RU2782613C2 - DRUG CONTAINING mTOR INHIBITOR FOR TREATMENT OR PREVENTION OF EYE SYMPTOMS, DISORDERS, OR DISEASES, AND ITS USE - Google Patents

Abstract

FIELD: medicine; ophthalmology.

SUBSTANCE: group of inventions relates to the field of medicine, namely to ophthalmology; it is intended for prevention or treatment of a pathological condition of corneal endothelium. A composition including rapamycin as a single active ingredient is used in prevention or treatment of Fuchs endothelial corneal dystrophy. The use of a composition including rapamycin for conservation and/or growth, or maintenance of growth of corneal endothelial cells is also presented.

EFFECT: use of the group of inventions provides prevention or treatment of Fuchs endothelial corneal dystrophy, as well as conservation and/or growth, or maintenance of growth of corneal endothelial cells.

4 cl, 21 dwg, 14 ex

Description

[Technical field]

[0001]

The present invention relates to a medicament for the treatment or prevention of ocular pathological conditions, disorders or diseases, containing an mTOR inhibitor (also called mechanistic target of rapamycin, mammalian target of rapamycin), and its use.

[Prior Art]

[0002]

Visual information is recognized when light transmitted to the cornea, which is the transparent tissue at the front of the eyeball, reaches the retina and excites nerve cells in the retina, and the electrical signal generated is transmitted through the optic nerve to the visual cortex. For good vision, the cornea must be transparent. The transparency of the cornea is maintained by maintaining a constant water content using the pumping and barrier functions of the corneal endothelial cells.

[0003]

Human corneal endothelial cells are present at a density of approximately 3000 cells/mm ² at birth. After injury, human corneal endothelial cells have an extremely limited ability to regenerate. Fuchs endothelial corneal dystrophy is a disease that causes abnormalities in endothelial cells within the cornea and greatly reduces the density of corneal endothelial cells, resulting in corneal edema. The etiology of this disease is unknown. In Fuchs endothelial corneal dystrophy, an extracellular matrix such as collagen or fibronectin is deposited on part of the posterior surface of the Descemet's membrane in the posterior part of the cornea, resulting in the appearance of gutta (corneal gutta) and hypertrophy of the Descemet's membrane. Guttas (corneal gutta) and Descemet's membrane hypertrophy are the cause of photophobia or blurred vision in patients with Fuchs endothelial corneal dystrophy, which significantly impairs the quality of life of patients. Thus, extracellular matrix, such as fibronectin, is associated with conditions that cause reduced visual acuity, such as guttas on the surface of the corneal endothelium or cloudy guttas, and may be the main cause of corneal endothelial disorders associated with corneal opacities, such as opacities, clouding cornea or thorn. It should be understood that there is no effective therapeutic method for Fuchs endothelial corneal dystrophy other than corneal transplantation. However, there is a shortage of corneal donation in Japan, where the number of patients waiting for corneal transplantation is about 2600, while the number of corneal transplants performed in Japan is about 1700 per year.

[0004]

In the case of Fuchs corneal endothelial dystrophy, culturing (Non-Patent Sources 1 and 3) and immortalization (Non-Patent Source 2) of corneal endothelial cells from patients with Fuchs' corneal dystrophy have been reported, but cells suitable for screening a therapeutic drug or a drug that prevents the progression of the disease , which retain signs of the disease, such as overproduction of extracellular matrices, have not been reported. Therefore, there is a limitation on the development of an appropriate therapeutic drug. Currently, there is no therapeutic agent in clinical practice, so the therapy is dependent on corneal transplantation.

[List of sources]

[Non-Patent Sources]

[0005]

[NPL 1] Zaniolo K, et al. Exp Eye Res.; 94(1):22-31. 2012

[NPL 2] Azizi B, et al. Invest Ophthalmol Vis Sci. 2; 52(13): 9291-9297. 2011

[NPL 3] Kelliher C. et al. Exp Eye Res Vol. 93(6), 880-888, 2011

[Summary of the Invention]

[The solution of the problem]

[0006]

The inventors have found that inhibition of mTOR suppresses cell death (apoptosis) of the eye, especially corneal endothelial cells, and can be used to treat or prevent eye diseases such as corneal endothelial disorders (especially corneal endothelial disorders in Fuchs endothelial corneal dystrophy) mediated by transforming growth factor β (TGF-β). In addition, the inventors unexpectedly found that the overexpression of extracellular matrix (ECM), such as fibronectin, can be suppressed by inhibition of mTOR. The inventors have thus found that an mTOR inhibitor can be used to alleviate, treat, or prevent corneal endothelial diseases mediated by extracellular matrix overexpression (e.g., gutta, Descemet's membrane hypertrophy, corneal opacification, leukoma, other pathological conditions associated with opacification, and etc.). Since cell death and extracellular matrix are independent events, it is preferable that both of them can be suppressed.

[0007]

Thus, the present invention relates, for example, to the following objects.

(Paragraph 1)

A composition for use in the prevention or treatment of an ocular pathological condition, disorder or disease, comprising an mTOR inhibitor.

(Point 2)

The composition of claim 1, wherein the ocular condition, disorder, or disease is a pathological condition, disorder, or disease of the corneal endothelium.

(Point 3)

The composition of claim 1 or 2, wherein the ocular condition, disorder or disease is a transforming growth factor β (TGF-β) mediated corneal endothelial disease, disorder or disease.

(Item 4)

The composition according to any one of claims 1 to 3, wherein the pathological condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, corneal transplant disorder, corneal endothelitis, trauma, eye surgery, eye laser surgery disorder, aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus corneal endothelitis.

(Item 5)

The composition according to any one of paragraphs 1-4, wherein the pathological condition, disorder or disease of the corneal endothelium is mediated by overexpression of the extracellular matrix (ECM).

(Item 6)

The composition of claim 5, wherein the pathological condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, guttitis, Descemet's membrane hypertrophy, corneal hypertrophy, turbidity, scarring, corneal opacity, corneal spot, corneal leukoma, photophobia, and fuzzy vision.

(Item 7)

The composition according to any one of claims 1 to 6, wherein the pathological condition, disorder or disease comprises Fuchs endothelial corneal dystrophy.

(Item 8)

The composition according to any one of paragraphs 1-7, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus, everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalizib, Ridaforolimus, NVP-BEZ235, CZ415, Torkiniba, Torina 1, Omipalizib, OSI-027, PF-04691502, Apitoliziba, WYE-354, Vistucertiba, Torina 2, Tacrolimus, GSK1059615, Gedatoliziba, WYE-125132, BGT226, Palomida 529, PP121, WYE-687, CH51327 -600, ETP-46464, GDC-0349, XL388, Zotarolimus and chrysophanoic acid.

(Item 9)

The composition according to any one of paragraphs 1-7, wherein the mTOR inhibitor is a substance that suppresses the expression of the mTOR gene.

(Item 10)

The composition according to claim 9, wherein the substance that suppresses the expression of the mTOR gene is miRNA, an antisense nucleic acid, or a ribozyme against the mTOR gene.

(Item 11)

The composition according to paragraph 9 or 10, in which the substance that suppresses the expression of the mTOR gene is miRNA against the mTOR gene, where the miRNA includes a sense strand consisting of the nucleic acid sequence presented in SEQ ID NO: 1, or a nucleic acid sequence, where 1- 3 nucleotide bases are removed, replaced, inserted and/or added and an antisense strand consisting of the nucleic acid sequence shown in SEQ ID NO: 2 or a nucleic acid sequence where 1-3 nucleotide bases are removed, replaced, inserted and/or added.

(Item 12)

The composition according to any one of paragraphs 1-8, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus and everolimus.

(Item 13)

A composition according to any one of paragraphs 1-12, wherein the composition is an eye drop.

(Item 14)

A composition according to any one of paragraphs 1-8, wherein the mTOR inhibitor is rapamycin and is present in the composition at a concentration of at least about 0.1 nM.

(Item 15)

A composition according to any one of paragraphs 1-8, wherein the composition is an eye drop wherein the mTOR inhibitor is rapamycin and is present in the eye drops at a concentration of at least about 0.1 mM.

(Item 16)

A composition according to any one of items 1-8, wherein the mTOR inhibitor is temsirolimus and is present in the composition at a concentration of at least about 0.01 nM.

(Item 17)

A composition according to any one of 1-8, wherein the composition is an eye drop wherein the mTOR inhibitor is temsirolimus and is present in the eye drops at a concentration of at least about 0.01 mM.

(Item 18)

A composition according to any one of paragraphs 1-8, wherein the mTOR inhibitor is everolimus and is present in the composition at a concentration of at least about 0.1 nM.

(Item 19)

A composition according to any one of paragraphs 1-8, wherein the composition is an eye drop wherein the mTOR inhibitor is everolimus and is present in the eye drops at a concentration of at least about 0.1 mM.

(Item 1A)

A method for preventing or treating an ocular condition, disorder, or disease in a patient, the method comprising administering to the patient an effective amount of an mTOR inhibitor.

(Item 2A)

The method of claim 1A, wherein the ocular condition, disorder, or disease is a pathological condition, disorder, or disease of the corneal endothelium.

(Item 3A)

The method of claim 1A or 2A, wherein the ocular condition, disorder, or disease is a transforming growth factor-β (TGF-β) mediated corneal endothelial disease, disorder, or disease.

(Clause 4A)

The method of any one of 1A-3A, wherein the pathological condition, disorder, or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, post-corneal transplant disorder, corneal endothelitis, trauma, eye surgery, post-ocular laser surgery disorder, aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus corneal endothelitis.

(Clause 5A)

The method according to any one of items 1A-4A, wherein the pathological condition, disorder or disease of the corneal endothelium is mediated by extracellular matrix (ECM) overexpression.

(Clause 6A)

The method according to item 5A, wherein the pathological condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, guttitis, Descemet's membrane hypertrophy, corneal hypertrophy, turbidity, scarring, corneal opacity, corneal spot, leukoma, photophobia, and fuzzy vision.

(Clause 7A)

The method of any one of 1A-6A, wherein the pathological condition, disorder, or disease comprises Fuchs endothelial corneal dystrophy.

(Clause 8A)

The method according to any one of paragraphs 1A-7A, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus, everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalizib, Ridaforolimus, NVP-BEZ235, CZ415, Torkinib , Torina 1, Omipalizib, OSI-027, PF-04691502, Apitoliziba, WYE-354, Vistucertiba, Torina 2, Tacrolimus, GSK1059615, Gedatoliziba, WYE-125132, BGT226, Palomida 529, PP121, WYE-6899, CH5, WAY-7 600, ETP-46464, GDC-0349, XL388, Zotarolimus and chrysophanoic acid.

(Item 9A)

The method according to any one of items 1A-7A, wherein the mTOR inhibitor is a substance that suppresses the expression of the mTOR gene.

(Clause 10A)

The method according to item 9A, where the substance that suppresses the expression of the mTOR gene is miRNA, antisense nucleic acid or ribozyme against the mTOR gene.

(Clause 11A)

The method according to paragraph 9A or 10A, where the substance that suppresses the expression of the mTOR gene is siRNA against the mTOR gene, where the siRNA includes a sense strand consisting of a nucleic acid sequence presented in SEQ ID NO: 1, or a nucleic acid sequence, where 1-3 nucleotide bases are removed, replaced, inserted and/or added, and an antisense strand consisting of the nucleic acid sequence shown in SEQ ID NO: 2 or a nucleic acid sequence where 1-3 nucleotide bases are removed, replaced, inserted and/or added .

(Clause 12A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus, and everolimus.

(Clause 13A)

The method of any one of 1A-12A, wherein the mTOR inhibitor is administered in the form of eye drops.

(Clause 14A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is rapamycin and is administered at a concentration of at least about 0.1 nM.

(Clause 15A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is administered in the form of eye drops, wherein the mTOR inhibitor is rapamycin and is present in the eye drops at a concentration of at least about 0.1 mM.

(Clause 16A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is temsirolimus and is administered at a concentration of at least about 0.01 nM.

(Clause 17A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is administered in the form of eye drops, wherein the mTOR inhibitor is temsirolimus and is present in the eye drops at a concentration of at least about 0.01 mM.

(Clause 18A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is everolimus and is administered at a concentration of at least about 0.1 nM.

(Clause 19A)

The method of any one of 1A-8A, wherein the mTOR inhibitor is administered in the form of eye drops, wherein the mTOR inhibitor is everolimus and is present in the eye drops at a concentration of at least about 0.1 mM.

(Clause 20A)

The method of any one of 1A-19A, wherein the mTOR inhibitor is administered topically.

(Clause 21A)

The method of any one of 1A-20A, wherein the mTOR inhibitor is administered topically to the eye.

(Clause 22A)

The method of any one of 1A-21A, wherein the mTOR inhibitor is administered such that it contacts the cornea.

(Item 1B)

The use of an mTOR inhibitor for the manufacture of a medicament for the prevention or treatment of an ocular condition, disorder or disease.

(Item 2B)

The use of claim 1B, wherein the ocular condition, disorder, or disease is a pathological condition, disorder, or disease of the corneal endothelium.

(Item 3B)

Use according to 1B or 2B, wherein the ocular condition, disorder or disease is a transforming growth factor β (TGF-β) mediated corneal endothelial condition, disorder or disease.

(Item 4B)

Use according to any one of 1B-3B, wherein the pathological condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, corneal transplant disorder, corneal endotheliitis, trauma, eye surgery, eye laser surgery disorder, aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus corneal endothelitis.

(Item 5B)

The use of any one of 1B-4B, wherein the pathological condition, disorder, or disease of the corneal endothelium is mediated by extracellular matrix (ECM) overexpression.

(Item 6B)

Use according to item 5B, wherein the pathological condition, disorder or disease of the corneal endothelium is selected from the group consisting of Fuchs endothelial corneal dystrophy, guttitis, Descemet's membrane hypertrophy, corneal hypertrophy, turbidity, scarring, corneal opacity, corneal spot, leukoma, photophobia, and fuzzy vision.

(Item 7B)

The use according to any one of 1B-6B, wherein the pathological condition, disorder or disease comprises Fuchs endothelial corneal dystrophy.

(Item 8B)

Use according to any one of 1B-7B, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus, everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalizib, Ridaforolimus, NVP-BEZ235, CZ415, Torkinib , Torina 1, Omipalizib, OSI-027, PF-04691502, Apitoliziba, WYE-354, Vistucertiba, Torina 2, Tacrolimus, GSK1059615, Gedatoliziba, WYE-125132, BGT226, Palomida 529, PP121, WYE-6899, CH5, WAY-7 600, ETP-46464, GDC-0349, XL388, Zotarolimus and chrysophanoic acid.

