Novel treatment of cornea using laminin

Abstract

A technique for treating cornea is provided. More specifically, the present invention is an agent for treating or preventing a state of corneal endothelial disease, said agent comprising at least one factor selected from the group consisting of laminin and fragments thereof, whereby said problem is solved by also providing a technique characterized in that the agent is administered together with corneal endothelial cells. In particular, the present invention may comprise laminin 511 ([alpha] 5 [beta] 1 [gamma] 1), laminin 521 ([alpha] 5 [beta] 2 [gamma] 1), or fragments of these.

Description

[0001] This application is a divisional application. The original application was filed on October 30, 2015, with application number 201580059642.1 (international application number PCT / JP2015 / 005473), entitled "A novel treatment of the cornea using laminin". Technical Field

[0002] This invention relates to novel therapies using laminin. More specifically, this invention relates to ophthalmic treatments using laminin, and still more specifically to the treatment and prevention of corneal endothelial growth. Background Technology

[0003] At birth, the corneal endothelial cells number approximately 3000 cells / mm². 2 The density of corneal endothelial cells exists. Once damaged, human corneal endothelial cells do not have the ability to regenerate. Therefore, corneal endothelial cells are considered difficult to culture. Due to the difficulty in culturing and proliferating them in current transplantation techniques, treatment and surgery for corneal endothelial cells are practically impossible. Japan faces a shortage of corneal donations, with approximately 2,600 patients waiting for corneal transplants each year, while only about 1,700 corneal transplants are performed in Japan.

[0004] Patent documents 1 and 2 are known regarding the relationship between laminin and ophthalmology.

[0005] Reference List Patent documents Patent Document 1: Japanese National Phase PCT Publication 2004-500012 Patent Document 2: Japanese National Phase PCT Publication 2003-532647 Summary of the Invention

[0006] Solution to the problem The inventors have discovered that certain laminin proteins can be used in ophthalmic treatments, particularly corneal endothelial therapy, and this invention is based on this discovery. Therefore, this invention representatively provides the following: (1) A treatment or preventive agent for diseases, disorders or conditions of the corneal endothelium, said agent comprising at least one agent selected from laminin and fragments thereof.

[0007] (2) The therapeutic or preventive agent of item 1, wherein the laminin contains the RGD sequence.

[0008] (3) The therapeutic or preventive agent described in item 1 or 2, wherein the laminin comprises an α5 chain and / or a γ1 chain.

[0009] (4) The therapeutic or preventive agent of any one of items 1-3, wherein the laminin comprises laminin 511 (α5β1γ1) and laminin 521 (α5β2γ1).

[0010] (5) The therapeutic or preventive agent of any one of items 1-4, wherein the fragment has the ability to adhere to corneal endothelial cells.

[0011] (6) The therapeutic or preventive agent of any one of items 1-5, wherein the agent is laminin 511, laminin 521 or laminin 511-E8 fragment.

[0012] (7) Any one of items 1-6, wherein the corneal endothelium is derived from primates.

[0013] (8) The treatment or preventive agent of any one of items 1-7, wherein the disease, disorder or condition of the corneal endothelium is selected from Fuchs' corneal endothelial dystrophy, corneal endotheliitis, trauma and disorder and condition in ophthalmic surgery.

[0014] (9) Any one of the treatments or preventive agents of the corneal endothelium, wherein the disease, disorder or condition of the corneal endothelium is selected from photophobia, blurred vision, visual impairment, eye pain, epiphora, congestion, pain, bullous keratopathy, eye discomfort, diminished contrast, glare, corneal stromal edema, bullous keratopathy and corneal opacity.

[0015] (10) The therapeutic or preventive agent of any one of items 1-9, wherein the corneal endothelium comprises the corneal endothelial layer, the posterior elastic membrane, or both.

[0016] (11) The therapeutic or preventive agent of any one of items 1-10, wherein the corneal endothelium has a detached posterior elastic membrane.

[0017] (12) Any of the therapeutic or preventive agents described in any of items 1-11 further comprises corneal endothelial cells.

[0018] (13) Any of the therapeutic or preventive agents described in any one of items 1-11 further comprises a ROCK inhibitor.

[0019] (14) Any of the therapeutic or preventive agents described in any of items 1-11 further comprises corneal endothelial cells and ROCK inhibitors.

[0020] (15) The therapeutic or preventive agent described in item 13 or 14, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

[0021] (16) The therapeutic or preventive agent of any one of items 1-15, wherein the agent is injected into the eye to come into contact with the tissues within the eye.

[0022] (17) The therapeutic or preventive agent of any one of items 1-16, wherein the agent is present at about 21 nM or more.

[0023] (18) The treatment or preventive agent of any one of items 1-17, wherein corneal endothelial cells are also administered.

[0024] (19) The therapeutic or preventive agent of any one of items 1-18, wherein the agent is provided in a mixture with corneal endothelial cells, and at least one agent selected from laminin and fragments thereof is injected into the eye to come into contact with the tissues within the eye.

