a culture medium described in any one of [6] to [8] above.

[0023]

[13] A method of manufacturing described in

[12] above, wherein the corneal endothelial cells are of human origin.

[0024]

[14] Use of a Rho kinase inhibitor for producing an adhesion promoter for corneal endothelial cells.

[0025]

[15] The use described in

[14] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0026]

[16] Uses described in

[14] above, in which corneal endothelial cells are of human origin.

[0027]

[17] An application described in any one of

[14] to

[16] above, wherein a corneal endothelial cell adhesion promoter is a preparation for the prevention or treatment of corneal endothelial damage.

[0028]

[18] The use described in

[17] above, in which a corneal endothelial cell adhesion promoter is in the form of an anterior chamber injection solution or ocular irrigation solution.

[0029]

[19] Use of a Rho kinase inhibitor for preparing a culture medium of corneal endothelial cells.

[0030]

[20] The use described in

[19] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0031]

[21] Uses described in

[19] above, in which corneal endothelial cells are of human origin.

[0032]

[22] Use of a Rho kinase inhibitor for preparing a corneal preservation solution.

[0033]

[23] The use described in

[22] , wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0034]

[24] Uses described in

[22] above, in which the cornea is of human origin.

[0035]

[25] A method for promoting the adhesion of corneal endothelial cells, comprising contacting an effective amount of a Rho kinase inhibitor to a target or corneal endothelial cell that requires the promotion of corneal endothelial cell adhesion.

[0036]

[26] The promoting method described in

[25] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0037]

[27] The method of promotion described in

[25] above, in which the corneal endothelial cells are of human origin.

[0038]

[28] A method of promotion described in

[25] above for the purpose of preventing or treating corneal endothelial damage.

[0039]

[29] The method of promotion described in

[25] above, which involves administering an effective amount of a Rho kinase inhibitor in the form of an anterior injection solution or ocular irrigation solution.

[0040]

[30] A method for culturing corneal endothelial cells, comprising the step of culturing corneal endothelial cells using a culture medium containing a Rho kinase inhibitor.

[0041]

[31] A culture method described in

[30] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0042]

[32] A culture method described in

[30] above in which corneal endothelial cells are of human origin.

[0043]

[33] A method for protecting the cornea, comprising the step of maintaining a corneal flap or corneal endothelial cell in a corneal protective solution containing a Rho kinase inhibitor.

[0044]

[34] The protection method described in

[33] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0045]

[35] A protection method described in

[33] above, in which the cornea is of human origin.

[0046]

[36] A method for preparing a corneal endothelial preparation, comprising the step of culturing corneal endothelial cells using a culture medium containing a Rho kinase inhibitor.

[0047]

[37] A method of preparation described in

[36] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0048]

[38] A method of manufacturing as described in

[36] above, wherein the corneal endothelial cells are of human origin.

[0049]

[39] A step of culturing corneal endothelial cells using a culture medium containing a Rho kinase inhibitor, and

[0050] A step of transplanting the corneal endothelial preparation obtained by the above culture step to a subject requiring corneal endothelial transplantation.

[0051] A method for treating bullous keratosis, corneal edema, or corneal leukoplakia, including

[0052]

[40] A treatment method described in

[39] wherein the Rho kinase inhibitor is at least one selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof.

[0053]

[41] A treatment method described in

[39] above in which the corneal endothelial cells are of human origin. Effects of the invention

[0054] The corneal endothelial cell adhesion promoter of the present invention contains a Rho kinase inhibitor as an active ingredient. The corneal endothelial cell adhesion promoter of the present invention promotes the adhesion of corneal endothelial cells, thereby enabling the formation of a corneal endothelial cell layer with good cell morphology and high cell density. Accordingly, it is useful as a therapeutic agent or preventive agent for diseases accompanied by corneal endothelial damage, such as bullous keratosis and corneal endothelitis. The corneal endothelial cell adhesion promoter of the present invention is useful as a corneal endothelial protective agent in the treatment or prevention of diseases accompanied by corneal endothelial damage. Furthermore, according to the corneal endothelial cell adhesion promoter of the present invention, it can be used as a corneal endothelial protective agent in the treatment or prevention of corneal endothelial damage caused by intraocular surgery such as cataract surgery or vitreous surgery, corneal endothelial damage caused by an increase in intraocular pressure (particularly glaucoma attack), or corneal endothelial damage caused by oxygen deficiency due to contact lens wear. According to the culture medium or corneal preservation medium of the present invention, since it contains a Rho kinase inhibitor, corneal endothelial cells can be cultured, maintained, or preserved well, and stable supply, maintenance, or preservation of corneal endothelial preparations becomes possible. Brief explanation of the drawing