(Item 9B)

The use according to any one of 1B-7B, wherein the mTOR inhibitor is a substance that suppresses the expression of the mTOR gene.

(Clause 10B)

The use of item 9B, wherein the mTOR gene suppressor is an siRNA, an antisense nucleic acid, or an anti-mTOR gene ribozyme.

(Clause 11B)

Use according to item 9B or 10B, wherein the mTOR gene repressive agent is an siRNA against the mTOR gene, where the siRNA comprises a sense strand consisting of the nucleic acid sequence shown in SEQ ID NO: 1 or a nucleic acid sequence where 1-3 nucleotide bases are removed, replaced, inserted and/or added, and an antisense strand consisting of the nucleic acid sequence shown in SEQ ID NO: 2 or a nucleic acid sequence where 1-3 nucleotide bases are removed, replaced, inserted and/or added .

(Clause 12B)

The use of any one of 1B-8B, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus, and everolimus.

(Clause 13B)

Use according to any one of 1B-12B, wherein the drug is eye drops.

(Clause 14B)

Use according to any one of items 1-8, wherein the mTOR inhibitor is rapamycin and is present in the drug at a concentration of at least about 0.1 nM.

(Clause 15B)

The use of any one of 1B-8B wherein the drug is eye drops, wherein the mTOR inhibitor is rapamycin and is present in the eye drops at a concentration of at least about 0.1 mM.

(Clause 16B)

The use of any one of 1B-8B, wherein the mTOR inhibitor is temsirolimus and is present in the drug at a concentration of at least about 0.01 nM.

(Clause 17B)

The use of any one of 1B-8B, wherein the drug is eye drops, wherein the mTOR inhibitor is temsirolimus and is present in the eye drops at a concentration of at least about 0.01 mM.

(Clause 18B)

The use of any one of 1B-8B, wherein the mTOR inhibitor is everolimus and is present in the drug at a concentration of at least about 0.1 nM.

(Clause 19B)

The use of any one of 1B-8B wherein the drug is eye drops, wherein the mTOR inhibitor is everolimus and is present in the eye drops at a concentration of at least about 0.1 mM.

(Item 1C)

An mTOR inhibitor for use in the prevention or treatment of an ocular condition, disorder or disease.

(Item 2C)

An mTOR inhibitor according to item 1C, comprising one or more of the features from the items above.

(Item 1D)

Composition for conservation of corneal endothelial cells, including an mTOR inhibitor.

(Item 2D)

The composition of item 1D, including one or more features of the items above.

(Clause 1E)

A method for preserving corneal endothelial cells, comprising contacting an effective amount of an mTOR inhibitor with corneal endothelial cells.

(Clause 2E)

The method according to paragraph 1E, including one or more of the features of the paragraphs above.

(Item 1F)

A method for growing corneal endothelial cells or stimulating the growth of corneal endothelial cells, comprising contacting an effective amount of an mTOR inhibitor with corneal endothelial cells.

(Item 2F)

The method according to paragraph 1E, including one or more of the features of the paragraphs above.

[0008]

The present invention contemplates that one or more of the above features could be provided not only in the specific combinations described, but also in other combinations thereof. Additional embodiments and advantages of the present invention will be apparent to those skilled in the art upon reading and understanding the following detailed description as appropriate.

[Beneficial Effects of the Invention]

[0009]

In the present invention, it has been surprisingly found that an mTOR inhibitor can treat or prevent a disease caused by a transforming growth factor β (TGF-β) mediated disorder or disease in Fuchs endothelial corneal dystrophy or the like, and can provide a drug that can treat or prevent an ocular condition, disorder or disease, including such disease. The present invention also relates to a drug that can treat or prevent a disease mediated by an endothelial corneal disorder caused by excess production of an extracellular matrix (eg, fibronectin), such as gutta, Descemet's membrane hypertrophy, corneal opacification, leukoma, or other conditions associated with opacification. . The present invention also relates to a composition for preserving corneal endothelial cells or a composition for promoting the growth of corneal endothelial cells, comprising an mTOR inhibitor, as well as a method for preserving and/or growing or promoting the growth of corneal endothelial cells.

[Brief Description of Drawings]

[0010]

[Figure 1] Figure 1 shows microscopic images of iFECD cells. The upper left image shows the control group that did not receive TGF-β2, the upper right image shows the group that received TGF-β2, the lower left image shows the group that added TGF-β2 and rapamycin, and the lower right The image shows a group in which TGF-β2 and Z-VD-FMK, which is a caspase inhibitor, were added.

[Figure 2] Figure 2 shows Western blot results of total caspase 3, cleaved caspase 3, PARP and GAPDH. The figure shows, from left to right, a control group in which no TGF-β2 was added, a group in which TGF-β2 was added, a group in which TGF-β2 and rapamycin were added, and a group in which TGF-β2 and Z- were added. VD-FMK, which is a caspase inhibitor.

[Figure 3] Figure 3 shows the results of Western blotting of Akt, p-Akt, S6K, p-S6K and GAPDH. The figure shows, from left to right, a control group in which no TGF-β2 was added, a group in which TGF-β2 was added, a group in which TGF-β2 and rapamycin were added, and a group in which TGF-β2 and Z- were added. VD-FMK, which is a caspase inhibitor.

[Figure 4] Figure 4 shows the results of Western blotting of Smad2, p-Smad2, Smad3, p-Smad3 and GAPDH. The figure shows, from left to right, a control group in which no TGF-β2 was added, a group in which TGF-β2 was added, a group in which TGF-β2 and rapamycin were added, and a group in which TGF-β2 and Z- were added. VD-FMK, which is a caspase inhibitor.

[Figure 5] Figure 5 shows the results of Western blotting of fibronectin and GAPDH (n=3). The figure shows, from left to right, a control group in which no TGF-β2 was added, a group in which TGF-β2 was added, a group in which TGF-β2 and rapamycin were added, and a group in which TGF-β2 and Z- were added. VD-FMK, which is a caspase inhibitor.

[Figure 6] Figure 6 shows the results of agarose gel electrophoresis and Western blotting. The panel above shows the result of agarose gel electrophoresis of mTOR and GAPDH, and the panel below shows the results of Western blotting of mTOR, S6K, p-S6K and GAPDH. In the figure, each panel, from left to right, shows the random siRNA added group, the TGF-β2+random siRNA added group, the mTOR siRNA added group, and the TGF-β2+mTOR added group. miRNA.

[Figure 7] Figure 7 shows microscopic images of iFECD cells. The top image on the left shows the random siRNA added group, the top right image shows the mTOR siRNA added group, the bottom left image shows the TGF-β2 + random siRNA group, and the bottom right image shows the group in which TGF-β2+mTOR siRNA was added.

[Figure 8] Figure 8 shows Western blot results of total caspase 3, cleaved caspase 3, PARP and GAPDH. The figure, from left to right, shows the group in which random siRNA was added, the group in which TGF-β2+random siRNA was added, the group in which mTOR siRNA was added, and the group in which TGF-β2+mTOR siRNA was added.

[Figure 9] Figure 9 shows the results of Western blotting of fibronectin and GAPDH. The figure, from left to right, shows the group in which random siRNA was added, the group in which TGF-β2+random siRNA was added, the group in which mTOR siRNA was added, and the group in which TGF-β2+mTOR siRNA was added.

[Figure 10] Figure 10 shows a graph of caspase 3/7 activity. The figure, from left to right, shows the group that did not add TGF-β2, the control group, TGF-β2 (10 ng/ml) + rapamycin (0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM), and TGF-β2 (10 ng/ml) + Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 11] Figure 11 shows a graph of caspase 3/7 activity. The figure, left, shows the group in which no TGF-β2 was added, control group, TGF-β2 (10 ng/ml) + everolimus (0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM), and TGF-β2 (10 ng/ml) + Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 12] Figure 12 shows a graph of caspase 3/7 activity. The figure, from left to right, shows the group in which TGF-β2 was not added, control group, TGF-β2 (10 ng/ml) + temsirolimus (0.000001 μM, 0.00001 μM, 0.0001 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM), and TGF-β2+Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 13] Figure 13 shows a graph of caspase 3/7 activity. The figure, from left to right, shows the group that did not add TGF-β2, the control group, TGF-β2 (10 ng/ml) + Pl-103 (0.001 μm, 0.01 μm, 0.1 μm, 1 μm) , and TGF-β2 (10 ng/ml) + Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 14] Figure 14 shows a graph of caspase 3/7 activity. The figure shows, from left to right, the group in which TGF-β2 was not added, the control group, TGF-β2 (10 ng / ml) + CC-223 (0.001 μm, 0.01 μm, 0.1 μm, 1 μm) , and TGF-β2 (10 ng/ml) + Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 15] Figure 15 shows a graph of caspase 3/7 activity. The figure shows, from left to right, the group in which no TGF-β2 was added, the control group, TGF-β2 (10 ng/ml) + INK128 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β2 (10 ng/ml) + Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 16] Figure 16 shows a graph of caspase 3/7 activity. The figure shows, from left to right, the group in which no TGF-β2 was added, the control group, TGF-β2 (10 ng/ml) + AZD8055 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β (10 ng/ml) 2+Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 17] Figure 17 shows a graph of caspase 3/7 activity. The figure shows, from left to right, the group in which TGF-β2 was not added, the control group, TGF-β2 (10 ng/ml) + KU 0063794 (0.001 μM, 0.01 μM, 0.1 μM, 1 μM), and TGF-β2 (10 ng/ml) + Z-VD-FMK (10 μM). Statistical significance was tested using Dunnett's t-test (* denotes p<0.05 and ** denotes p<0.01, n=5).

[Figure 18] Figure 18 shows a schematic diagram of the relationship of vision from corneal endothelial cells and ECM deposits. The diagram shows that vision deteriorates as corneal endothelial cells die and ECM deposition increases. Descemet's membrane hypertrophy or gutta formation caused by ECM deposition usually begins to develop at the age of 30-40 years in patients with Fuchs endothelial corneal dystrophy and progresses throughout the life of patients. Progression results in visual impairment such as blurry vision, halo, sensitivity to bright light, or blurred vision. As corneal endothelial cell death progresses in parallel, corneal transparency is maintained by the remaining corneal endothelium compensating for pumping until the corneal endothelial cell density falls below about 1000 cells/mm ² . If the density becomes lower than about 1000 cells/mm ² , aqueous humor infiltration from the anterior segment into the cornea results in corneal edema leading to severe visual impairment. The existing technique can preserve visual function by suppressing ECM deposition and death of corneal endothelial cells.

[Figure 19] Figure 19 shows a typical example of corneal endothelial cell imaging observed by corneal contact endothelial reflectance microscopy using a mouse FECD model in which 2 μl of mTOR inhibitor eye drops (1 mM) were dripped into the left and right eyes twice per day, morning and evening, for two months. An image where saline (base) was dripped is shown as a control.

[Figure 20] Figure 20 shows a plot of corneal endothelial cell density (cell/mm ² ) in a mouse FECD model dripped with mTOR inhibitor eye drops. A graph where saline (base) was dripped is shown as a control. Statistical significance was tested using Student's t-test (* denotes p<0.05, n=3).

[Figure 21] Figure 21 shows a plot of area (%) of gutta in a FECD mouse model dripped with mTOR inhibitor eye drops. A graph where saline (base) was dripped is shown as a control. Statistical significance was tested using Student's t-test (** denotes p<0.01, n=3).

[Description of Embodiments]

[0011]

The present invention is described below. Throughout the text of the description, the expression in the form of a singular should be understood as covering its concept in the plural, unless expressly stated otherwise. Thus, it is to be understood that the singular forms also cover their concept in the plural, unless expressly stated otherwise. In addition, it should be understood that the terms used in the scope of the invention are used in the meaning that is commonly used in the art, unless expressly indicated otherwise. Thus, unless otherwise specified, all terminology and scientific and technical terms that are used in the framework of the invention have a meaning that is within the general knowledge of experts in the field of technology to which the present invention pertains. In case of conflict, the present description (including definitions) shall prevail.

[0012]

(Definition)

When used within the scope of the invention, "approximately", in front of a numerical value, means ±10% of the next numerical value.

[0013]

As used herein, "mTOR inhibitor" refers to any agent that inhibits mTOR signaling. The mTOR inhibitor is preferably water soluble. This is because if the mTOR inhibitor is not water soluble, it may be necessary to use a solvent that is not highly biocompatible. Solubility in water can be classified based on the definition of solubility in the pharmacopoeia. In other words, the amount of solvent required to dissolve 1 g or 1 ml of the solute is defined as highly soluble: less than 1 ml; easily soluble: 1 ml or more and less than 10 ml; highly soluble: 10 ml or more and less than 30 ml; slightly soluble: 30 ml or more and less than 100 ml; sparingly soluble: 100 ml or more and less than 1000 ml; poorly soluble: 1000 ml or more and less than 10000 ml; and almost insoluble: 10,000 ml or more. Solubility within the scope of the invention is evaluated in a similar manner. It should be understood that solubility in water means that any solubility of a substance can be used, provided that an effective amount can be dissolved if water is used as the solvent. Such a water-soluble component is preferably used in the form of eye drops.

[0014]

mTOR (mammalian target of rapamycin) is a serine/threonine kinase identified as the target molecule of rapamycin and is believed to play a major role in regulating cell division, cell survival, and the like. Also mTOR is known as SKS; FRAP; FRAP1; FRAP2; RAFT1; RAPT1, as well as 2475 as the NCBI gene identification number. Based on such information, specialists can develop and manufacture various mTOR inhibitors.

[0015]

The mTOR inhibitors that can be used in the present invention are not particularly limited as long as they are compounds having mTOR inhibitory activity. Relevant examples include: rapamycin, temsirolimus, everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalizib (XL765, SAR245409), Ridaforolimus (Deforolimus, MK-8669), NVP-BEZ235, CZ415, Torkinib (PP242 ), Torin 1, Omipalizib (GSK2126458, GSK458), OSI-027, PF-04691502, Apitolizib (GDC-0980, RG7422), WYE-354, Vistusertib (AZD2014), Torin 2, Tacrolimus (FK506), GSK1059615, Gedatolisib ( PF-05212384, PKI-587), WYE-125132 (WAY 132), BGT226 (NVP-BGT226), Palomide 529 (P529), PP121, WYE-687, CH5132799, WAY-600, ETP-46464, GDC-0349, XL388, Zotarolimus (CA. 578) and chrysophanoic acid.