[0025] (20) Any of the therapeutic or preventive agents described in any one of items 1-19 further comprises a ROCK inhibitor.

[0026] (21) Any one of the therapeutic or preventive agents described in items 1-20, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

[0027] (22) The therapeutic or preventive agent of any one of items 1-21, wherein the agent mixed with corneal endothelial cells is about 2.1 nM or more, and the agent to be injected is about 21 nM or more.

[0028] (23) At least one reagent selected from laminin and its fragments, for the treatment or prevention of diseases, disorders or conditions of the corneal endothelium.

[0029] (24) The reagent described in item 23 further includes one or more of the features described in items 2-22.

[0030] (25) A method for treating or preventing a disease, disorder or condition of the corneal endothelium, the method comprising administering an effective amount of at least one reagent selected from laminin and fragments thereof to a subject in need of the treatment or prevention.

[0031] (26) The method described in item 25 further includes one or more of the features described in items 2-11.

[0032] (27) The method of item 26 or 26 further includes administering corneal endothelial cells to the subject.

[0033] (28) The method of any one of items 25-27, further comprising administering a ROCK inhibitor to the subject.

[0034] (29) The method described in item 28, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

[0035] The method of any one of items 25-29 (30) further includes administering corneal endothelial cells and a ROCK inhibitor to the subject.

[0036] (31) The method of any one of items 25-30, wherein the reagent is injected into the eye of the subject so as to come into contact with the tissues inside the eye.

[0037] (32) The method of any one of items 25-31, wherein the reagent is present at about 21 nM or more.

[0038] The method of any one of items 25-32 (33) further includes administering corneal endothelial cells and the reagent separately.

[0039] (34) The method of any one of items 25-33, wherein the reagent is provided in combination with corneal endothelial cells, and at least one reagent selected from laminin and fragments thereof is injected into the eye to come into contact with the tissues within the eye.

[0040] The method of any one of items 25-34 (35) further includes administering the ROCK inhibitor and the reagent separately.

[0041] (36) The method of any one of items 25-35, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

[0042] (37) The method of any one of items 25-32, wherein the reagent mixed with the corneal endothelial cells is about 2.1 nM or more, and the reagent to be injected is about 21 nM or more.

[0043] (38) The use of at least one reagent selected from laminin and fragments thereof for the preparation of a medicament for the treatment or prevention of diseases, disorders or conditions of the corneal endothelium.

[0044] (39) The use described in item 38 also includes one or more of the features described in items 2-22.

[0045] (40) The use of at least one agent selected from laminin and fragments thereof for the treatment or prevention of diseases, disorders or conditions of the corneal endothelium.

[0046] (41) The use described in item 40 also includes one or more of the features described in items 2-22.

[0047] It should be understood that one or more of the above features may be provided in combinations other than those explicitly shown in this invention. Other embodiments and advantages of the invention will be recognized by those skilled in the art upon reading and understanding the following detailed description as needed.

[0048] Beneficial effects of the invention This invention enables novel ophthalmic treatments, particularly those targeting new corneal endothelial cells (especially human corneal endothelial cells). Specifically, this invention enables near-complete recovery from bullous keratopathy. In a preferred embodiment, the posterior elastic membrane is cured. Such an effect is remarkable and cannot be achieved using conventional techniques. Attached Figure Description

[0049] [ Figure 1 ] Figure 1 Images of the anterior segment of corneal endothelial cells cultured in a rabbit bullous keratopathy model using the laminin 511-E8 fragment are shown. From left to right: Control: anterior segment of rabbit corneal endothelial cells mechanically scraped off as a control; RCEC: anterior segment of the model in which cultured rabbit corneal endothelial cells were injected into the anterior chamber and held face down for 3 hours; RCEC+E8: anterior segment of the model in which cultured rabbit corneal endothelial cells were injected into the anterior chamber together with DMEM containing the laminin 511-E8 fragment (concentration adjusted to 2.1 nM) and held face down for 3 hours. The top row shows images after 1 week, and the bottom row shows images after 2 weeks.

[0050] [ Figure 2 ] Figure 2 The changes in corneal thickness following transplantation of cultured corneas in a rabbit bullous keratopathy model using the laminin 511-E8 fragment are shown. The vertical axis represents corneal thickness (μm) measured by an ultrasonic pachymeter. The horizontal axis represents the number of days after treatment. Standard error is expressed in bars.

[0051] [ Figure 3 ] Figure 3Histological examination results of corneal endothelial transplantation using a culture of laminin 511-E8 fragment are shown. Figure 3 The anti-Na diagram is shown from left to right. + / K + -Staining with antibodies against ATPase, anti-ZO-1, anti-N-cadherin, and phalloidin.