[0055] Figure 1 is a phase-contrast microscope image of a first-stage cultured rabbit corneal endothelial cell (24 hours after the start of culture, magnification is 100x). Figure 2 is a graph investigating the proliferation of primary cultured rabbit corneal endothelial cells by MTS assay. The vertical axis represents the relative value to the absorbance of the Rock inhibitor (-) group on day 1. Figure 3 is a graph showing the adhesion of primary cultured rabbit corneal endothelial cells 24 hours after the start of culture. The vertical axis represents the relative value to the absorbance of the Rock inhibitor (-) group 24 hours after the start of culture. Figure 4 is a phase-contrast microscope image of primary cultured monkey corneal endothelial cells on the 3rd day after the start of culture. The magnification is 100x. Figure 5 is a phase-contrast microscope image of a first-generation cultured human corneal endothelial cell (day 7, magnification 100x). Figure 6 is a photograph of primary cultured monkey corneal endothelial cells (A: ROCK inhibitor (-), B: ROCK inhibitor (+), magnification is 20x) and a graph (C) showing the total area of ​​the cultured cells. Figure 7 is a graph showing the number of live cells in primary cultured monkey corneal endothelial cells. Figure 8 is a graph investigating the effect of Y-27632 on the passage culture of monkey corneal endothelial cells. Figure 9 is a phase-contrast microscope image (magnification is 100x) investigating the effect of Y-27632 on cell morphology during passage of monkey corneal endothelial cells. Figure 10 is a micrograph (phaloidine fluorescence staining, magnification is 200x) investigating the effect of Y-27632 on cell morphology during passage of monkey corneal endothelial cells. Figure 11 is a micrograph (anti-Ki67 antibody staining, magnification 200x) investigating the effect of Y-27632 on the cell cycle of monkey corneal endothelial cells. Figure 12 shows the results of flow cytometry investigating the effect of Y-27632 on the cell cycle of monkey corneal endothelial cells. Figure 12A shows day 1 of passage of the ROCK inhibitor (-) group, B shows day 2 of passage of the ROCK inhibitor (-) group, C shows day 1 of passage of the ROCK inhibitor (+) group, and D shows day 2 of passage of the ROCK inhibitor (+) group. Figure 13 is a graph showing the results of the flow cytometry analysis of Figure 12 as the anti-Ki67 antibody positive cell rate. Figure 14 is a graph showing the effect of Y-27632 on the apoptosis of monkey corneal endothelial cells. Figure 15 is a graph showing the effect of Y-27632 on the apoptosis of monkey corneal endothelial cells by the number of dead cells. Figure 16 is a graph showing the effect of Y-27632 on the density of cultured monkey corneal endothelial cells. Figure 17 is a graph investigating the adhesion of cultured monkey corneal endothelial cells 24 hours after subculture. The vertical axis represents the relative value of the fasudil group to the luminescence intensity of the control group 24 hours after the start of subculture. Specific details for implementing the invention

[0056] The corneal endothelial cell adhesion promoter of the present invention (hereinafter abbreviated as “adhesion promoter of the present invention”) contains a Rho kinase inhibitor as an active ingredient.

[0057] In the present invention, Rho kinase refers to a serine / threonine kinase that is activated upon the activation of Rho. Examples include ROKα (ROCKII: Leung, T. et al., J. Biol. Chem., 270, 29051-29054, 1995), p160ROCK (ROKβ, ROCK-I: Ishizaki, T. et al., The EMBO J., 15(8), 1885-1893, 1996) and other proteins having serine / threonine kinase activity.

[0058] In the present invention, one type of Rho kinase inhibitor may be included alone, or several types may be included in combination as needed.

[0059] As for Rho kinase inhibitors, the following literature:

[0060] US4678783, patent 3421217, WO99 / 20620, WO99 / 61403, WO02 / 076976, WO02 / 076977, WO02 / 100833, WO03 / 059913, WO03 / 062227, WO2004 / 009555, WO2004 / 022541, WO2004 / 108724, WO2005 / 003101, WO2005 / 039564, WO2005 / 034866, WO2005 / 037197, WO2005 / 037198, WO2005 / 035501, WO2005 / 035503, WO2005 / 035506, Examples of compounds disclosed in WO2005 / 080394, WO2005 / 103050, WO2006 / 057270, WO2007 / 026664, etc. These compounds can be prepared by the methods described in each disclosed document. As one specific example, 1-(5-isoquinolinesulfonyl)homopiperazine (fasudyl), (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane (Y-27632), etc., may be provided, and commercially available products may be suitably used for these compounds.

[0061] Among these, (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine, and pharmacologically acceptable salts thereof are preferably used because they are particularly excellent for promoting the adhesion of corneal endothelial cells. As salts of the compounds, pharmaceutically acceptable acid addition salts are preferred, and examples of such acids include inorganic acids such as hydrochloric acid, hydrobromide, and sulfuric acid, and organic acids such as methanesulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, tartaric acid, and salicylic acid. (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane·2-hydrochloride and 1-(5-isoquinolinesulfonyl)homopiperazine hydrochloride are more preferred.

[0062] In the present invention, "promotion of adhesion of corneal endothelial cells" may include, for example, both promotion of adhesion between corneal endothelial cells and promotion of adhesion between corneal endothelial cells and a culture substrate.

[0063] The adhesion promoter of the present invention exerts an adhesion-promoting effect on corneal endothelial cells isolated from corneal tissue derived from mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, etc.) or on isolated and serially subcultured corneal endothelial cells. Since the adhesion promoter of the present invention exhibits excellent adhesion-promoting effects, particularly on human-derived corneal endothelial cells which are considered difficult to culture and serially subculture, it is preferable to target human-derived corneal endothelial cells.

[0064] Corneal endothelial cells play a role in maintaining the transparency of the cornea. If the density of these endothelial cells decreases beyond a certain limit, corneal edema occurs, making it impossible to maintain corneal transparency and leading to corneal endothelial damage. The adhesion promoter of the present invention promotes the adhesion of corneal endothelial cells, enabling the formation of a corneal endothelial cell layer with good cell morphology and high cell density. Furthermore, because it possesses an apoptosis-inhibiting effect on corneal endothelial cells, it is useful as a treatment or preventive agent for diseases accompanied by corneal endothelial damage, such as bullous keratosis and corneal endothelitis. Additionally, the adhesion promoter of the present invention is useful as a treatment or preventive agent for corneal endothelial damage resulting from endophthalmic surgeries such as cataract surgery or vitreous surgery, corneal endothelial damage caused by increased intraocular pressure (particularly glaucoma attacks), or corneal endothelial damage caused by oxygen deficiency due to contact lens wear.

[0065] The adhesion promoter of the present invention is not particularly limited as long as it is in a formulation suitable for local administration to the eye, but it is preferable to formulate it in the form of an anterior chamber injection solution or an ocular irrigation solution. These can be prepared using conventional techniques commonly used in the field. When administered locally to the eye in the form of an anterior chamber injection solution or an ocular irrigation solution, the Rho kinase inhibitor comes into contact with corneal endothelial cells in vivo, thereby promoting the adhesion of corneal endothelial cells.