[0016]

Preferred mTOR inhibitors include, but are not limited to, rapamycin, temsirolimus, and everolimus. Without being bound by any theory, this is because these drugs are approved by the FDA, PMDA, and the like, and the risks associated with safety and toxicity aspects are minimized. An even more preferred mTOR inhibitor is rapamycin. Another preferred mTOR inhibitor is temsirolimus. Another preferred mTOR inhibitor, without limitation, is everolimus.

[0017]

Other examples of mTOR inhibitors that can be used in the present invention include neutralizing anti-mTOR antibodies, compounds that inhibit mTOR activity, compounds that inhibit transcription of the gene encoding mTOR (e.g., antisense nucleic acids, siRNAs, ribozymes), peptides, compounds with a plant component , a component of traditional medicines such as Campo traditional medicine, or other components, and the like.

[0018]

The antisense nucleic acids used in the present invention can inhibit the expression and/or function of a gene (nucleic acids) encoding a member of the mTOR signaling pathway and the like by any of the above-described actions. As one embodiment, the creation of an antisense sequence complementary to the untranslated region near the 5' end of the mRNA of the gene encoding the above mTOR is considered to be effective in inhibiting translation of the gene. In addition, a sequence that is complementary to the 3' untranslated region or coding region can also be used. Thus, the antisense nucleic acids used in the present invention not only include a translation region of a gene encoding the above mTOR or the like, but also include nucleic acids containing an antisense sequence of a non-translated region sequence. The antisense nucleic acid used is placed after a suitable promoter and, preferably, a sequence containing a transcription termination signal is fused to the 3' end. The nucleic acid thus obtained can be converted into the desired animal (animal cell) using a known method. The antisense nucleic acid sequence is preferably a sequence that is complementary to the gene encoding the mTOR of the transformed animal (animal cell) or a portion thereof. However, such a sequence need not be completely complementary if gene expression can be efficiently suppressed. The RNA to be transcribed preferably has a complementarity that is 90% or more, and most preferably 95% or more, with respect to the target gene transcript. In order to effectively inhibit the expression of a target gene when using an antisense nucleic acid, it is preferable that the length of the antisense nucleic acid is at least 12 bases and less than 25 bases. However, the antisense nucleic acid of the present invention need not be necessarily limited to this length. For example, the length may be 11 bases or less, 100 bases or more, or 500 bases or more. The antisense nucleic acid may consist of only DNA, but may contain a nucleic acid other than DNA, such as a closed nucleic acid (LNA). In one embodiment, the antisense nucleic acid used in the present invention may be an LNA containing an antisense nucleic acid comprising an LNA at the 5' end or an LNA at the 3' end. In an embodiment using an antisense nucleic acid in the present invention, an antisense sequence can be generated from an mTOR nucleic acid sequence using the method described in, for example, Hirashima and Inoue, Shin-seikagaku Jikkenn Kouza 2 [New Biochemical Experiment Course 2] Kakusan IV Idenshi 25 no Fukusei to Hatsugen [Duplication and Expression of Gene of Nucleic Acid IV], Ed. by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1993, 319-347.

[0019]

Expression of mTOR can also be inhibited using a ribozyme or DNA encoding a ribozyme. A ribozyme refers to an RNA molecule that has catalytic activity. Although there are ribozymes with different activities, research focusing in particular on ribozymes as an RNA cleaving enzyme has led to the design of a ribozyme that cleaves RNA site-specifically. There are ribozymes with a size of 400 nucleotides or larger, such as the group I and M1 intron ribozymes contained in RNase P, but there are also ribozymes with an active domain of approximately 40 nucleotides, called hammerhead ribozymes or ribozymes containing hairpin structure (Makoto Koizumi and Eiko Otsuka, Protein, 5 Nucleic Acid and Enzyme, 1990, 35, 2191).

[0020]

For example, the self-cleaving domain of the hammerhead ribozyme cleaves the 3' C15 region of a sequence called G13U14C15. Base pairing of U14 and A9 is considered important for their activity. It has also been demonstrated that cleavage can be performed at A15 or U15 instead of C15 (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) Target RNAs can be designed by constructing their substrate-binding regions complementary to the RNA sequence near the target site (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein, Nucleic Acid and Enzyme, 1990, 35, 2191., Koizumi, M. et al., Nucl. Acids Res., 1989, 17, 7059.)

[0021]

Hairpin ribozymes can also be used for the purposes of the present invention. Such a ribozyme is found, for example, in the negative strand satellite RNA of the tobacco ring spot virus (Buzayan, J M., Nature, 1986, 323, 349). It has been demonstrated that target-specific RNA-cleaving ribozymes can also be generated from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl. Acids Res, 1991, 19, 6751, Yo Kikuchi, Kagaku to Seibutsu [Chemistry and Biology ], 1992, 30, 112). Thus, the expression of a gene encoding mTOR, etc. can be inhibited by specific cleavage of the gene transcript by a ribozyme.

[0022]

Expression of the endogenous mTOR gene can also be suppressed by RNA interference (hereinafter abbreviated as "RNAi") using double-stranded RNA having a sequence identical or similar to that of the target gene. RNAi is a technique currently attracting attention that can suppress the expression of a gene having a sequence that is homologous to double-stranded RNA (dsRNA) when the dsRNA enters the cell directly. In mammalian cells, short chain dsRNA (siRNA) can be used to induce RNAi. RNAi has many advantages over knockout mice, such as stable performance, ease of experimentation, and low cost. miRNAs are discussed in detail in other parts of the description.

[0023]

As used herein, "miRNA" is an RNA molecule having a double-stranded RNA region of 15-40 bases, where the siRNA has the function of cleaving the target gene mRNA with a sequence complementary to the antisense strand of the siRNA to suppress expression of the target gene. In particular, the siRNA in the present invention is an RNA containing a double-stranded RNA region consisting of an RNA sense strand consisting of a sequence homologous to consecutive RNA sequences in mTOR mRNA and an antisense RNA strand consisting of a sequence complementary to the sense RNA sequence. The development and production of such siRNA and mutant siRNA, discussed below, is within the technical purview of those skilled in the art. Any consecutive RNA regions in the mRNA that are a transcript of the mTOR sequence can be appropriately selected to produce a double-stranded RNA corresponding to that region, which is within the normal procedure of those skilled in the art. In addition, those skilled in the art can appropriately select an siRNA sequence having a stronger RNAi effect from mRNA sequences that are transcripts of the sequence by a known method. In addition, if one of the chains is determined, those skilled in the art can easily find the base sequence of the other chain (complementary chain). MiRNA can be appropriately obtained using a commercially available nucleic acid synthesizer. The public synthesis service can also be used to synthesize the desired RNA.

[0024]

With regard to bases, the length of the double-stranded portion of the RNA is 15 to 40 bases, preferably 15 to 30 bases, more preferably 15 to 25 bases, even more preferably 18 to 23 bases, and most preferably 19 to 21 bases. It should be understood that the upper limits and lower limits are not limited to such specific limits and may be any combination of these limits. The end structure of the sense strand or antisense strand of siRNA is not particularly limited, and may be appropriately selected according to purpose. For example, such an end structure may have a blunt end or a sticky end (protruding end). The type in which the 3' end is projecting is preferred. An siRNA having a protruding end that consists of several bases, preferably 1 to 3 bases, and more preferably 2 bases at the 3' end of the sense RNA strand and the antisense RNA strand, is preferred because it has a strong effect of suppressing the expression of the target gene in many cases. The type of bases at the protruding end is not particularly limited, and they may be either a base included in RNA or a base included in DNA. An example of a preferred overhang includes dTdT at the 3' end (2 bp deoxy-T) and the like. Examples of preferred siRNAs include, but are not limited to, all siRNAs with dTdT (2 bp deoxy-T) at the 3' end of the sense or antisense strands of the siRNA.

[0025]

In addition, it is also possible to use an siRNA in which one to more nucleotides are removed, replaced, inserted and/or added to one or both of the sense strand and antisense strand of the siRNA described above. One to more bases used herein are not specifically limited, but are preferably 1-4 bases, more preferably 1-3 bases, and most preferably 1-2 bases. Specific examples of such mutations include, but are not limited to, mutations that give rise to 0-3 bases in the 3' overhang, mutations that change the base sequence of the 3' overhang to give a different base sequence, mutations in which the lengths of the sense strand described above RNA and antisense strand RNA differ by 1-3 bases as a result of insertion, addition or deletion of bases, base substitution mutations in the sense strand and/or antisense strand with another base, and the like. However, it is necessary that the sense strand and the antisense strand be able to hybridize with such mutant siRNAs, and such mutant siRNAs have an ability to suppress gene expression that is equivalent to that of an siRNA without any mutations.

[0026]

An miRNA can also be a molecule with a structure in which one end is closed, such as a hairpin miRNA (short hairpin RNA; shRNA). shRNA is an RNA containing a sense strand RNA with a specific target gene sequence, an antisense strand RNA consisting of a sequence complementary to the sense strand, and a linker sequence for connecting the two strands, where a portion of the sense strand hybridizes with the antisense strand to form a double-stranded portion of the RNA .

[0027]

It is desirable that the siRNA does not exhibit a so-called off-target effect in clinical use. The off-target effect refers to the effect of suppressing the expression of a gene other than the target gene that is partially homologous to the siRNA used. In order to avoid an off-target effect, it can be confirmed that the candidate siRNA does not have cross-reactivity by using a DNA chip and the like. In addition, the off-target effect of confirming the presence of a gene containing a fragment that has high homology with a candidate miRNA sequence other than the target gene can be avoided by using a well-known database provided by NCBI (U.S. National Center for Biotechnology Information) and the like.

[0028]

To obtain siRNA according to the present invention, a known method such as a method using chemical synthesis or a method using gene recombination technology can be suitably used. With the synthesis method, double-stranded RNA can be synthesized based on the sequence information using a standard method. With the gene recombination method, siRNA can be produced by constructing an expression vector encoding a sense strand sequence or an antisense strand sequence, and introducing the vector into a host cell, and then obtaining each of the sense RNA strand and antisense RNA strand resulting transcription. It is also possible to obtain the desired double-stranded RNA by expressing shRNA forming a hairpin structure containing the sense strand of a particular target gene sequence, an antisense strand consisting of a sequence complementary to the sense strand, and a linker sequence to connect the two strands.

[0029]

With respect to siRNA, all or part of the nucleic acid constituting the siRNA may be a natural or modified nucleic acid, provided that such nucleic acid has an activity to suppress the expression of the target gene.

[0030]

The siRNA according to the present invention need not necessarily be a double-stranded RNA pair to the target sequence. It may be a mixture of multiple pairs ("multiple" is not specifically limited, but preferably refers to a small number, about 2-5) of double-stranded RNAs per region comprising the target sequence. In this regard, those skilled in the art can suitably obtain siRNA as a mixture of nucleic acids corresponding to the target sequence using a commercially available nucleic acid synthesizer and the DICER enzyme. The public synthesis service can also be used to synthesize the desired RNA. It should be noted that the siRNA according to the present invention encompasses the so-called "miRNA mixture". With respect to miRNAs of the present invention, not all nucleotides need to be ribonucleotides (RNA). In other words, in the present invention, one or more ribonucleotides constituting an siRNA may be the corresponding deoxyribonucleotide. The term "corresponding" refers to the content of the same type of base (adenine, guanine, cytosine, thymine (uracil)), but with a different sugar residue structure. For example, a deoxyribonucleotide corresponding to a ribonucleotide containing adenine refers to a deoxyribonucleotide containing adenine.

[0031]

In addition, a DNA (vector) that can express the above-described RNA of the present invention is also included as a preferred embodiment of a nucleic acid that can suppress the expression of mTOR and the like. For example, a DNA (vector) that can express the above double-stranded RNA according to the present invention is a DNA having a structure in which the DNA encoding one of the double-stranded RNA strands and the DNA encoding the other strand of the double-stranded RNA are connected to a promoter, in whereby each of these DNAs can be expressed. The above-described DNA according to the present invention can be properly obtained by those skilled in the art using standard genetic engineering techniques. More specifically, an expression vector according to the present invention can be obtained by appropriate insertion of DNA encoding an RNA of interest into various known expression vectors.

[0032]

In the present invention, the modified nucleic acid can be used as a nucleic acid for suppressing the expression of a target gene. A modified nucleic acid refers to a nucleic acid that has a nucleoside (base residue, sugar residue) and/or internucleoside binding site modification, and has a structure that is different from that of a natural nucleic acid. Examples of the "modified nucleoside" included in the modified nucleic acid include: abasic nucleosides; arabinonucleoside, 2'-deoxyuridine, α-deoxyribonucleoside, β-L-deoxyribonucleoside and other nucleosides containing a sugar modification; peptido nucleic acids (PNAs), phosphate group binding peptido nucleic acids (PHONAs), closed nucleic acids (LNAs), morpholino nucleic acids, and the like. The above nucleosides having a sugar modification include 2'-O-methylribose, 2'-deoxy-2'-fluororibose, 3'-O-methylribose and other substituted pentoses; 1',2'-deoxyribose; arabinose; substituted arabinose sugar; and a nucleoside having an alpha-anomer and hexose sugar modification. Such nucleosides may be a modified base in which the base residue is modified. Examples of such modified bases include pyrimidine such as 5-hydroxycytosine, 5-fluorouracil and 4-thiouracil; a purine such as 6-methyladenine and 6-thioguanosine; other heterocyclic bases; and the like.

[0033]

Examples of a "modified internucleoside bond" that is included in a modified nucleic acid include an alkyl linker, a glyceryl linker, an amino linker, a poly(ethylene glycol) bond, an internucleoside methylphosphonate bond; and bonds between non-natural nucleosides such as methylphosphonothioate, phosphotriester, phosphothiotryester, phosphothioate, phosphodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphate, and phosphoramidate.

[0034]

The nucleic acid sequence contained in the double-stranded siRNA of the present invention includes siRNAs to mTOR, other members of the mTOR signaling pathway, and the like.