[0066] For example, when the adhesion promoter of the present invention is used as an anterior injection solution or ocular irrigation solution, a stabilizer (e.g., sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.), a solubilizing agent (e.g., glycerin, propylene glycol, macrogol, polyoxyethylene hydrogenated castor oil, etc.), a suspending agent (e.g., polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, etc.), an emulsifier (e.g., polyvinylpyrrolidone, soybean lecithin, egg yolk lecithin, polyoxyethylene hydrogenated castor oil, polysorbate 80, etc.), and a buffer (e.g., phosphate buffer, acetic acid buffer, boric acid buffer, carbonic acid buffer, citric acid buffer, Tris buffer, glutamic acid, Epsilon-aminocaproic acid, etc.), viscosity enhancers (e.g., water-soluble cellulose derivatives such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose; sodium chondroitin sulfate; sodium hyaluronate; carboxyvinyl polymer; polyvinyl alcohol; polyvinylpyrrolidone; macrogol, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, sodium dihydroacetate, para-hydroxybenzoic acid esters, sodium edetate, boric acid, etc.), isotonic agents (e.g., sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.), pH adjusters (e.g., hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid Other additives such as 1-menthol, d-camphor, d-borneol, peppermint oil, etc., and cooling agents (e.g., 1-menthol, d-camphor, d-borneol, peppermint oil, etc.) may be added. The amount of these additives added varies depending on the type and use of the additive, but it is sufficient to add them at a concentration that achieves the purpose of the additive.

[0067] The amount of the active ingredient in the adhesion promoter of the present invention is typically 0.00001 w / v% to 1 w / v%, preferably 0.00001 w / v% to 0.1 w / v%, and more preferably 0.0001 w / v% to 0.01 w / v%. The dosage and frequency of administration vary depending on symptoms, age, body weight, and form of administration, but typically for adults, for example when used as an anterior injection solution, a preparation containing 0.0001 w / v% to 0.1 w / v%, preferably 0.0001 w / v% to 0.01 w / v% of the active ingredient, may be administered several times a day, preferably once or twice, more preferably once, at a dose of 0.01 mL to 0.1 mL per dose.

[0068] The adhesion promoter of the present invention may be added to the culture medium when corneal endothelial cells are cultured in a test tube. By adding a Rho kinase inhibitor to the culture medium and continuing the culture, the Rho kinase inhibitor comes into contact with the corneal endothelial cells in vitro, thereby promoting the adhesion of the corneal endothelial cells.

[0069] The present invention provides a culture medium for corneal endothelial cells containing a Rho kinase inhibitor. The Rho kinase inhibitor included in the culture medium of the present invention is as described above.

[0070] The culture medium of the present invention may include a medium commonly used for culturing endothelial cells (e.g., DMEM (GIBCO BRL)), serum (e.g., fetal bovine serum (FBS)), growth factor (e.g., b-FGF), antibiotic (e.g., penicillin, streptomycin), etc.

[0071] The concentration of the Rho kinase inhibitor in the culture medium of the present invention is typically 1 μM to 100 μM, preferably 5 μM to 20 μM, more preferably 10 μM.

[0072] The culture medium of the present invention is suitable for use in the method for manufacturing the corneal endothelial preparation of the present invention described below, because it prevents cell shedding by promoting the adhesion of corneal endothelial cells, thereby enabling the formation of a corneal endothelial cell layer with good cell morphology and high cell density. In addition, the culture medium of the present invention is also used to maintain corneal endothelial cells.

[0073] The present invention provides a corneal preservation solution containing a Rho kinase inhibitor. The Rho kinase inhibitor included in the corneal preservation solution of the present invention is as described above. A corneal preservation solution is a liquid preparation for preserving a corneal fragment extracted from a donor during the period until it is transplanted to a receptant.

[0074] Examples of the corneal preservation solution of the present invention include a preservation solution commonly used during corneal transplantation (corneal flap preservation solution (Optisol GS: registered trademark), corneal transplant eye preservation solution (EPII: registered trademark)), physiological saline, phosphate-buffered physiological saline (PBS), etc., containing a Rho kinase inhibitor.

[0075] The concentration of the Rho kinase inhibitor in the corneal preservation solution of the present invention is typically 1 μM to 100 μM, preferably 5 μM to 20 μM, more preferably 10 μM.

[0076] The corneal preservation solution of the present invention is used as a corneal preservation solution for organ transplantation, etc., because it prevents cell shedding by promoting the adhesion of corneal endothelial cells, thereby enabling the formation of a corneal endothelial cell layer with good cell morphology and high cell density. In addition, the preservation solution of the present invention is also used as a preservation solution for cryopreserving corneal endothelial cells. For cryopreservation, glycerol, dimethyl sulfoxide, propylene glycol, acetamide, etc. may be further added to the preservation solution of the present invention.

[0077] The present invention provides a method for preparing a corneal endothelial preparation, comprising the step of culturing corneal endothelial cells using the culture medium of the present invention.

[0078] The corneal endothelial preparation is characterized by containing a substrate and a layer of corneal endothelial cells cultured in a test tube on the substrate.

[0079] In the present invention, the substrate is not particularly limited as long as it supports a cultured corneal endothelial cell layer and can maintain its shape in vivo for at least 3 days after transplantation. Furthermore, the substrate may serve as a scaffold when culturing corneal endothelial cells in a test tube, or it may serve only to support the corneal endothelial cell layer after culture. Preferably, the substrate serves as a scaffold that is used for culturing corneal endothelial cells and can be provided for transplantation as is after the completion of culture.

[0080] Examples of the above-mentioned materials include, for instance, polymer materials derived from natural products such as collagen, gelatin, and cellulose; synthetic polymer materials such as polystyrene, polyester, polycarbonate, and poly(N-isopropylacrylamide); biodegradable polymer materials such as polylactic acid and polyglycolic acid; hydroxyapatite; and amniotic membrane.

[0081] The shape described above is not particularly limited as long as it is a shape suitable for transplantation that supports the corneal endothelial cell layer, but it is preferably in the shape of a sheet. When the formulation of the present invention is in the shape of a sheet, it can be cut to a size suitable for the application site during transplantation. In addition, it is also possible to roll the sheet into a small, round shape and insert it through the opening. As a preferred embodiment, a circular shape covering approximately 80% of the area of ​​the damaged corneal endothelium is exemplified. In addition, it is also preferable to form a slit in the periphery of the circular shape so that it can adhere closely to the application site.

[0082] In a preferred form, the above description is collagen. As collagen, the collagen sheet described in Japanese Patent Publication No. 2004-24852 may be suitably used. Such a collagen sheet may be prepared, for example, from an amniotic membrane according to the method described in Japanese Patent Publication No. 2004-24852.

[0083] The corneal endothelial cell layer above preferably has at least one of the following features. More preferably, it has two or more of the following features, and more preferably, all of them.

[0084] (1) The cell layer has a single-layer structure. This is one of the characteristics of the corneal endothelial cell layer in living organisms.

[0085] (2) The cell density in the cell layer is about 1,000 cells / mm² to about 4,000 cells / mm². In particular, when an adult is used as the recipient, it is preferable to have about 2,000 cells / mm² to about 3,000 cells / mm².

[0086] (3) The shape of the cells constituting the cell layer when viewed from a plane is approximately hexagonal. This is one of the characteristics of the cells constituting the corneal endothelial cell layer in living organisms. The formulation of the present invention is similar to the corneal endothelial cell layer in living organisms and performs the same functions as the innate corneal endothelial cell layer, and can also exhibit proliferative ability in living organisms.

[0087] (4) The cells in the cell layer are regularly aligned. In the endothelial cell layer of the living cornea, the cells constituting it are regularly aligned, and it is believed that the normal function and high transparency of the corneal endothelial cells are maintained, and the moisture regulation function of the cornea is also properly exercised. Therefore, by possessing these morphological characteristics, the formulation of the present invention is expected to perform the same function as the endothelial cell layer of the living cornea.

[0088] The manufacturing method of the present invention includes the step of culturing corneal endothelial cells using the culture medium of the present invention, and can be carried out, for example, by the following method.

[0089] <1> Collection and in vitro culture of corneal endothelial cells

[0090] Corneal endothelial cells are collected by conventional methods from the cornea of ​​the recipeant themselves or a suitable donor. Considering the transplantation conditions in the present invention, allogeneic corneal endothelial cells may be prepared. For example, after separating the Descemet's membrane and the endothelial cell layer of the corneal tissue from the corneal stroma, they are transferred to a culture dish and treated with a dispasate or the like. Accordingly, the corneal endothelial cells are detached from the Descemet's membrane. Corneal endothelial cells remaining in the Descemet's membrane can be detached by pipetting or the like. After removing the Descemet's membrane, the corneal endothelial cells are cultured in the culture medium of the present invention. As the culture medium, for example, commercially available DMEM (Dulbecco's Modified Eagle's Medium) with appropriate additions of FBS (fetal bovine serum), β-FGF (basic-fibloblast growth factor), and antibiotics such as penicillin and streptomycin, and additionally Y-27632 or Fasudil, may be used. It is preferable to use a culture vessel (culture dish) with its surface coated with type I collagen, type IV collagen, fibronectin, laminin, or the extracellular matrix of bovine corneal endothelial cells. Alternatively, a conventional culture vessel treated with a commercially available coating agent, such as FNC coating mix (registered trademark), may be used. This is because the combined use of such a coating and the culture medium of the present invention promotes the adhesion of corneal endothelial cells to the surface of the culture vessel, thereby enabling good proliferation.

[0091] The temperature conditions for culturing corneal endothelial cells are not particularly limited as long as the corneal endothelial cells grow, but for example, about 25°C to about 45°C, preferably about 30°C to about 40°C, and more preferably about 37°C when considering proliferation efficiency. The culture method is carried out in a conventional cell culture incubator under humidification and in an environment with a CO2 concentration of about 5% to 10%.

[0092] <2> Subculture

[0093] Subculture may be performed after the corneal endothelial cells provided for culture have proliferated. Preferably, subculture is performed when the cells become subconfluent or confluent. Subculture may be performed as follows. First, the cells are detached from the surface of the culture vessel by treatment with trypsin-EDTA or the like, and then the cells are recovered. The culture medium of the present invention is added to the recovered cells to form a cell suspension. It is preferable to perform centrifugation when or after recovering the cells. By such centrifugation, a cell suspension with a high cell density can be prepared. A preferred cell density is approximately 1 to 2 × 10⁶. 6 It is per mL. In addition, the centrifugation conditions here may include, for example, 500 rpm (30 g) to 1000 rpm (70 g) and 1 minute to 10 minutes.

[0094] The cell suspension is seeded into a culture vessel and provided for culture, just as in the initial culture described above. The dilution ratio during subculturing varies depending on the condition of the cells, but is approximately 1:2 to 1:4, preferably approximately 1:3. Subculturing can be performed under the same culture conditions as the initial culture described above. The culture time varies depending on the condition of the cells used, but is, for example, 7 to 30 days. The above subculturing can be performed multiple times as needed. By using the culture medium of the present invention, cell adhesion in the early stages of culture is enhanced, thereby enabling a reduction in the culture period.

[0095] <3> Preparation of the corneal endothelial cell layer

[0096] The cell suspension is seeded onto a substrate, such as a collagen sheet, and provided for culture. At this time, the number of seeded cells is adjusted so that a cell layer with a desired cell density is formed in the corneal endothelial preparation finally produced. Specifically, cells are seeded so that a cell layer with a cell density of approximately 1,000 cells / mm² to approximately 4,000 cells / mm² is formed. Culture can be performed under the same conditions as the initial culture described above. The culture time varies depending on the condition of the cells used, but is, for example, 3 days to 30 days.

[0097] By performing culture as described above, a corneal endothelial preparation is obtained in which a layer of corneal endothelial cells cultured in a test tube is formed on the substrate.

[0098] In the present invention, the corneal endothelial preparation may include the culture medium of the present invention to maintain corneal endothelial cells. Additionally, the corneal endothelial preparation may include the corneal preservation solution of the present invention until it is provided for transplantation. The present invention also provides a combination of the corneal endothelial preparation and the culture medium or preservation solution of the present invention.