[0035]

Also, the nucleic acid or agent of the present invention can be introduced into a phospholipid endoplasmic reticulum, such as a liposome, and the endoplasmic reticulum can be introduced. The endoplasmic reticulum, which retains the siRNA or shRNA, can be introduced into a given cell using lipofection. The resulting cell is then administered systemically, for example, intravenously, intra-arterially, and the like. It can also be administered topically to a desired area of the eye, and the like. Although siRNA shows very good specific activity after in vitro transcription, siRNA is rapidly degraded in vivo due to serum nuclease activity, so its duration of action is limited. Thus, there is a need to develop a better and more efficient delivery system. As an example, Ochiya, T et al., Nature Med., 5: 707-710, 1999, Curr. Gene Ther., 1: 31-52, 2001 report that the biocompatible material atelocollagen, when mixed with a nucleic acid to form a complex, is a carrier that protects the nucleic acid from a degrading enzyme in vivo and is particularly suitable as a carrier for siRNA. Although such a form may be used, the method for administering a nucleic acid, therapeutic or prophylactic drug according to the present invention is not limited thereto. Thus, due to the rapid degradation when exposed to nuclease in serum in a living body, it becomes possible to ensure continued action over a long period of time. For example, Takeshita F. PNAS, (2003) 102 (34) 12177-82, Minakuchi Y Nucleic Acids Research (2004) 32 (13) e109 reports that atelocollagen derived from bovine skin forms a complex with a nucleic acid that provides protection of the nucleic acid from the degrading enzyme in vivo and is particularly suitable as a carrier for siRNA. Such technology can be used.

[0036]

As used herein, "iFECD" (Fuchs immortalized corneal endothelial dystrophy line) is an abbreviation for Fuchs immortalized corneal endothelial dystrophy cells. A method for producing iFECD is described, for example, in WO 2015/015655.

[0037]

As used herein, "HCEC" (human corneal endothelial cells) is an abbreviation for human corneal endothelial cells. In addition, "iHCEC" is an abbreviation for immortalized human corneal endothelial cells.

[0038]

As used herein, "programmed cell death" refers to the phenomenon in which cells spontaneously die at a certain time or in a certain environment, as if they were programmed to die. Programmed cell death is used with a meaning that includes, for example, "apoptosis".

[0039]

When used within the scope of the invention, "transforming growth factor β (also abbreviated as TGF-β)" is used in the same sense as used in the prior art. It is a homodimeric multifunctional cytokine with a molecular weight of 25 kDa, which exhibits a variety of biological activities, for example, it is responsible for the pathogenesis of various sclerotic diseases, rheumatoid arthritis and proliferative vitreoretinopathy, actively participates in alopecia, suppression of the function of immunocompetent cells with simultaneous suppression of excessive protease production to prevent destruction lung tissue, leading to emphysema, and in suppressing the growth of cancer cells. "TGF-β signal" refers to a signal mediated by TGF-β that causes TGF-β. Examples of TGF-β signals include signals mediated by TGF-β2 in addition to signals mediated by TGF-β1, TGF-β3, and the like. In humans, TGF-β has three isoforms, TGF-β1 through β3, which share approximately 70% homology and similar activity. TGF-β is synthesized in an inactive latent form with a molecular weight of approximately 300 kDa, which is unable to bind to the receptor. Its action is manifested when activated on the surface of the target cell or in its environment, while it passes into an active form that can bind to the receptor.

[0040]

Without being limited by any theory, the action of TGF-β in the target cell is thought to be mediated by phosphorylation of a series of information-transmitting proteins called Smad. Initially, when activated TGF-β binds to a type II TGF-β receptor on the surface of the target cell, a receptor complex is formed consisting of two type II receptor molecules and two type I TGF-β receptor molecules, with type II receptors phosphorylating type I receptors. type. It must be understood that when phosphorylated type I receptors phosphorylate Smad2 or Smad3, the phosphorylated Smad2 or Smad3 forms a complex with Smad4, where the complex moves into the nucleus and binds to a target sequence called the CAGA box, which is present in the promoter region of the target gene, causing induction of transcription and expression of the target gene with a coactivator.

[0041]

The transforming growth factor-β (TGF-β) signaling pathway can modulate various cellular activities such as cell growth and differentiation, growth arrest, programmed cell death, and epithelial-mesenchymal transition (EMT) by modulating the target gene. Members of the TGF-β family, including TGF-β itself (e.g., TGF-β1, TGF-β2, and TGF-β3), activin, and bone morphogenetic protein (BMP), are potent modulators of growth, differentiation, cell migration, programmed cell death, and etc.

[0042]

TGF-β is an approximately 24 kDa protein produced by many cells, including B-lymphocytes, T-lymphocytes and activated macrophages, and many other cell types. The effects of TGF-β on the immune system include induction of the IL-2 receptor, inhibition of IL-1-induced thymocyte growth, and blocking of IFN-γ-induced macrophage activation. TGF-β is believed to be involved in a variety of pathological conditions (Border et al. (1992) J. Clin. Invest. 90:1) and has been fully shown to function either as a tumor suppressor or as a facilitator. tumors.

[0043]

TGF-β signaling is mediated by two cell surface serine/threonine kinase receptors, TGF-βRII and ALK5. TGF-β signaling is initiated by ligand-induced receptor dimerization, which allows TGF-βRII to phosphorylate the ALK5 receptor. Phosphorylation activates ALK5 kinase activity, and activated ALK5 then phosphorylates the downstream effector protein Smad (the vertebrate homologue of the MAD protein or "Mothers against DPP (decapentaplegic mutations)"), Smad2 or Smad3. The p-Smad2/3 complex with Smad4 enters the nucleus and activates transcription of the target gene.

[0044]

Smad3 is a member of the R-Smad (receptor-activated Smad) subgroup of the Smad family and a direct mediator of transcriptional activation by the TGF-β receptor. Stimulation of TGF-β leads to phosphorylation and activation of Smad2 and Smad3, which form a complex with Smad4 ("total Smad" or "co-Smad" in vertebrates). It accumulates in the nucleus and modulates transcription of the target gene. R-Smad is localized in the cytoplasm and forms a complex with co-Smad through ligand-induced phosphorylation by the TGF-β receptor, migrates to the nucleus, where it modulates the expression of genes associated with the cooperative transcription factor and chromatin. Smad6 and Smad7 are inhibitory Smad ("I-Smad"), i.e. they are transcriptionally induced by TGF-β and function as an inhibitor of TGF-β signaling (Feng et al. (2005) Annu. Rev. Cell. Dev. Biol 21: 659). Smad6/7 blocks the receptor-mediated activation of R-Smad, as a result of which it exerts its inhibitory effect; and they bind to the type I receptor, which competitively interferes with the mobilization and phosphorylation of R-Smad. Smad6 and Smad7 are known to replenish E3 ubiquitin ligase, which induces ubiquitination and degradation of proteins interacting with Smad6/7.

[0045]

TGF-β signaling pathways also include other pathways using BMP-7 transition and the like that go through ALK-1/2/3/6 to express function through Smad1/5/8. For TGF-β signaling, see J. Massagu'e, Annu. Rev. Biochem. 1998. 67: 753-91; Vilar JMG, Jansen R, Sander C (2006) PLoS Comput Biol 2(1): e3; Leask, A., Abraham, D. J. FASEB J. 18, 816-827 (2004); Coert Margadant & Arnoud Sonnenberg EMBO reports (2010)11, 97-105; Joel Rosenbloom et al., Ann Intern Med. 2010; 152: 159-166 etc.

[0046]

As used herein, the term "ocular condition, disorder or disease" refers to any ocular condition, disorder or disease. An ocular condition, disorder, or disease is a condition, disorder, or disease, for example, in the retina, vitreous fluid, lens, cornea, sclera, or other part of the eye. The mTOR inhibitor of the present invention can be particularly effective against a pathological condition, disorder or disease of the corneal endothelium.

[0047]

As used herein, "transforming growth factor β (TGF-β) mediated corneal endothelial condition, disorder or disease" refers to any corneal endothelial condition, disorder or disease caused by TGF-β in corneal endothelial cells. In the present invention, exposure to TGF-β2 on corneal endothelial cells, such as cells in the Fuchs model of corneal endothelial dystrophy (eg, iFECD), unexpectedly resulted in various disorders (eg, programmed cell death). This phenomenon has not been sufficiently understood. The inventors, after further analysis of the pathological condition, disorder or disease of the corneal endothelium mediated by the TGF-β signal, unexpectedly found that such a disorder can be suppressed by an mTOR inhibitor. A pathological condition, disorder or disease of the corneal endothelium mediated by the TGF-β signal is associated with another mTOR signaling pathway. Examples of conditions, disorders, or diseases of the corneal endothelium mediated by the TGF-β signal include, but are not limited to, Fuchs' corneal endothelial dystrophy, corneal transplant disorder, corneal endothelitis, trauma, post-ocular surgery disorder, ocular laser surgery disorder, aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disease, cytomegalovirus corneal endothelitis, and the like, in which TGF-β expression is observed. Since the disorder found in the present invention, or a disorder associated therewith, is considered to be severe or elevated, especially in corneal endothelial cells or corneal endothelial tissue with increased, compared to normal, expression of TGF-β2, any pathological condition, disorder or disease of the endothelium of the cornea, in which such observable endothelial cells or endothelial tissue of the cornea is particularly intended as a target according to the present invention.

[0048]

As used herein, "overexpression of extracellular matrix in corneal endothelial cells" refers to expression of extracellular matrix at a pathological level compared to extracellular levels of matrix expression in normal corneal endothelial cells. "Expression of extracellular matrix at a pathological level" refers to the production of extracellular matrix proteins, such as fibronectin, in excess of the amount produced in the extracellular matrix in normal form. The production status does not include stimulation, as well as an increase in expression levels due to a response to transforming growth factor (TGF) β, if necessary. For example, it may be about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, or about 2.0 times or more relative to the amount of extracellular matrix under normal circumstances for human corneal endothelial cells. The difference from normal values is preferably, but not necessarily, statistically significant. It is enough that the difference is a medically significant difference.

[0049]

When used within the scope of the invention, "endothelial corneal disorder due to overexpression of extracellular matrix (ECM)" or the corresponding "condition" is basically a disorder associated with hypertrophy, deposition, opacification caused by extracellular matrix, etc., or a corresponding condition, which leads to the formation of gutta on the surface of the corneal endothelium, hypertrophy of the Descemet's membrane such as cloudy gutta of the Descemet's membrane, and the like, and is associated with a condition that causes visual impairment. In endothelial disorders of the cornea, such as Fuchs's corneal dystrophy, excess production of extracellular matrix impairs vision or visual sensation even without a decrease in the number of cells, in contrast to the exacerbation of the condition caused by the death (especially apoptosis) of corneal endothelial cells. Thus, even if cell death can be suppressed, this must be taken into account. "Endothelial corneal disorder due to excessive production of extracellular matrix (ECM)" and the corresponding "condition" include, but are not limited to, opacification, scar, corneal opacity, corneal patch, corneal leukoma, and the like.

[0050]

In a preferred embodiment, the pathological condition, disorders or diseases that are the object of the present invention are disorders associated with Fuchs endothelial corneal dystrophy. It has been demonstrated that the induction of TGF-β in corneal endothelial cells plays a role in the development of Fuchs endothelial corneal dystrophy. It has also been demonstrated that TGF-β induction may be involved in cell loss in FECD. Therefore, inhibition of the TGF-β signaling pathway is naturally expected to be an effective therapy in FECD. However, the inventors surprisingly found that an mTOR inhibitor can suppress the TGF-β signal-mediated disorder.

[0051]

Since the drug according to the present invention can treat cell damage and the like, which is induced by TGF-β2, which may be one of the main causes of abnormalities or disorders in Fuchs endothelial corneal dystrophy, it should be understood that the drug can be used for treatment or prevention Fuchs endothelial corneal dystrophy. In particular, the present invention is able to suppress cell damage or programmed cell death induced by TGF-β2 in the Fuchs model of corneal endothelial dystrophy, in Examples, therefore, the present invention can be considered suitable for use in the treatment of patients with severe TGF-β2-associated disease in the model corneal dystrophy of the Fuchs endothelium. The drug of the present invention can also unexpectedly suppress extracellular matrix (ECM) overexpression, so the drug can treat or prevent a disorder, and the like. in the corneal endothelium, such as the deposition of ECM in the Descemet's membrane. Thus, the present invention can treat or prevent damage to corneal endothelial cells in Fuchs endothelial corneal dystrophy, corneal endothelial density reduction, gutta formation, Descemet's membrane hypertrophy, corneal hypertrophy, corneal epithelial disorder, turbidity, scarring, corneal stromal turbidity, photophobia, blurred vision , visual impairment, ophthalmalgia, epiphora, hyperemia, pain, bullous keratopathy, discomfort in the eyes, decreased visual contrast, halo, sensitivity to bright light, corneal stromal edema, etc.

[0052]

(General methods)

Molecular biology techniques, biochemical techniques, microbiological techniques used in the context of the invention are well known and traditionally used in this field, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and 3rd Ed. it (2001); Ausubel, F.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M.A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. associates; Ausubel, F.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. associates; Innis, M.A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J.J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Gait, M.J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M.J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G.T. (1996). Bioconjugate Techniques, Academic Press, Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume], Idenshi Donyu & Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis & Expression Analysis], Yodosha, 1997, etc. Messages from Nancy Joyce et al. {Joyce, 2004 #161} and {Joyce, 2003 #7} are well known in the field of corneal endothelial cells. However, as discussed above, long-term culturing or sub-culturing results in fibroblast-like transformation, and research is ongoing in search of an effective culturing method. The relevant portions thereof (which may be the entire document) are incorporated herein by reference.

[0053]

(Description of Preferred Embodiments)

The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are illustrative examples of the present invention, thus the scope of the present invention is not limited to such preferred embodiments. It is to be understood that those skilled in the art may refer to the following preferred embodiments in order to easily make modifications or changes within the scope of the present invention. Those skilled in the art may appropriately combine any of these embodiments of the present invention.

[0054]

<Remedy>

In one aspect, the present invention provides a composition for use in the prevention or treatment of an ocular condition, disorder or disease, comprising an mTOR inhibitor. In particular, an mTOR inhibitor is effective against a pathological condition, disorder or disease in the corneal endothelium.

[0055]

Although it is believed that mTOR is involved in intracellular signaling and is responsible for the regulation of cell mitosis, cell survival, etc., its mechanism of action in the corneal endothelium has not been studied. Thus, it turned out to be unexpected and surprising that an mTOR inhibitor is effective in the treatment and prevention of eye diseases, disorders or pathological conditions, in particular of the corneal endothelium.

[0056]

In one embodiment, the endothelial pathological condition, disorder or disease of the cornea mediated by transforming growth factor β (TGF-β) in corneal endothelial cells is selected from the group consisting of Fuchs endothelial corneal dystrophy, corneal transplant disorders, corneal endothelitis, trauma, disorders after eye surgery, eye laser surgery disorders, aging, posterior polymorphic dystrophy (PPD), congenital hereditary endothelial dystrophy (CHED), idiopathic corneal endothelial disorder, and cytomegalovirus corneal endothelitis.