[0099] The corneal endothelium preparation obtained by the manufacturing method of the present invention can be used as a graft in the treatment of diseases requiring corneal endothelium transplantation, such as bullous keratosis, corneal edema, corneal leukoplakia, particularly corneal dystrophy, and bullous keratosis caused by corneal endothelium damage resulting from trauma or intraocular surgery.

[0100] The subjects for administration of the adhesion promoter and corneal endothelial preparation of the present invention may include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, etc.).

[0101] Examples

[0102] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. When using experimental animals, the International Guiding Principles for Biomedical Research involving Animals, the Animal Welfare and Care Act, and the standards regarding the rearing and storage of experimental animals were followed. In addition, this experiment was conducted in accordance with the Guidelines of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmic and Vision Research.

[0103] Example 1

[0104] Review of the Effects of ROCK Inhibitors on Rabbit Corneal Endothelial Cell Culture

[0105] Descemet's membranes with attached corneal endothelial cells were isolated from rabbit corneal tissue excised immediately after euthanasia. The Descemet's membranes were incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. After centrifugation, the isolated corneal endothelial cells were stirred to equal concentrations using corneal endothelial medium containing 10 μM Y-27632 for the ROCK inhibitor (+) group and corneal endothelial medium without Y-27632 for the ROCK inhibitor (-) group, and seeded into 96-well plates at a density of approximately 2000 cells per well. As a medium for corneal endothelial cells, a culture medium (DMEM, Gibco Invitrogen) supplemented with 1% fetal bovine serum and 2 mg / ml bFGF (Gibco Invitrogen) was used. The plates were pre-treated with FNC coating mix (Athena ES) for 10 minutes. The culture medium was changed at 72 hours after the start of culture, and from 72 hours onwards, both the ROCK inhibitor (+) group and the ROCK inhibitor (-) group were cultured until the 5th day using a standard corneal endothelial medium without Y-27632. A phase-contrast micrograph of the first culture of rabbit corneal endothelial cells 24 hours after the start of culture is shown in Figure 1. From day 1 to day 3 after the start of culture, the number of cells was measured every 24 hours by MTS ([3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay using CellTiter 96 (registered trademark) AQueous One Solution Cell Proliferation (Promega) (Fig. 2).

[0106] In the ROCK inhibitor (+) group (Fig. 1B), cell adhesion at 24 hours (day 1) after cell seeding was significantly enhanced by about 2.8 times compared to the ROCK inhibitor (-) group (Fig. 1A) (Figs. 2, 3). At the 3rd day, when confluence was reached, no significant difference was observed between the two groups (Fig. 2). In this regard, it became clear that Y-27632 has the effect of enhancing early cell adhesion after passage in rabbit corneal endothelial cells in primary culture. Furthermore, the same results were obtained in an examination using rabbit corneal endothelial cells that were subcultured, so it was thought that Y-27632 acts on early cell adhesion after passage in both primary culture and subculture.

[0107] Example 2

[0108] Review of the effects of Y-27632 on monkey corneal endothelial cell culture

[0109] Descemet's membranes with attached corneal endothelial cells were isolated from corneal tissue extracted from crab-eating monkeys euthanized for other purposes. In the ROCK inhibitor (+) group, Descemet's membranes were placed in corneal endothelial medium supplemented with 10 μM Y-27632 and incubated for 10 minutes at 37°C and 5% CO2. In the ROCK inhibitor (-) group, they were placed in corneal endothelial medium without Y-27632 and incubated for 10 minutes under the same conditions. As the corneal endothelial medium, the same cell culture medium as in Example 1 was used. After incubation, the Descemet's membranes were incubated with 1.2 U / ml Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. After centrifugation, the isolated corneal endothelial cells were stirred to equal concentrations in corneal endothelial media containing Y-27632(+) and Y-27632(-), and seeded into 12-well plates at a density of approximately 20,000 cells per well. The plates were pre-treated with FNC coating mix (Athena ES) for 10 minutes. The culture medium was changed at 72 hours after the start of culture, and after 72 hours, both the ROCK inhibitor(+) group and the ROCK inhibitor(-) group were cultured using standard corneal endothelial media without the addition of Y-27632.

[0110] From Fig. 4, in the initial culture of corneal endothelial cells of crab-eating monkeys 24 hours after the start of culture, clearly more cells were adhered to the plate in the case of ROCK inhibitor (+) (Fig. 4B) compared to the case of ROCK inhibitor (-) (Fig. 4A). The number of days to reach confluence was 3 days for the case of ROCK inhibitor (+), whereas it was 7 days for the case of ROCK inhibitor (-). In this regard, Y-27632 was found to be a useful agent that enables cell culture by promoting early cell adhesion after the start of culture, even in the case of monkey corneal endothelial cells, which are thought to be difficult to culture in vitro compared to rabbits.

[0111] Example 3

[0112] Review of the effects of Y-27632 on human corneal endothelial cell culture

[0113] Of the human corneal tissue obtained from the U.S. Eye Bank, the remaining peripheral corneal tissue was used after the central portion with a diameter of 7 mm was used for corneal transplantation. Descemet's membranes to which corneal endothelial cells adhered were separated. In the ROCK inhibitor (+) group, the Descemet's membranes were placed in a corneal endothelial medium containing 10 μM of Y-27632 and incubated for 10 minutes at 37°C and 5% CO2. In the ROCK inhibitor (-) group, the membranes were placed in a corneal endothelial medium without Y-27632 and incubated for 10 minutes under the same conditions. The same culture medium as in Example 1 was used as the corneal endothelial medium. After incubation, the Descemet membrane was incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and stirred to equal concentrations in corneal endothelial media of Y-27632(+) and Y-27632(-), and seeded at a density of approximately 10,000 cells per well in a 48-well plate pretreated with FNC coating mix (Athena ES). Since human corneal endothelial cells obtained from peripheral corneal tissue are very few in number, cell culture was performed in one well from the donor cornea of ​​one eye. The donor corneas used in this review were from the ROCK inhibitor(+) group at age 69 years and 6 days from the start of culture after donor death, and from the ROCK inhibitor(-) group at age 51 years and 7 days from the start of culture after donor death. It is thought that age and the time elapsed from donor death to cell culture have almost the same effect on cell culture.