[0057]

In another preferred embodiment, the present invention provides a medicament having an effect and efficacy in the treatment or prevention of a pathological condition caused by extracellular matrix overexpression in Fuchs endothelial corneal dystrophy, for use in the treatment or prevention of such a condition or in a method for the treatment or prevention of such a condition. Examples of such a condition include guttae on the surface of the corneal endothelium, cloudy guttae of Descemet's membrane, hypertrophy of the Descemet's membrane, blurred vision, halo, sensitivity to bright light, blurred vision, corneal clouding, leukoma, disturbances in visual sensation, and the like. Pathological conditions caused by excess extracellular matrix production are discussed further below.

[0058]

In yet another aspect, the present invention relates to a medicament for the treatment or prevention of a pathological condition, disorder or disease of the corneal endothelium caused by overexpression of extracellular matrix in corneal endothelial cells, containing an mTOR inhibitor. As discussed above, an mTOR inhibitor can treat or prevent corneal endothelial disease and the like mediated by the TGF-β signal, however, unexpectedly, an mTOR inhibitor can also suppress extracellular matrix overexpression in corneal endothelial cells. This suggests that an mTOR inhibitor can simultaneously treat corneal endothelial disorders mediated by TGF-β signaling and extracellular matrix overexpression in corneal endothelial cells. In particular, Fuchs' corneal endothelial dystrophy is a disease in which the density of corneal endothelial cells is significantly reduced due to TGF-β signal, and extracellular matrix is deposited in the Descemet's membrane, resulting in the formation of corneal gutta and Descemet's membrane hypertrophy. For this reason, the suppression of extracellular matrix overexpression means that the therapy and prevention of Fuchs endothelial corneal dystrophy can be greatly improved, and in some cases a complete cure is possible. It is also possible to reduce, treat or prevent the formation of corneal gutta and Descemet's membrane hypertrophy, as well as other conditions associated with opacification or deposition (irreversible opacification of the corneal stroma due to prolonged corneal edema, etc.) that may occur due to excessive production of extracellular matrix during corneal endothelial disorders such as Fuchs endothelial corneal dystrophy.

[0059]

In one embodiment, a pathological condition, disorder or disease of the corneal endothelium caused by overexpression of extracellular matrix in corneal endothelial cells may be due to overexpression of fibronectin in corneal endothelial cells.

[0060]

In one embodiment, the pathological condition, disorder or disease of the corneal endothelium caused by overexpression of extracellular matrix in corneal endothelial cells is selected from the group consisting of Fuchs endothelial corneal dystrophy, gutt formation, Descemet's membrane hypertrophy, corneal hypertrophy, turbidity, scar, stromal turbidity corneal epithelial edema, corneal epithelial disorder, photophobia and blurred vision.

[0061]

In another aspect, the present invention provides a medicament for use in the treatment or prevention of an endothelial pathological condition, disorder or disease of the cornea mediated by a TGF-β signal and extracellular matrix overexpression in corneal endothelial cells, comprising an mTOR inhibitor. an mTOR inhibitor can simultaneously treat or prevent corneal endothelial disorders mediated by TGF-β signaling and extracellular matrix overexpression in corneal endothelial cells.

[0062]

In one embodiment, the corneal endothelial condition, disorder or disease mediated by TGF-β signal and extracellular matrix overexpression in corneal endothelial cells is selected from the group consisting of Fuchs endothelial corneal dystrophy, other corneal endothelial dystrophy, and drug-induced endothelial corneal disorder. , surgery, trauma, infection, uveitis, etc.

[0063]

In one embodiment, the corneal endothelial condition, disorder or disease mediated by TGF-β signal and extracellular matrix overexpression in corneal endothelial cells comprises Fuchs' corneal endothelial dystrophy. Fuchs endothelial corneal dystrophy is a disease in which the density of corneal endothelial cells is significantly reduced due to the TGF-β signal, with extracellular matrix being deposited in the Descemet's membrane, leading to a disorder such as corneal gutta and Descemet's membrane hypertrophy. Therefore, suppression of extracellular matrix overexpression means that therapy can significantly reduce Fuchs endothelial corneal dystrophy and cause a complete cure in some cases. Reduction of a disorder and the like such as corneal gutta and Descemet's membrane hypertrophy in Fuchs' endothelial corneal dystrophy using an mTOR inhibitor causes a qualitative reduction in eye disease and provides an unconventional therapeutic effect against a disease such as Fuchs' endothelial corneal dystrophy that has been a threat.

[0064]

In one embodiment, examples of uses of the present invention include, but are not limited to, eye drops, as well as administration methods such as anterior chamber injection, controlled release impregnation, subconjunctival injection, and systemic administration (oral and intravenous injection).

[0065]

In one embodiment, the mTOR inhibitor used in the present invention may be any type of mTOR inhibitor, as long as it is effective in treating or preventing the given ocular condition, disorder, or disease (eg, corneal endothelium). Certain mTOR inhibitors include at least one selected from the group consisting of rapamycin, temsirolimus, everolimus, PI-103, CC-223, INK128, AZD8055, KU 0063794, Voxtalizib (XL765, SAR245409), Ridaforolimus (Deforolimus, MK-8669 ), NVP-BEZ235, CZ415, Torkinib (PP242), Torina 1, Omipalizib (GSK2126458, GSK458), OSI-027, PF-04691502, Apitoliziba (GDC-0980, RG7422), WYE-354, Vistusertiba (AZD2014), Torina 2, Tacrolimus (FK506), GSK1059615, Gedatolizib (PF-05212384, PKI-587), WYE-125132 (WAY 132), BGT226 (NVP-BGT226), Palomida 529 (P529), PP121, WYE-687, CH5132799, WAY -600, ETP-46464, GDC-0349, XL388, Zotarolimus (CA. 578) and chrysophanoic acid.

[0066]

The above mTOR inhibitors can be used alone or in combination in the medicament of the present invention. The concentration of the mTOR inhibitor used in the present invention can be appropriately changed depending on the type of mTOR inhibitor. For example, the concentration may be, without limitation, at least about 0.0001 nM (nmol/L), at least about 0.001 nM, at least about 0.01 nM, at least about 0.1 nM, at least at least about 1 nM, at least about 10 nM, at least about 100 nM, or at least about 1000 nM. The upper concentration limit of mTOR used in the present invention includes, without limitation, approximately 100 μM (μmol/L), approximately 10 μM, approximately 1 μM, or approximately 0.5 μM. Examples of the range of concentrations of the mTOR inhibitor used in the present invention include, without limitation, from about 0.01 nM to about 100 μm, from about 0.1 nM to about 100 μm, from about 1 nM to about 100 μm, from about 10 nM to about 100 µM, from about 100 nM to about 100 µM, from about 1 µM to about 100 µM, from about 0.01 nM to about 10 µM, from about 0.1 nM to about 10 µM, from about 1 nM to about 10 µM, from about 10 nM to about 10 µM, from about 100 nM to about 10 µM, from about 1 µM to about 10 µM, from about 0.01 nM to about 1 µM, from about 0.1 nM to about 1 µM, from about 1 nM to about 1 µM, from about 10 nM to about 1 µM, from about 100 nM to about 1 µM, from about 0.01 nM d about 100 nM, from about 0.1 nM to about 100 nM, from about 1 nM to about 100 nM, and from about 10 nM to about 100 nM. When using two or more types of mTOR inhibitors in combination, the concentration of each mTOR inhibitor can be adjusted accordingly.

[0067]

In a preferred embodiment, the mTOR inhibitor is selected from the group consisting of, for example, rapamycin, temsirolimus, everolimus and salts thereof. Without wishing to be bound by theory, this is because it has been found that treatment with mTOR inhibitors such as rapamycin, temsirolimus, and everolimus has shown a significantly better therapeutic outcome compared to other mTOR inhibitors, and in particular, results in the cure of endothelial disease or Transforming growth factor β2 (TGF-β2) associated corneal disorders such as Fuchs endothelial dystrophy or endothelial disease or extracellular matrix (ECM) overexpression corneal disorders were significantly improved. In addition, this is because these mTOR inhibitors have already been approved by the FDA, PMDA, etc., and are expected to be used as an ophthalmic drug, as a pharmaceutical product, even considering aspects such as safety .

[0068]

The above compounds can be used alone or in combination in the medicament of the present invention. The concentration of the compound used in the present invention is about 0.01 nM to about 100 µM (µmol/L), or about 0.1 nM to 100 µM, typically about 1 nM to 100 µM, about 10 nM to 100 μM, preferably from about 0.1 to 30 μM, and more preferably from about 1 to 10 μM. The upper limits and lower limits of these ranges can be appropriately set in combination, and when two or more types of compounds are used in combination, the concentration can be changed accordingly. Examples of other concentration ranges include, without limitation, usually about 0.01 nM to 100 µM, about 0.1 nM to 100 µM, or about 0.001 to 100 µM, preferably about 0.01 to 75 µM, about 0 .05 to 50 µM, about 1 to 10 µM, about 0.01 to 10 µM, about 0.05 to 10 µM, about 0.075 to 10 µM, about 0.1 to 10 µM, about 0 .5 to 10 µM, about 0.75 to 10 µM, about 1.0 to 10 µM, about 1.25 to 10 µM, about 1.5 to 10 µM, about 1.75 to 10 µM , about 2.0 to 10 µM, about 2.5 to 10 µM, about 3.0 to 10 µM, about 4.0 to 10 µM, about 5.0 to 10 µM, about 6, 0 to 10 µM, about 7.0 to 10 µM, about 8.0 to 10 µM, about 9.0 to 10 µM, about 0.01 to 50 µM, about 0.05 to 5.0μM, approximately 0.075 to 5.0 μM, approximately 0.1 to 5.0 μM, approximately 0.5 to 5.0 μM, approximately 0.75 to 5.0 μM, approximately 1.0 to 5.0 µM, about 1.25 to 5.0 µM, about 1.5 to 5.0 µM, about 1.75 to 5.0 µM, about 2.0 to 5.0 µM, about 2.5 to 5.0 µM, about 3.0 to 5.0 µM, about 4.0 to 5.0 µM, about 0.01 to 3.0 µM, about 0.05 to 3 .0 µM, about 0.075 to 3.0 µM, about 0.1 to 3.0 µM, about 0.5 to 3.0 µM, about 0.75 to 3.0 µM, about 1, 0 to 3.0 µM, approximately 1.25 to 3.0 µM, approximately 1.5 to 3.0 µM, approximately 1.75 to 3.0 µM, approximately 2.0 to 3.0 µM , approximately 0.01 to 1.0 μM, approximately 0.05 to 1.0 μM, approximately 0.075 to 1.0 μM, approximately 0.1 to 1.0 μM, approximately 0.5 to 1 .0 µM, approximately 0.75 to 1 .0 µM, about 0.09 to 35 µM, about 0.09 to 3.2 µM, more preferably about 0.05 to 1.0 µM, about 0.075 to 1.0 µM, about 0, 1 to 1.0 µM, about 0.5 to 1.0 µM, about 0.75 to 1.0 µM.

[0069]

In the case of application in the form of eye drops, the concentration of the composition can be determined using about 1-10000 times, preferably about 100-10000 times, such as about 1000 times the concentration, relative to the above effective concentration as a reference, taking into account dilution tear fluid, etc., and paying attention to toxicity. You can also set a higher concentration. For example, the concentration is from about 0.01 µM (µmol/L) to 1000 mM (mmol/L), from about 0.1 µM to 100 mM, from about 1 µM to 100 mM, from about 10 µM to 100 mM, or about 0.1 µM to 30 mM, about 1 µM to 30 mM, more preferably about 1 µM to 10 mM, about 10 µM to 10 mM, about 100 µM to 10 mM, about 10 µM to 100 mm, from about 100 μm to 100 mm, or may be from about mM to 10 mm, from about 1 mm to 100 mm. The upper limits and lower limits of these ranges can be appropriately set in combination, and when two or more types of compounds are used in combination, the concentration can be changed accordingly.

[0070]

In another embodiment, the mTOR inhibitor is rapamycin. The concentration of rapamycin to be used is at least about 0.1 nM, at least about 1 nM, at least about 10 nM, preferably about 100 nM. In a preferred embodiment, rapamycin is used in the form of eye drops and the concentration of rapamycin to be used in such a case is at least about 0.1 mM, at least about 1 mM, at least about 10 mM, preferably at least approximately 100 mm. A saturation amount of 1000 mM is provided as an example of an upper limit of concentration.

[0071]

In another embodiment, the mTOR inhibitor is temsirolimus. The concentration of temsirolimus to be used is at least about 0.01 nM, at least about 0.1 nM, preferably about 1 nM, preferably about 10 nM, more preferably about 100 nM, more preferably about 1 μM, more preferably approximately 10 μM. Temsirolimus is used in the form of eye drops, wherein the concentration of temsirolimus to be used in such a case is at least about 0.01 mM, at least about 0.1 mM, at least about 1 mM, preferably at least about 10 mm. A saturation amount of 1000 mM is provided as an example of an upper limit of concentration. In another embodiment, the mTOR inhibitor is everolimus. The concentration of everolimus to be used is at least about 0.1 nM, at least about 1 nM, at least about 10 nM, preferably about 100 nM. Everolimus is used in the form of eye drops, wherein the concentration of everolimus to be used in such a case is at least about 0.1 mM, at least about 1 mM, at least about 10 mM, preferably at least about 100 mM . A saturation amount of 1000 mM is provided as an example of an upper limit of concentration.

[0072]

In one embodiment, the therapeutic or prophylactic drug of the present invention may be for any animal with corneal endothelium, such as a mammal. Such a drug is preferably for the treatment or prevention of primate corneal endothelium. The object of therapy or prophylaxis is preferably the human corneal endothelium.

[0073]

In another embodiment, the mTOR inhibitor may be a substance that suppresses the expression of the mTOR gene. Examples of a substance that suppresses the expression of the mTOR gene include, without limitation, siRNA, antisense nucleic acid, or ribozyme.

[0074]

In a specific embodiment, the mTOR inhibitor is an siRNA against the mTOR gene. Typical examples of miRNA used in the present invention include, without limitation:

the semantic chain presented in

CAUUCGCAUUCAGUCCAUAtt (SEQ ID NO: 1); and

antisense strand presented in

UAUGGACUGAAUGCGAAUGat (SEQ ID NO: 2).