[0114] After 72 hours of culture initiation, the culture medium was changed, and after 72 hours, both the ROCK inhibitor (+) group and the ROCK inhibitor (-) group were cultured using standard corneal endothelial medium without the addition of Y-27632. To examine the effects on early cell adhesion and cell morphology after the initiation of culture, cells were observed using a phase-contrast microscope and photographs were taken with a digital camera after the initiation of culture (Fig. 5).

[0115] From Fig. 5, on the 7th day after the start of culture, in the ROCK inhibitor (+) group, a confluent, high-density monolayer of cells similar to normal corneal endothelial cells, consisting of uniform polygonal cells, was formed (Fig. 5B), whereas in the ROCK inhibitor (-) group, only endothelial cells in the shape of fibroblasts with low cell density were engrafted in an island shape (Fig. 5A). From these results, it was thought that in the initial culture of human corneal endothelial cells, which is known to be very difficult to culture, it is possible to form a corneal endothelial cell layer with good cell morphology and high cell density by adding Y-27632, which has the effect of promoting early cell adhesion after the start of culture, to the culture medium.

[0116] Preparation Example 1

[0117] Intravenous injection solution containing a Rho kinase inhibitor

[0118] Prepare the anterior injection solution shown below by the usual method.

[0119] Y-27632 10 mg

[0120] 0.1 g sodium dihydrogen phosphate

[0121] 0.9 g sodium chloride

[0122] Appropriate amount of sodium hydroxide

[0123] Appropriate amount of sterile purified water

[0124] Total volume 100 mL

[0125] (pH7)

[0126] Y-27632 uses the one manufactured by Wako Junyaku High School.

[0127] Preparation Example 2

[0128] Ocular irrigation solution containing a Rho kinase inhibitor

[0129] Prepare the ophthalmic irrigation solution shown below by the usual method.

[0130] Y-27632 1.0 mg

[0131] Opegaard MA appropriate amount

[0132] Total volume 100 mL

[0133] The Opegaard MA uses the one manufactured by Senju Seiyaku Co., and the Y-27632 uses the one manufactured by Wako Junyaku Kogyo Co.

[0134] Preparation Example 3

[0135] Culture medium for preparing corneal endothelial sheets containing a Rho kinase inhibitor

[0136] Prepare the culture solution shown below by the usual method.

[0137] Y-27632 0.5 mg

[0138] FBS 10 mL

[0139] 1 mL of penicillin-streptomycin solution

[0140] FGF basic 200 ng

[0141] Appropriate amount of DMEM

[0142] Total volume 100 mL

[0143] FBS manufactured by Invitrogen, penicillin-streptomycin solution manufactured by Nakarai Task (containing penicillin 5000 u / mL, streptomycin 5000 μg / mL), FGF basic manufactured by Invitrogen, Y-27632 manufactured by Wako Junyaku Kogyo Co., Ltd., and DMEM manufactured by Invitrogen are used.

[0144] Preparation Example 4

[0145] Corneal preservation solution containing Rho kinase inhibitor

[0146] Prepare the preservation solution shown below by the usual method.

[0147] Y-27632 0.2 mg

[0148] Optisol-GS appropriate amount

[0149] Total volume 100 mL

[0150] Optisol-GS manufactured by Bausch & Lomb and Y-27632 manufactured by Wako Junyaku Kogyo Co., Ltd. are used.

[0151] Example 4

[0152] Review of the effect of Y-27632 on the primary culture of monkey corneal endothelial cells

[0153] Descemet's membranes with attached corneal endothelial cells were isolated from corneal tissue excised from euthanized crab-eating monkeys. As in Example 2, in the ROCK inhibitor (+) group, Descemet's membranes were placed in corneal endothelial medium containing 10 μM Y-27632 and incubated for 10 minutes at 37°C and 5% CO2. In the ROCK inhibitor (-) group, they were placed in corneal endothelial medium without Y-27632 and incubated for 10 minutes under the same conditions. The same cell culture medium as in Example 1 was used as the corneal endothelial medium. After incubation, the Descemet's membranes were incubated with 1.2 U / ml Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. After centrifugation, the isolated corneal endothelial cells were stirred to equal concentrations in corneal endothelial media containing Y-27632(+) and Y-27632(-), and seeded into 96-well plates at a density of approximately 2,000 cells per well. The culture medium was changed at 72 hours after the start of culture, and thereafter, both the ROCK inhibitor(+) and ROCK inhibitor(-) groups were cultured using standard corneal endothelial media without the addition of Y-27632. On the 10th day of culture, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes and stained with toluidine blue (Figs. 6A, 6B). The total cell area was measured and analyzed using Image J (National Institutes of Health, USA) (Fig. 6C).

[0154] In the primary culture of crab-eating monkey corneal endothelial cells on the 10th day after the start of culture, the ROCK inhibitor (+) (Fig. 6B) significantly increased the total area of ​​the cultured cells by about 1.6 times compared to the ROCK inhibitor (-) (Fig. 6A) by promoting adhesion in the early stages of culture (Fig. 6C). In this regard, Y-27632 was found to be a useful agent for primary culture in monkey corneal endothelial cells, which are thought to be difficult to culture in vitro.

[0155] Example 5

[0156] Review of the optimal concentration of Y-27632 for monkey corneal endothelial cells

[0157] As in Example 2, recovered monkey corneal endothelial cells (primary culture) were placed in corneal endothelial media containing 1, 10, 33, and 100 μM of Y-27632, respectively, and corneal endothelial media without Y-27632, stirred to achieve the same concentration, and seeded in a 96-well plate at a density of approximately 2,000 cells per well. At 24 hours after the start of culture, the number of live cells was measured using CellTiter-Glo (registered trademark) (Promega) (Fig. 7).