Any sequence is acceptable as long as there is an antisense effect or an RNAi sense effect against the mTOR gene. 1-3 nucleotide bases may be deleted, replaced, inserted and/or added to such sense strand and antisense strands.

[0075]

In another aspect, the present invention provides a method of treating or preventing an ocular (e.g., corneal endothelial) condition, disorder, or disease (pathological condition, disorder, or disease of the corneal endothelium mediated by TGF-β signal and/or extracellular matrix overexpression in corneal endothelial cells), comprising administering an effective amount of an mTOR inhibitor to a patient in need thereof.

[0076]

When used within the scope of the invention, "patient" refers to the object of application (transplantation) of a therapeutic or prophylactic drug or method of the present invention. Patient examples include mammals (eg, human, mouse, rat, hamster, rabbit, cat, dog, cow, horse, sheep, monkey, etc.), however, primates are preferred and humans are most preferred.

[0077]

An effective amount of a drug of the present invention that is effective in treating a particular disease, disorder, or condition may vary depending on the properties of the disorder or condition, and such an effective amount can be determined by one of skill in the art using standard clinical techniques based on information in this description. Also, if necessary, you can use the analysis in vitro to determine the optimal dose range. Since the exact dose to be used in the composition may vary depending on the route of administration and the severity of the disease or disorder, the dose should be determined according to the judgment of the physician and the condition of each patient. However, the dose, although not specifically limited, may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight, or between any two such values per dose. The administration interval, although not specifically limited, may be, for example, one or two doses every 1, 7, 14, 21 or 28 days, or one or two doses over a number of days between any two such values. The dose, the number of doses, the administration interval, and the route of administration may be appropriately selected depending on the age or body weight of the patient, condition, dosage form, target organ, and the like. For example, the present invention may be used in the form of eye drops. The drug according to the present invention can also be administered into the anterior chamber of the eye. The therapeutic drug preferably contains a therapeutically effective amount or an effective amount of active substances, which exhibits the desired effect. The presence of a therapeutic effect can be determined when the therapeutic marker is significantly reduced after administration. An effective amount can be estimated from a dose-response curve obtained using an in vitro testing system or in an animal model.

[0078]

<Conservation and growth of corneal endothelial cells>

In another aspect, the present invention provides a corneal endothelial cell preservation composition comprising an mTOR inhibitor. The present invention also provides a method for preserving corneal endothelial cells, comprising contacting an effective amount of an mTOR inhibitor with corneal endothelial cells. It should be understood that an embodiment of an mTOR inhibitor and the like used for conserving corneal endothelial cells may use any of the embodiments described in the <Drug> section herein. Contact with corneal endothelial cells can be carried out in vivo , ex vivo or in vitro , and can be used in the manufacture of a cell composition.

[0079]

In another aspect, the present invention provides a composition for growing or promoting growth of corneal endothelial cells, comprising an mTOR inhibitor. The present invention also provides a method for growing or promoting growth of corneal endothelial cells, comprising contacting an effective amount of an mTOR inhibitor with corneal endothelial cells. It should be understood that an embodiment of an mTOR inhibitor and the like used to grow or stimulate the growth of corneal endothelial cells may use any of the embodiments described in the <Drug> section herein. Contact with corneal endothelial cells can be carried out in vivo , ex vivo or in vitro , and can be used in the manufacture of a cell composition.

[0080]

Reference literature, such as scientific literature, patents, and patent applications cited within the scope of the invention, is incorporated within the scope of the invention by reference to the same extent that each document is specifically described in its entirety.

[0081]

The present invention has been described through preferred embodiments for ease of understanding. The present invention is further explained on the basis of Examples. The above description and the following examples are not intended to limit the present invention, but for the sole purpose of explanation. Thus, the scope of the present invention is not limited to the embodiments and examples specifically disclosed within the scope of the invention, but is limited only by the scope of the claims.

[Examples]

[0082]

The following describes examples of the present invention. Biological samples, etc., as appropriate, were processed in accordance with the standards adopted by the Ministry of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, etc., and, as appropriate, on on the basis of the Declaration of Helsinki or ethical standards prepared on its basis. For the donation of the eyes used in the study, consent was obtained from close relatives of all deceased donors. This study was approved by the ethics committee or appropriate body of the University of Erlangen-Nuremberg (Germany) and the SightLife ^™ Eye Bank (Seattle, Washington).

[0083]

(Derivation Example: Generation of a model based on an immortalized corneal endothelial cell line derived from patients with Fuchs' endothelial corneal dystrophy (iFECD))

In this example, the immortalized corneal endothelial cell line (iFECD) was derived from corneal endothelial cells from patients with Fuchs endothelial corneal dystrophy.

[0084]

(Cultivation method)

Corneal endothelial cells were mechanically separated with basement membrane from research cornea obtained from the Seattle Eye Bank. After using collagenase to separate and harvest corneal endothelial cells from the basement membrane, the cells were subjected to primary culture. As for the medium, Opti-MEM I serum reduced medium, liquid (INVITROGEN part number: 31985-070) to which 8% FBS (BIOWEST part number: S1820-500), 200 mg/ml CaCl ₂ ⋅ 2H ₂ O (SIGMA part number: C7902-500G), 0.08% chondroitin sulfate (SIGMA part number: C9819-5G), 20 µg/mL ascorbic acid (SIGMA part number: A4544-25G), 50 µg /ml gentamicin (INVITROGEN catalog number: 15710-064) and 5 ng/ml EGF (INVITROGEN catalog number: PHG0311) and conditioned for 3T3 feeder cells were used as basal medium. In addition, cells were cultured in a basal medium supplemented with SB431542 (1 µmol/L) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazole<4-[ 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine) (1 µmol/L) (also referred to in the context of the invention as "conditioned medium SB203580+SB431542+3T3").

[0085]

(image analysis method)

Corneal endothelial cells were obtained with ethics committee approval and written consent from 3 patients who suffered from bullous keratopathy consistent with the clinical diagnosis of Fuchs' corneal endothelial dystrophy and underwent corneal endothelial transplantation (Descemet's membrane endothelial keratoplasty=DMEK). For DMEK, pathological corneal endothelial cells were mechanically peeled off with basement membrane, ie Descemet's membrane, and immersed in Optisol-GS corneal preservation solution (Bausch&Lomb). Then, a collagenase treatment was applied to enzymatically harvest corneal endothelial cells, and the cells were cultured in conditioned medium SB203580+SB431542+3T3. For cultured corneal endothelial cells from a patient with Fuchs' corneal endothelial dystrophy, the SV40 large T antigen and the hTERT gene were amplified by PCR and introduced into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc.). The lentiviral vector was then used to infect 293T cells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) using a transfection reagent (Fugene HD; Promega Corp., Madison, WI) and three types of helper plasmids (pLP1, pLP2, pLP/VSVG; Life Technologies Inc.). Culture supernatant containing viruses was collected 48 hours after infection. 5 μg/ml of polybrene was used, which was added to the culture solution of cultured corneal endothelial cells from a patient with Fuchs endothelial corneal dystrophy, and the SV40 large T antigen and the hTERT gene were introduced. We studied images of an immortalized corneal endothelial cell line (iFECD) of patients with Fuchs endothelial corneal dystrophy obtained using a phase contrast microscope. Cultured corneal endothelial cells from test corneas obtained from the Seattle Eye Bank were immortalized in the same manner to obtain an immortalized normal corneal endothelial cell line (iHCEC) as a control. When imaging an immortalized corneal endothelial cell line (iFECD) and an immortalized healthy donor corneal endothelial cell line (iHCEC) using a phase contrast microscope, iHCEC cells, like iFECD, had a polygonal layer, as in normal corneal endothelial cells. IHCEC and iFECD were maintained and cultured in Dulbecco's Modified Eagle's Medium (DMEM) +10% fetal bovine serum (FBS).

[0086]

(Example 1: Effect of suppression of cell damage caused by TGF-β2 by rapamycin)

This Example demonstrated the effect of rapamycin, which is a typical example of an mTOR inhibitor, on cell damage.

[0087]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0088]

iFECD were seeded in a 12-well plate at a ratio of 1×10 ⁵ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (DMEM + 10% FBS + 1% P/S was used as the medium). After 24 hours, the medium was removed. Rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, the medium was removed. Medium containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) and rapamycin was added to culture cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0089]

(Results)

(Rapamycin inhibits cell damage caused by TGF-β2),

Figure 1 shows the results. Upon stimulation of iFECD cells with recombinant human TGF-β2 without rapamycin, the cells were found to be significantly damaged. On the other hand, it was observed that damage to corneal endothelial cells was suppressed in the case of pre-treatment with rapamycin. Therefore, rapamycin was found to suppress cell damage caused by recombinant human TGF-β2.

[0090]

(Example 2: Inhibition Effect of Rapamycin on TGF-β2-Induced Caspase Activity)

This Example demonstrated the effect of rapamycin, which is a typical example of an mTOR inhibitor, on caspase activity.

[0091]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0092]

iFECD were seeded in a 12-well plate at a ratio of 1×10 ⁵ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (DMEM + 10% FBS + 1% P/S was used as the medium). After 24 hours, the medium was removed. Rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, the medium was removed. Medium containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) and rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0093]

After microscopy, Western blotting of proteins was performed according to the following procedure.

[0094]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect the protein supernatant.

[0095]

2) Western blotting

8 μg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Rabbit anti-caspase 3 (Cell Signaling, 9662), rabbit anti-PARP (Cell Signaling, 9542), and mouse anti-GAPDH (MBL, M171-3) were used as primary antibodies. Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. As for the primary antibodies, rabbit anti-caspase 3: 1000-fold dilution, rabbit anti-PARP: 2000-fold dilution, and mouse anti-GAPDH: 5000-fold dilution, while secondary antibodies were diluted 5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0096]

(Results)

(Rapamycin inhibits TGF-β2 induced caspase activity)

The results of Western blotting of caspase are shown in Figure 2 . Upon stimulation of iFECD cells with recombinant human TGF-β2 without rapamycin, the presence of cleaved caspase 3 (approximately 17 kDa), which is the active form, was observed. On the other hand, the activated form of cleaved caspase 3 was almost absent in the rapamycin supplemented group. Thus, as was found in the analysis, rapamycin suppressed the activation of caspase by recombinant human TGF-β2. mTOR is not associated with a caspase signal, nor is an mTOR inhibitor known to suppress caspase activity. Thus, it was unexpected that rapamycin could inhibit caspase activity.

[0097]

(Example 3: Effect of Suppression of S6K Phosphorylation Activity Induced by Recombinant Human TGF-β2 by Rapamycin)

This Example demonstrated the effect of rapamycin, which is a typical example of an mTOR inhibitor, on S6K phosphorylation activity induced by recombinant human TGF-β2.

[0098]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, 1×PBS (-) was added to the cells, which was preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0099]

iFECD cells were seeded in a 12-well plate at a ratio of 7×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (DMEM + 10% FBS + 1% P/S was used as the medium) . After 24 hours, the medium was removed. Rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, the medium was removed. Medium containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) and rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0100]

After microscopy, Western blotting of proteins was performed according to the following procedure.

[0101]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect the protein supernatant.

[0102]

2) Western blotting

8 μg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies used were rabbit anti-Akt1 (Cell Signaling, 2938), mouse anti-phospho-Akt (Cell Signaling, 4051), rabbit anti-S6K (Cell Signaling, 9202), rabbit anti-phospho-S6K (Cell Signaling, 9204) and a mouse anti-GAPDH antibody (MBL, M171-3). Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. Regarding primary antibodies, rabbit anti-Akt: 1000-fold dilution, mouse anti-phospho-Akt: 1000-fold dilution, rabbit anti-S6K: 1000-fold dilution, rabbit anti-phospho-S6K: 1000-fold dilution and mouse anti-GAPDH antibody: 5000-fold dilution, while the secondary antibody was diluted 5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0103]

(Results)

(Rapamycin inhibits TGF-β2 induced S6K phosphorylation activity)

The results are shown in Figure 3. Upon stimulation of iFECD cells with recombinant human TGF-β2 without rapamycin, Akt phosphorylation activity was detected. On the other hand, S6K phosphorylation activity was suppressed in the rapamycin supplemented group. Rapamycin was found to have an inhibitory effect on the mTOR signaling pathway according to Western blot analysis in the presence of rapamycin. Akt is active before mTOR and S6K is active after mTOR. Thus, given that upstream Akt was phosphorylated while downstream S6K phosphorylation was downregulated by an mTOR inhibitor, rapamycin was found to inhibit the mTOR pathway.

[0104]

(Example 4: Effect of inhibition of TGF-β2-induced Smad2/3 phosphorylation activity by rapamycin)

This Example demonstrated the suppression effect of rapamycin, which is a typical example of an mTOR inhibitor, on TGF-β2-induced Smad2/3 phosphorylation activity.

[0105]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0106]

iFECD were seeded in a 12-well plate at a ratio of 8×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (DMEM + 10% FBS + 1% P/S was used as medium). After 24 hours, the medium was removed. Rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, the medium was removed. Medium containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) and rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0107]

After microscopy, Western blotting of proteins was performed according to the following procedure.

[0108]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect protein supernatant.

[0109]

2) Western blotting

8 μg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies used were rabbit anti-Smad2 (Cell Signaling, 5339), mouse anti-phospho-Smad2 (Cell Signaling, 3108), rabbit anti-Smad3 (Cell Signaling, 9523), rabbit anti-phospho-Smad3 (Cell Signaling, 9520) and a mouse anti-GAPDH antibody (MBL, M171-3). Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. Regarding primary antibodies, rabbit anti-Smad2: 1000-fold dilution, mouse anti-phospho-Smad2: 1000-fold dilution, rabbit anti-Smad3: 1000-fold dilution, rabbit anti-phospho-Smad3: 1000-fold dilution and mouse anti-GAPDH antibody: 5000-fold dilution, while the secondary antibody was diluted 5000-fold. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0110]

(Results)

(Rapamycin does not inhibit TGF-β2-induced Smad2/3 phosphorylation activity)

The results are shown in Figure 4 . When stimulated with recombinant human TGF-β2 without rapamycin, Smad2 and Smad3 phosphorylation activity was detected. Thus, as found in Western blot analysis, rapamycin did not inhibit the Smad2/3 phosphorylation activity induced by recombinant human TGF-β2. Rapamycin's suppression of cell damage was shown not to be due to inhibition of the TGF-β signal, as the effect of TGF-β was thought to be mediated by Smad phosphorylation. This indicates that rapamycin suppresses corneal endothelial damage and the like. due to the TGF-β signal without direct inhibition of the TGF-β signal, which was an unexpected result.