[0158] In corneal endothelial cells 24 hours after the start of culture, when cell culture was performed in a medium containing 10 μM of Y-27632, the number of cells adhering to the plate was significantly higher compared to when cell culture was performed in a corneal endothelial medium without Y-27632 or in a corneal endothelial medium containing other concentrations of Y-27632 (Fig. 7). In this regard, Y-27632 at a concentration of 10 μM was found to be the most effective in monkey corneal endothelial cells.

[0159] Example 6

[0160] Review of the effect of Y-27632 on the passage of monkey corneal endothelial cells

[0161] Descemet's membranes, to which corneal endothelial cells were attached, were isolated from corneal tissue excised from euthanized crab-eating monkeys. The Descemet's membranes were incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and seeded into corneal endothelial medium. Corneal endothelial cells that had reached confluence were incubated with 0.05% trypsin at 37°C and 5% CO2 for 10 minutes, and subcultured. After performing 4 to 6 passages, corneal endothelial cells were placed in 96-well plates at dilutions of 1 / 2, 1 / 6, and 1 / 8, and passage culture was continued using corneal endothelial medium supplemented with 10 μM Y-27632 for the ROCK inhibitor (+) group and corneal endothelial medium supplemented without Y-27632 for the ROCK inhibitor (-) group. At 24 hours after passage, the cell count was measured using CellTiter-Glo (registered trademark) (Promega) as in Example 5 (Fig. 8).

[0162] In the case of cultured corneal endothelial cells of crab-eating monkeys 24 hours after passage, when passage was performed in a medium containing Y-27632, the number of cells adhering to the plate was significantly higher compared to a corneal endothelial medium not containing Y-27632 (Fig. 8). In this respect, Y-27632 was found to be effective in the passage of monkey corneal endothelial cells in addition to the initial culture.

[0163] Example 7

[0164] Review of the effect of Y-27632 on cell morphology during passage of monkey corneal endothelial cells

[0165] Corneal endothelial cells of crab-eating monkeys, which had been subcultured as in Example 6, were subcultured onto a slide glass to a dilution of 1 / 4. At 24 hours of subculture, cell morphology was observed using a phase-contrast microscope (Figs. 9A, 9B). Additionally, after fixing the corneal endothelial cells on the slide glass with 4% paraformaldehyde at room temperature for 10 minutes, the actin fibers were stained with phalloidin fluorescent stain (Figs. 10A, 10B).

[0166] In cultured corneal endothelial cells from crab-eating monkeys 24 hours after passage, when subculture was performed in a medium containing Y-27632 (Fig. 9B) compared to a corneal endothelial medium without Y-27632 (Fig. 9A), cell adhesion to the slide glass was enhanced, flattening into the shape of corneal endothelial cells was promoted, and cell aggregation was also enhanced. In addition, in phaloidin fluorescence staining, when subculture was performed in a medium containing Y-27632 (Fig. 10B) compared to a corneal endothelial medium without Y-27632 (Fig. 10A), actin stress fibers were clearly visible, indicating that Y-27632 promotes cytoskeleton formation.

[0167] Example 8

[0168] Review of the effects of Y-27632 on the cell cycle of monkey corneal endothelial cells

[0169] Corneal endothelial cells of crab-eating monkeys, which had been subcultured as in Example 6, were subcultured onto slide glasses to a 1 / 4 dilution. For the ROCK inhibitor (+) group, corneal endothelial medium containing 10 μM Y-27632 was used, and for the ROCK inhibitor (-) group, corneal endothelial medium without Y-27632 was used. On days 1, 2, and 14 of subculture, corneal endothelial cells on slide glasses were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then immunostained using an anti-Ki67 antibody (Fig. 11). In addition, corneal endothelial cells of crab-eating monkeys were similarly subcultured in the ROCK inhibitor (+) and ROCK inhibitor (-) groups, respectively, to a 1 / 4 dilution, and after harvesting using 0.05% trypsin on the 1st and 2nd days of subculture, the cell cycle was examined by flow cytometry using an anti-Ki67 antibody (Figs. 12, 13).

[0170] Compared to the ROCK inhibitor (-) group, the ROCK inhibitor (+) group had more Ki67-positive cells on both the 1st and 2nd days of subculture, whereas when confluence was nearly reached on the 14th day, the number of positive cells in the ROCK inhibitor (+) group was lower than that of the ROCK inhibitor (-) group (Fig. 11). In flow cytometry analysis as well, more Ki67-positive cells were observed in the ROCK inhibitor (+) group on both the 1st and 2nd days of subculture compared to the ROCK inhibitor (-) group (Figs. 12, 13).

[0171] Given that the Ki67 antigen is found in the nucleus of cells during the G1 phase of the cell proliferation cycle as well as from the S to M phases, Y-27632 was found to be a useful agent for efficient cell culture, as it has the function of promoting the cell cycle early after passage of crab-eating monkey corneal endothelial cells.

[0172] Example 9

[0173] Review of the effects of Y-27632 on the apoptosis of monkey corneal endothelial cells

[0174] Corneal endothelial cells of crab-eating monkeys, which had been subcultured as in Example 6, were subcultured to a dilution of 1 / 4. For the ROCK inhibitor (+) group, corneal endothelial medium supplemented with 10 μM Y-27632 was used, and for the ROCK inhibitor (-) group, corneal endothelial medium supplemented without Y-27632 was used. On the first day of subculture, all cells, including those in the medium, were collected using 0.05% trypsin, and then flow cytometry was performed using Annexin V and PI (Propidium Iodide) to examine apoptosis (Figs. 14, 15).

[0175] Compared to the ROCK inhibitor (-) group, the ROCK inhibitor (+) group showed a significant decrease in the rate of apoptotic cells in whole cells on day 1 of subculture (Fig. 14). In addition, 1×10 4 When comparing the number of apoptotic cells per cell, the ROCK inhibitor (+) group showed a significant decrease compared to the ROCK inhibitor (-) group (Fig. 15).

[0176] In this regard, Y-27632 was shown to have the function of inhibiting apoptosis during passage of corneal endothelial cells from crab-eating monkeys.

[0177] Example 10

[0178] Review of the effect of Y-27632 on the density of cultured monkey corneal endothelial cells

[0179] Descemet's membranes, to which corneal endothelial cells were attached, were isolated from corneal tissue excised from euthanized crab-eating monkeys. The Descemet's membranes were incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and seeded into corneal endothelial medium. Corneal endothelial cells that reached confluence were incubated with 0.05% trypsin at 37°C and 5% CO2 for 10 minutes, followed by subculture. For the ROCK inhibitor (+) group, corneal endothelial medium supplemented with 10 μM Y-27632 was used, while for the ROCK inhibitor (-) group, corneal endothelial medium without Y-27632 was used. The culture medium was changed at 72 hours after the start of culture, and after 72 hours, both the ROCK inhibitor (+) group and the ROCK inhibitor (-) group were cultured using standard corneal endothelial medium without Y-27632. At each passage, images of the cells were taken using a phase-contrast microscope, and cell density was measured to examine the effect of Y-27632 on the density of cultured monkey corneal endothelial cells.

[0180] As a result of repeating passages 7 times, a higher cell density was observed in the ROCK inhibitor (+) group compared to the ROCK inhibitor (-) group throughout the entire period of passage culture (Fig. 16). In this regard, it was thought that using Y-27632 made it possible to culture corneal endothelial cells at a high density, and that it would be useful for regenerative medicine, such as cultured corneal endothelial sheet transplantation, in the future.

[0181] Example 11

[0182] Review of the effects of Fasudil on monkey corneal endothelial cell culture

[0183] Descemet's membranes, to which corneal endothelial cells were attached, were isolated from corneal tissue excised from euthanized crab-eating monkeys. The Descemet's membranes were incubated with 1.2 U / ml of Dispase II (Roche Applied Science) at 37°C and 5% CO2 for 45 minutes, after which the corneal endothelial cells were mechanically isolated by pipetting. The isolated corneal endothelial cells were centrifuged and then subcultured in corneal endothelial medium. As the corneal endothelial medium, the same cell culture medium as in Example 1 was used. After isolating cultured monkey corneal endothelial cells recovered by 0.05% trypsin treatment during subculture and centrifuging them, the Fasudil group was treated with corneal endothelial medium containing 10 μM Fasudil (SIGMA-ALDRICH), and the control group was treated with corneal endothelial medium without Fasudil. The cells were stirred to achieve the same concentration and seeded in 96-well plates at a density of approximately 2,000 cells per well. After 24 hours of culture initiation, the number of adhered cells was measured using the CellTiter-Glo (registered trademark) Luminescent Cell Viability Assay (Promega) (Fig. 17). Fasudil, a ROCK inhibitor, also demonstrated an effect of promoting cell adhesion of corneal endothelial cells.

[0184] Preparation Example 5

[0185] Intravenous injection solution containing a Rho kinase inhibitor

[0186] Prepare the anterior injection solution shown below by the usual method.

[0187] Pasudil 10 mg

[0188] 0.1 g sodium dihydrogen phosphate

[0189] 0.9 g sodium chloride

[0190] Appropriate amount of sodium hydroxide

[0191] Appropriate amount of sterile purified water

[0192] Total volume 100 mL

[0193] (pH7)

[0194] Preparation Example 6

[0195] Culture medium for preparing corneal endothelial sheets containing a Rho kinase inhibitor

[0196] Prepare the culture solution shown below by the usual method.

[0197] Pasudil 0.5 mg

[0198] FBS 10 mL

[0199] 1 mL of penicillin-streptomycin solution

[0200] FGF basic 200 ng

[0201] Appropriate amount of DMEM

[0202] Total volume 100 mL

[0203] FBS manufactured by Invitrogen, penicillin-streptomycin solution manufactured by Nakarai Task (containing penicillin 5000 u / mL, streptomycin 5000 μg / mL), FGF basic manufactured by Invitrogen, and DMEM manufactured by Invitrogen are used.

[0204] This application is based on Japanese patent application 2007-223141 (filing date: August 29, 2007) and Japanese patent application 2008-016088 (filing date: January 28, 2008), the contents of which are all incorporated herein.

Claims

Claim 1 A formulation for use in a method for treating corneal endothelial damage comprising a Rho kinase inhibitor, wherein the method comprises (i) identifying a subject currently suffering from corneal endothelial damage and (ii) administering the formulation to the subject's eye to treat the corneal endothelial damage in the subject's eye. Claim 2 A formulation according to claim 1, wherein the Rho kinase inhibitor is one or more selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof. Claim 3 In paragraph 1, a formulation in which the eye is a human eye. Claim 4 A formulation according to claim 1, wherein the Rho kinase inhibitor has corneal endothelial cell adhesion-promoting activity. Claim 5 A formulation according to claim 1, wherein the Rho kinase inhibitor has an activity that promotes the proliferation of corneal endothelial cells or an activity that inhibits the apoptosis of corneal endothelial cells. Claim 6 A formulation according to any one of claims 1 to 5, administered in the form of an anterior injection or an endovascular perfusion fluid. Claim 7 In claim 6, a formulation comprising 0.001 to 0.01 w / v% of a Rho kinase inhibitor. Claim 8 A formulation according to any one of claims 1 to 5, further comprising cultured human corneal endothelial cells. Claim 9 A formulation that does not contain FGF-2 in any one of claims 1 to 5. Claim 10 A preparation for treating corneal endothelial disorders comprising a Rho kinase inhibitor. Claim 11 A preparation according to claim 10, wherein the Rho kinase inhibitor is one or more selected from the group consisting of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane, 1-(5-isoquinolinesulfonyl)homopiperazine and pharmacologically acceptable salts thereof. Claim 12 A preparation that is an anterior injection solution or ocular irrigation solution in paragraph 10 or 11.

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