[0111]

(Example 5: Effect of inhibition of TGF-β2-induced fibronectin production by rapamycin)

This Example demonstrated the suppression effect of rapamycin, which is a typical example of an mTOR inhibitor, on TGF-β2-induced fibronectin production.

[0112]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0113]

iFECD cells were seeded in a 6-well plate at a ratio of 2×10 ⁵ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (DMEM + 10% FBS + 1% P/S was used as a medium) . After 24 hours, the medium was removed. Rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, the medium was removed. Medium containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) and rapamycin was added to culture the cells for 24 hours (DMEM+2% FBS+1% P/S was used as medium). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0114]

After microscopy, Western blotting of proteins was performed according to the following procedure.

[0115]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect the protein supernatant.

[0116]

2) Western blotting

5 μg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Mouse anti-fibronectin antibody (BD Bioscience, 610077) and mouse anti-GAPDH antibody (MBL, M171-3) were used as primary antibodies. Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. For mouse anti-fibronectin antibody: 15,000-fold dilution, and mouse anti-GAPDH antibody: 5,000-fold dilution, while the secondary antibody was diluted 5,000 times. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0117]

(Results)

(Rapamycin inhibits TGF-β2-induced fibronectin production)

The results are shown in Figure 5 . Upon stimulation with recombinant human TGF-β2 without rapamycin, fibronectin production was observed in iFECD. On the other hand, almost no fibronectin production was observed in the rapamycin supplemented group. Thus, the level of fibronectin expression induced by recombinant human TGF-β2 was found to be suppressed by rapamycin in Western blot analysis. The mTOR inhibitor was not known to be involved in the production of an extracellular matrix such as fibronectin. Thus, it was unexpected that mTOR is able to suppress the production of extracellular matrix.

[0118]

In Fuchs endothelial corneal dystrophy, excess production of extracellular matrix such as fibronectin and its deposition on the Descemet's membrane leads to a disorder such as Descemet's membrane hypertrophy or gutta formation. Such a disorder in patients with Fuchs endothelial corneal dystrophy usually begins to develop at the age of 30-40 and progresses throughout the patient's life. Progression results in visual impairment such as blurry vision, halo, sensitivity to bright light, or blurred vision. While corneal endothelial cells in Fuchs' corneal endothelial dystrophy are constantly destroyed, the transparency of the cornea is maintained by the remaining corneal endothelium compensating for a pumping function until the density of corneal endothelial cells falls below about 1000 cells/mm ² . If the density becomes lower than about 1000 cells/mm ² , aqueous humor infiltration from the anterior segment of the eye into the cornea results in corneal edema leading to severe visual impairment (Figure 18 ). Thus, patients with Fuchs endothelial corneal dystrophy suffer from impaired visual function mainly due to two reasons, namely, excessive production of extracellular matrix and death of corneal endothelial cells. The caspase inhibitor of the present invention is involved in both the suppression of extracellular matrix production and the suppression of corneal endothelial cell death, which is particularly useful in the treatment of Fuchs' endothelial corneal dystrophy.

[0119]

(Example 6: Effect of suppression by mTOR siRNA of mTOR expression and S6K phosphorylation)

This Example demonstrated the effect of mTOR suppression of siRNA, which is another typical example of an mTOR inhibitor, mTOR expression and S6K phosphorylation.

[0120]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0121]

iFECD were seeded in a 12-well plate at a ratio of 3×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (medium: DMEM +10% FBS +1% P/S). After 24 hours, the medium was removed. Lipofectamine (Invitrogen, 92008) and 3 pmol mTOR siRNA (Ambion, AS026M0L) were added to culture the cells for 24 hours (medium: OptiMEM-I (Invitrogen, 31985-088)). The mTOR siRNA used had the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 2. After 24 hours, the medium was removed and the cells were cultured for 72 hours at 37°C (5% CO ₂ ) ( medium: DMEM +10% FBS +1% P/S). After 72 hours, the medium was removed. Media containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) was added to culture the cells for 24 hours (medium: DMEM+2% FBS+1% P/S). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0122]

After microscopy, amplification of the cDNA base sequence was carried out using the PCR method according to the following method.

[0123]

1) Collection of total RNA

The medium was removed from the culture dish in which the iFECD cells were cultured, 350 μl of RLT lysis buffer from the RNeasy miniKit (QIAGEN, M610A) was added to the cells and eluted. 350 μl of 70% EtOH was added to the cells and transferred to an RNeas mini centrifuge column (QIAGEN) which was then centrifuged for 15 seconds at 10,000 rpm to remove the breakthrough. Then, 30 μl of RNeasy RNase-free water (QIAGEN) was added to the RNeasy mini centrifuge column, followed by centrifugation for 1 minute at 10,000 rpm to isolate total RNA.

[0124]

2) Synthesis of cDNA

Isolated total RNA 450 ng, Rever Tra Ace (TOYOBO), 10 mM dNTP mix (TOYOBO), 5×RT buffer (TOYOBO), and random primer (Invitrogen) were added for cDNA synthesis using a T3000 cycler (biometra). The reaction conditions included an annealing reaction for 10 minutes at 30°C, a reverse transcription reaction for 60 minutes at 42°C, and a thermal denaturation reaction for 5 minutes at 99°C.

[0125]

3) PCR method

1 µl synthesized cDNA, 5 µl 2×GO Taq GreenMaster Mix (Promega), 1 µl custom-synthesized Forward Primer (Invitrogen) to mTOR, 1 µl custom-synthesized Reverse Primer (Invitrogen) and 2 µl H ₂ O were mixed for amplification complementary DNA base sequences using the T3000 amplifier (biometra). The reaction conditions included conducting an initial thermal denaturation reaction for 2 minutes at 94°C, followed by 26 cycles of the thermal denaturation reaction for 30 seconds at 95°C, an annealing reaction for 20 seconds at 53°C, and an elongation reaction for 25 seconds at 72°C. Then, an elongation reaction was carried out for 5 minutes at 72°C. After amplification, UV detection was performed using an Amersham ^™ 600 Imager (GE Healthcare Japan) and agarose gel electrophoresis.

[0126]

Then, after visualization, Western blotting of the proteins was performed according to the following procedure.

[0127]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect the protein supernatant.

[0128]

2) Western blotting

5 μg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies used were rabbit anti-mTOR (Cell Signaling, 2972), rabbit anti-S6K (Cell Signaling, 9202), rabbit anti-phospho-S6K (CellSignaling, 9204), and mouse anti-GAPDH (MBL, M171-3 ). Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. Regarding primary antibodies, rabbit anti-mTOR: 1000-fold dilution, rabbit anti-S6K: 1000-fold dilution, rabbit anti-phospho-S6K: 1000-fold dilution, and mouse anti-GAPDH: 5000-fold dilution, while secondary the antibody was diluted 5000 times. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0129]

(Results)

(mTOR miRNA downregulates mTOR expression and S6K phosphorylation)

The results are shown in Figure 6 . When conducting anti-mTOR RNAi in iFECD cells, downregulation of mTOR expression was found at both the RNA and protein levels. In addition, S6K phosphorylation was found to be suppressed by mTOR siRNA at the protein level. Thus, mTOR expression and S6K phosphorylation were found to be downregulated by mTOR siRNA in PCR and Western blot assays.

[0130]

(Example 7: Inhibition effect of mTOR siRNA on cell damage caused by TGF-β2)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0131]

iFECD were seeded in a 12-well plate at a ratio of 3×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (medium: DMEM +10% FBS +1% P/S). After 24 hours, the medium was removed. Lipofectamine (Invitrogen, 92008) and 3 pmol mTOR siRNA (Ambion, AS026M0L) were added to culture the cells for 24 hours (medium: OptiMEM-I (Invitrogen, 31985-088)). The mTOR siRNA used had the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 2. After 24 hours, the medium was removed and the cells were cultured for 72 hours at 37°C (5% CO ₂ ) ( medium: DMEM +10% FBS +1% P/S). After 72 hours, the medium was removed. Media containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) was added to culture the cells for 24 hours (medium: DMEM+2% FBS+1% P/S). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0132]

(Results)

(mTOR siRNA inhibits TGF-β2-induced cell damage)

The results are shown in Figure 7 . Upon stimulation of iFECD with recombinant human TGF-β2 without the use of mTOR siRNA, the cells were found to be significantly damaged. On the other hand, it was noted that damage to corneal endothelial cells was suppressed by the use of mTOR siRNA. Therefore, downregulation of mTOR expression was found to suppress TGF-β2-induced cell damage.

[0133]

(Example 8: Inhibition effect of mTOR siRNA on caspase activation induced by TGF-β2)

This Example demonstrated the suppression effect of mTOR siRNA, which is another exemplary mTOR inhibitor, of TGF-β2-induced caspase activation.

[0134]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0135]

iFECD were seeded in a 12-well plate at a ratio of 3×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (medium: DMEM +10% FBS +1% P/S). After 24 hours, the medium was removed. Lipofectamine (invitrogen, 92008) and 3 pmol mTOR siRNA (Ambion, AS026M0L) were added to culture the cells for 24 hours (medium: OptiMEM-I (invitrogen, 31985-088)). The mTOR siRNA used had the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 2. After 24 hours, the medium was removed and the cells were cultured for 72 hours at 37°C (5% CO ₂ ) ( medium: DMEM +10% FBS +1% P/S). After 72 hours, the medium was removed. Media containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) was added to culture the cells for 24 hours (medium: DMEM+2% FBS+1% P/S). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0136]

Then, after microscopy, Western blotting of the proteins was performed according to the following procedure.

[0137]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect the protein supernatant.

[0138]

2) Western blotting

6 µg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Rabbit anti-caspase 3 (Cell Signaling, 9662), rabbit anti-PARP (Cell Signaling, 9542), and mouse anti-GAPDH (MBL, M171-3) were used as primary antibodies. Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. As for the primary antibodies, rabbit anti-caspase 3: 1000-fold dilution, rabbit anti-PARP: 2000-fold dilution, and mouse anti-GAPDH: 5000-fold dilution, while the secondary antibody was diluted 5000 times. Chemi Lumi ONE Ultra (Nacalai Tesque, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0139]

(Results)

(mTOR siRNA suppresses TGF-β2-induced caspase activation)

The results are shown in Figure 8 . Upon stimulation of iFECD cells with recombinant human TGF-β2 without the use of mTOR siRNA, approximately 17 kDa cleaved caspase 3, which is the active form, was observed in iFECD. On the other hand, the activated form of cleaved caspase 3 was almost not observed in the mTOR siRNA supplemented group. Thus, in Western blot analysis, it was demonstrated that caspase activation induced by TGF-β2 is suppressed under conditions of suppression of the mTOR signal.

[0140]

(Example 9: Inhibition effect of mTOR siRNA on fibronectin production induced by TGF-β2)

This Example demonstrated the effect of suppressing mTOR siRNA, which is another exemplary mTOR inhibitor, of TGF-β2-induced fibronectin production.

[0141]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0142]

iFECD were seeded in a 12-well plate at a ratio of 3×10 ⁴ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (medium: DMEM +10% FBS +1% P/S). After 24 hours, the medium was removed. Lipofectamine (invitrogen, 92008) and 3 pmol mTOR siRNA (Ambion, AS026M0L) were added to culture the cells for 24 hours (medium: OptiMEM-I (invitrogen, 31985-088)). The mTOR siRNA used had the sense strand as shown in SEQ ID NO: 1 and the antisense strand as shown in SEQ ID NO: 2. After 24 hours, the medium was removed and the cells were cultured for 48 hours at 37°C (5% CO ₂ ) ( medium: DMEM +10% FBS +1% P/S). After 48 hours, the medium was removed. Media containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) was added to culture the cells for 24 hours (medium: DMEM+2% FBS+1% P/S). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0143]

Then, after microscopy, Western blotting of the proteins was performed according to the following procedure.

[0144]

1) Collection of protein

The medium was collected on ice to collect free and dead cells. The solution was also collected after washing the cells twice with 1×PBS (-). Centrifuged at 800 g and 4°C for 12 minutes. The supernatant was taken to obtain precipitation. Protein isolation buffer (RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% DOC, 1% NP-40) was added to the washed cells for ice to isolate proteins. The centrifugation pellets of free and dead cells were also then suspended together for extraction. The collected solution was sonicated three times for 30 seconds in cold water with an ultrasonicator (BIORUPTOR, TOSHO DENKI) and centrifuged for 10 min at 4°C and 15,000 rpm to collect the protein supernatant.

[0145]

2) Western blotting

7 μg of the isolated protein was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Mouse anti-fibronectin antibody (BDBioscience, 610077) and mouse anti-GAPDH antibody (MBL, M171-3) were used as primary antibodies. Peroxidase-labeled anti-rabbit immunoglobulin and anti-mouse immunoglobulin (GE Healthcare Biosciences, NA931V, NA934V) were used as secondary antibodies. As for the primary antibodies, rabbit anti-caspase 3: 1000-fold dilution, rabbit anti-PARP: 2000-fold dilution, and mouse anti-GAPDH: 5000-fold dilution, while the secondary antibody was diluted 5000 times. Chemi Lumi ONE Ultra (Nicolai tissue, 11644-40) was used for detection. The intensity of the detected bands was determined using a Lumino LAS-4000 mini image analyzer (Fuji Film) and ImageQuant ^™ software (GE Healthcare).

[0146]

(Results)

(mTOR siRNA suppresses fibronectin production induced by Recombinant human TGF-β2),

The results are shown in Figure 9 . Upon stimulation of iFECD cells with recombinant human TGF-β2 without the use of mTOR siRNA, fibronectin production was observed in iFECD. On the other hand, almost no fibronectin production was observed in the mTOR siRNA supplemented group. Thus, using Western blot analysis, it was found that in the presence of mTOR siRNA, the expression of fibronectin was suppressed.

[0147]

(Example 10: Inhibition effect of TGF-β2 induced caspase activity of each mTOR inhibitor)

This Example demonstrated the effect of each type of mTOR inhibitor in inhibiting TGF-β2 induced caspase activity.

[0148]

(Materials and methods)

The medium was removed from the culture dish in which the iFECD was cultured, added to the cells with 1×PBS (-) which had been preheated to 37°C, and washed. This was repeated twice. The cells were again added 1×PBS (-) and incubated for 3 minutes at 37°C (5% CO ₂ ). After removal of PBS (-), 0.05% trypsin-EDTA (Nacalai Tesque, 32778-34) was added to the cells and incubated for 5 minutes at 37°C (5% CO ₂ ). The cells were then suspended in the medium and collected by centrifugation at 1500 rpm for 3 minutes. DMEM (Nacalai Tesque, 08456-36) + 10% FBS (Biowest, S1820-500) + 1% P/S (Nacalai Tesque, 26252-94) was used as medium.

[0149]

Corneal endothelial cells from immortalized FECD patients (batch: iFECD3-5) were seeded into a 96-well plate at 4×10 ³ cells per well and cultured for 24 hours at 37°C (5% CO ₂ ) (medium: DMEM + 10% FBS +1% P/S). After 24 hours, the medium was removed. Each mTOR inhibitor was added to cell culture for 24 hours (medium: DMEM +2% FBS +1% P/S). After 24 hours, the medium was removed. Media containing 10 ng/ml recombinant human TGF-β2 (Wako, 200-19911) and each mTOR inhibitor was added to culture the cells for 24 hours (medium: DMEM+2% FBS+1% P/S). After 24 hours, cell morphology and apoptosis were observed using a phase contrast microscope.

[0150]

After microscopy, caspase 3/7 activity was measured using the Caspase-Glo® 3/7 assay according to the following procedure.

[0151]

The medium was discarded to obtain 50 μl per well. 50 µl/well Caspase Glo® 3/7 assay reagent (mixture of Caspase-Glo® 3/7 assay buffer and Caspase-Glo® 3/7 assay substrate) (Promega, G8091) was added to give a 1:1 ratio of co environment. Subsequent operations were performed in the dark. The shaker was used to thoroughly mix the contents for two minutes at 120 rpm, then left for 40 minutes at room temperature. After incubation, 80 μl was transferred to an assay plate (Corning, 3912, assay plate, 96-well, white polystyrene) and absorbance was measured using the GloMax-Multi Detection System (Promega, E7051).

[0152]

In this Example, the following mTOR inhibitors were used.

*Rapamycin (Wako, #53123-88-9)

*Everolimus (Cayman Chemical, #11597)

*Temsirolimus (Tocris Bioscience, #5264)

*PI-103 (Cayman Chemical, #10009209)

*CC-223 (Cayman Chemical, #19917)

*INK128 (Cayman Chemical, #11811)

*AZD8055 (Cayman Chemical, #16978)

*KU 0063794 (Tocris Bioscience, #3725)

[0153]

(Results)

(Rapamycin)

The results are shown in Figure 10 . The Caspase-Glo® 3/7 assay measures the activity of caspase 3/7, which is involved in the induction of apoptosis. The higher the activity of caspase 3/7, the more damage it causes to cells. In Figure 10, no significant difference was found in caspase 3/7 activity compared to control when 0.00001, 0.0001, 0.001 and 0.01 nM rapamycin was added. On the other hand, it was found that the activity of caspase 3/7 was significantly suppressed compared with the control group by adding 0.1, 1, 10 and 100 nM of rapamycin. Even at a concentration of 0.1 nM, which is very low, rapamycin has been shown to significantly inhibit caspase 3/7 activity.

[0154]

(Everolimus)

The results are shown in Figure 11 . It was found that the activity of caspase 3/7 was significantly suppressed compared to the control when adding 0.0001, 0.001, 0.01 μm and 0.1 μm everolimus. Even at a concentration of 0.0001 μM, which is very low, everolimus has been shown to significantly inhibit caspase 3/7 activity.

[0155]

(Temsirolimus)

The results are shown in Figure 12 . No significant difference in caspase 3/7 activity compared to control was found with the addition of 0.000001 μM temsirolimus. On the other hand, it was found that caspase 3/7 activity was significantly suppressed compared to the control when 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10 μM temsirolimus were added. Even at a concentration of 0.00001 μM, which is very low, everolimus has been shown to significantly inhibit caspase 3/7 activity.

[0156]

(PI-103)

The results are shown in Figure 13 . No significant difference in caspase 3/7 activity compared to control was found when 1 μM PI-103 was added. On the other hand, it was found that the activity of caspase 3/7 was significantly suppressed compared to the control by adding 0.001, 0.01, and 0.1 μM PI-103.

[0157]

(CC-223)

The results are shown in Figure 14 . It was found that the activity of caspase 3/7 was significantly suppressed compared to the control when adding 0.001, 0.01, 0.1 and 1 μm CC-223.

[0158]

(INK128)

The results are shown in Figure 15 . It was found that the activity of caspase 3/7 was significantly suppressed compared to the control when adding 0.001, 0.01, 0.1 and 1 μm INK128.

[0159]

(AZD8055)

The results are shown in Figure 16 . It was found that the activity of caspase 3/7 was significantly suppressed compared to the control when adding 0.01, 0.1 and 1 μm AZD8055.

[0160]

(KU 0063794)

The results are shown in Figure 17 . No significant difference in caspase 3/7 activity compared to control was found with the addition of 0.001 and 0.01 μM KU 0063794. On the other hand, it was found that caspase 3/7 activity was significantly suppressed compared with control with the addition of 0.1 and 1 μM KU 0063794.

[0161]

(Example 11: In vivo assessment in a mouse model)

This Example demonstrated the in vivo effect of an mTOR inhibitor in a scoring system using the Fuchs mouse model of corneal endothelial dystrophy.

Specifically, in this Example, an mTOR inhibitor to a collagen type 8 mouse (Col8a2 Q455K/Q455K), which is a Fuchs endothelial corneal dystrophy mouse model, was dripped to confirm the effect in vivo .

[0162]

(Materials and methods)

An in vivo evaluation was performed using an alpha-2 collagen VIII (Col8a2) Q455K knockout mouse (Hum Mol Genet. 2012 Jan 15; 21 (2): 384-93.), which is a model of Fuchs endothelial corneal dystrophy. Deposition of extracellular matrix (collagen, fibronectin, etc.) on the basement membrane of the corneal endothelium (Descemet's membrane), called gutta, and a decrease in cell density as a result of damage to the corneal endothelium were found in this mouse model similar to the manifestations of Fuchs endothelial corneal dystrophy in humans . Thus, this mouse model is considered to be a good model of Fuchs endothelial corneal dystrophy.

[0163]

It is possible to treat or prevent the occurrence of Fuchs endothelial corneal dystrophy or delay the progression of the pathology by inhibiting the corneal endothelial mTOR pathway by anterior chamber drip, intravitreal injection, subconjunctival administration, or systemic administration of an mTOR inhibitor, or gene therapy in such a mouse. Thus, in this example, such a mouse was used to confirm the effect.

[0164]

(Receiving mTOR inhibitor eye drops)

Torizel® 25 mg solution for intravenous infusion (Pfizer Inc.) (temsirolimus) was used as an mTOR inhibitor eye drop. This product and the solvent supplied with this product were mixed in a ratio of 2:3 to obtain 92.78 µl of a 9.7 mm mixture. Then, the 9.7 mM mixture was diluted with 807.22 μl of OTSUKA saline solution (Otsuka Pharmaceutical Co., Ltd.) to obtain 900 μl of the 1 mM mixture in a 1.5 ml tube. Then, 900 μl of a 10 μM mixture was prepared using 9 μl of the prepared 1 mM mixture and 891 μl of OTSUKA saline. After preparation of 1.5 ml, the tube was covered with aluminum foil to create darkness and then stored in a refrigerator at 4°C.

[0165]

(Mouse instillation test)

A mouse having collagen type 8 (Col8a2Q455K/Q455K), which is a mouse model of Fuchs endothelial corneal dystrophy (FECD) (obtained from Johns Hopkins University), was used. The severity of FECD was classified by pre-instillation corneal endothelial imaging using mice in the FECD model that were 20-24 weeks of age with the same degree of condition. 2 µl of a prepared mTOR inhibitor eye drop (1 mM, 10 µM) was dripped into each left and right eye of 45 mice, twice a day, morning and evening. OTSUKA saline was used as a control. The instillation period was 3 months, during which the person in charge of the experiments conducted a study under masking conditions against an mTOR inhibitor eye drop and a control eye drop (OTSUKA saline).

[00166]

(Evaluation of the effectiveness of an eye drop)

Before starting the instillation test, images of the corneal endothelium were visually examined using contact reflection of the corneal endothelium (wide-field slit-scan contact reflex microscope KSSP (Konan medical Inc., Hyogo, Japan)) to assess the grade. After the start of the corneal endothelial imaging instillation test, the mice were observed once every 4 weeks with corneal endothelial contact imaging to evaluate the effectiveness of the mTOR inhibitor eye drop.

[0167]

(Results)

Figure 19 shows a typical example of corneal endothelial cell imaging observed by corneal endothelial contact imaging in a mouse FECD model instilled with 2 µl of an mTOR inhibitor eye drop (1 mM) into each left and right eye twice a day, in the morning and in the evening for 2 months. An image of corneal endothelial cells in a mouse FECD model dripped with saline is shown as a control. Compared to controls, the size of corneal endothelial cells is smaller and the cell density is higher in an individual who received an eye drop of an mTOR inhibitor. In addition, the formation of outgrowths of the extracellular matrix, called gutta, which can be observed on the image as black spots using contact reflection of the corneal endothelium, was suppressed (Figure 19 ).

[0168]

The control corneal endothelial cell density averaged 1558 cells/ ^mm2 , while the mTOR inhibitor eye drop group averaged 1957 cells/ ^mm2 , which was significantly higher, thus reflecting the suppression of the decrease in corneal endothelial cell density found in a mouse FECD model (Figure 20 ). This indicates that the occurrence of corneal stromal edema, corneal epithelial edema, and the like, which is frequently encountered if the density of corneal endothelial cells is usually about 1000 cells/mm ² or less, can be prevented by suppressing the decrease in the number of corneal endothelial cells. by drip administration of an mTOR inhibitor to a patient with FECD.

[0169]

Furthermore, regarding the gutta range, the control had a mean of 3.02%, while the mTOR inhibitor instillation group had a mean of 0.58%, which was significantly lower, thus reflecting the inhibition of gutta formation found in mice in FECD models (Figure 21 ). When the same analysis was performed on 15 mice, statistically significant differences were found with respect to gutt range. Guttas are known to cause a decrease in visual function due to uneven light reflection and the like, even in an early stage FECD patient who does not have corneal stroma or corneal epithelium edema. Thus, this result indicates that the decrease in visual function due to irregular light reflection can be suppressed by suppressing the formation of gutta by instillation of an mTOR inhibitor in a patient with FECD.

[0170]

(Example 12: Diagnosis and treatment example)

The present invention is useful in diagnosing Fuchs endothelial corneal dystrophy or similar corneal endothelial disease (specific examples include: 1) observation of gutt formation, Descemet's membrane hypertrophy, corneal epithelial edema, or corneal stromal edema by slit lamp microscopy, 2) observation of images of gutta or endothelial corneal abnormalities using a reflective microscope, 3) observing corneal edema using Pentacam, OCT, ultrasonic corneal thickness gauge, etc., and 4) when determining high risk using genetic diagnosis). The present invention can be used in eye drop therapy, anterior chamber injection, controlled release administration, intravitreal injection, subconjunctival injection, and the like.

[0171]

A commercially available substance that complies with the requirements of the Japanese Pharmacopoeia, its equivalent product, etc., can be used as any other ingredient other than the active substance.

[0172]

(Example 13: Eye Drop Manufacturing Example)

As an example of the preparation of the composition, this Example describes the production of an eye drop containing an mTOR inhibitor as follows.

[0173]

The composition of the test substances at each concentration is shown below.

[0174]

Rapamycin Effective Amount (e.g. final concentration 0.1 mM) Sodium chloride Sodium dihydrogen phosphate dehydrate (Optional) Benzalkonium chloride sodium hydroxide Optimal dose Purified water Optimal dose Total 100 ml (pH 7.0)

The concentration can be diluted using a base consisting of the following components.

[0175]

Sodium chloride Sodium dihydrogen phosphate dehydrate (Optional) Benzalkonium chloride sodium hydroxide Optimal dose Purified water Optimal dose Total 100 ml (pH 7.0)

[0176]

(Example 14: Mouse instillation test)

Instillation of the mTOR inhibitor was done in a mouse having collagen type 8 (Col8a2 Q455K/Q455K), which is a mouse in the Fuchs endothelial corneal dystrophy model in this Example. This mouse used in the model demonstrates the deposition of extracellular matrix (collagen, fibronectin, etc.) characteristic of Fuchs endothelial corneal dystrophy.

(Materials and methods)

[0177]

As disclosed above, the present invention is illustrated using its preferred embodiments. However, it is to be understood that the scope of the present invention is to be interpreted solely on the basis of the Claims. It is also intended that any patent, any patent application, and any sources cited within the scope of the invention should be incorporated by reference in the same manner as the content specifically described within the scope of the invention. The present application claims priority from Japanese Patent Application 2017-118619 (filed June 16, 2017). All of its contents are incorporated herein by reference.

[Industrial applicability]

[0178]

The present invention relates to a medicament for use in the treatment or prevention of endothelial corneal disorder mediated by transforming growth factor β (TGF-β) and/or extracellular matrix overexpression in corneal endothelial cells, in particular a medicament for use in the treatment or prevention of endothelial corneal disorder with Fuchs endothelial corneal dystrophy. The present invention provides a technology available to industries (pharmaceutical or the like) using technologies related to the production of pharmaceutical compositions and the like based on such technology.

[List of sequences in plain text form]

[0179]

SEQ ID NO: 1: mTOR siRNA sense strand

SEQ ID NO: 2: mTOR siRNA antisense strand

--->

SEQUENCE LIST

<110> The Doshisha

<120> MEDICINE CONTAINING mTOR INHIBITOR FOR THE TREATMENT

OR PREVENTION OF EYE SYMPTOMS, DISTURBANCES OR DISEASES

AND ITS APPLICATION

<130> DU004

<150> JP 2017-118619

<151> 2017-06-16

<160> 2

<170>PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Sense strand miRNA

<400> 1

cauucgcauu caguccauat t 21

<210> 2

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Antisense miRNA strand

<400> 2

uauggacuga augcgaaugat 21

<---

Claims (4)

1. Use of a composition comprising rapamycin as the sole active ingredient in the prevention or treatment of Fuchs endothelial corneal dystrophy. 2. Use according to claim 1, wherein the composition is an eye drop. 3. Use according to claim 1 or 2 wherein rapamycin is present in the composition at a concentration of at least about 0.1 nM. 4. Use of a composition comprising rapamycin for the conservation and/or growth or maintenance of growth of corneal endothelial cells.

Images