[0052] [ Figure 4 ] Figure 4 The results of examining cultured corneal endothelial transplantation in a rabbit bullous keratopathy model using both laminin and a ROCK inhibitor are shown, illustrating the effects of the co-administered laminin and ROCK inhibitor on early cell adhesion in cultured corneal endothelium. The bullous keratopathy model was established by mechanical detachment of the rabbit corneal endothelium. Cell adhesion to the stroma was compared 24 hours later in the following individuals: those in which cultured rabbit corneal endothelial cells were injected into their anterior chamber along with the ROCK inhibitor Y-27632(+) (100 μM), and those in which cells were injected along with a laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM). Images of phalloidin and DAPI staining are shown on the left. The top row shows results with Y-27632(+) (100 μM) but without the laminin 511-E8 fragment, while the bottom row shows results with both the laminin 511-E8 fragment and Y-27632(+) (100 μM). Phalloidin staining showed that more cells adhered in individuals injected with both the laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM). The right side shows a plot of cell density data. The vertical axis represents cell density (cells / mm²). 2 In individuals injected with cell-laminin 511-E8 fragment (2.1 nM) and Y-27632 (+) (100 μM), the density of adherent cells was significantly higher.

[0053] [ Figure 5 ] Figure 5Images of the anterior segment of cultured corneal endothelial transplanted in a rabbit bullous keratopathy model are shown. The images also show cultured corneal endothelial transplanted in a rabbit bullous keratopathy model using laminin and ROCK inhibitors. Images of the anterior segment are shown, from left to right: an individual with detached corneal endothelial cells but not detached posterior elastic membrane, injected with cultured corneal endothelial cells and Y-27632(+) (100 μM); an individual with detached corneal endothelial cells but not detached posterior elastic membrane, injected with cells and laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM); an individual with detached posterior elastic membrane and injected with cells and Y-27632(+) (100 μM) in a bullous keratopathy model; and an individual with detached posterior elastic membrane and injected with cells and laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM) in a bullous keratopathy model. The top row represents the results on day 3, and the bottom row represents the results on day 7.

[0054] [ Figure 6 ] Figure 6 The cultured corneal endothelium was shown after transplantation. Figure 5 The corneal thickness (μm) shown in the four groups illustrates the changes in corneal thickness following cultured corneal endothelial transplantation in a rabbit bullous keratopathy model using a combination of laminin and ROCK inhibitors. The horizontal axis represents the number of days after treatment. Solid lines indicate no posterior elastic membrane detachment, and dashed lines indicate posterior elastic membrane detachment. Each solid circle represents the presence of laminin 511-E8 fragments, and hollow circles represent the absence of laminin 511-E8 fragments. Compared to the case without detachment, corneal thinning was delayed more significantly with posterior elastic membrane detachment.

[0055] [ Figure 7 ] Figure 7 The cultured corneal endothelium was shown after transplantation. Figure 5 The intraocular pressure (mmHg) in the four groups shown illustrates the changes in intraocular pressure following cultured corneal endothelial transplantation in a rabbit bullous keratopathy model using a combination of laminin and ROCK inhibitors. The horizontal axis represents the number of days after treatment. Solid lines indicate the absence of posterior elastic membrane detachment, and dashed lines indicate the presence of posterior elastic membrane detachment. Each solid circle represents the presence of laminin 511-E8 fragments, and hollow circles represent the absence of laminin 511-E8 fragments. No intraocular pressure elevation was observed in any group as a complication attributed to cell transplantation.

[0056] [ Figure 8 ] Figure 8 The image shows cultured corneal endothelium transplanted 14 days later. Figure 6The four groups of histological examinations shown illustrate the histological examination of a rabbit bullous keratopathy model using a combination of laminin and ROCK inhibitors. Figure 8 The anti-Na diagram is shown from left to right. + / K + -Staining with antibodies against ATPase, anti-ZO-1, anti-N-cadherin, and phalloidin. Figure 8 Images of the following individuals are shown from top to bottom: individuals with detached corneal endothelial cells but not detached elastic membrane, and injected with cultured corneal endothelial cells and Y-27632(+) (100 μM); individuals with detached corneal endothelial cells but not detached elastic membrane, and injected with cells and laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM); individuals with detached posterior elastic membrane and injected with cells and Y-27632(+) (100 μM) in a bullous keratopathy model; and individuals with detached posterior elastic membrane and injected with cells and laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM) in a bullous keratopathy model.

[0057] [ Figure 9 ] Figure 9 Images of the anterior segment of corneal endothelial cells cultured in a monkey bullous keratopathy model treated with a laminin 511-E8 fragment are shown. In a cynomolgus monkey model where corneal endothelial cells were mechanically dissected, cultured cynomolgus monkey corneal endothelial cells were injected into the anterior chamber and the monkey was kept face down for 3 hours. The top left image shows the result on day 1, the top right image shows the result on day 3, the bottom left image shows the result on day 7, and the bottom right image shows the result on day 14.

[0058] [ Figure 10 ] Figure 10 Images of the anterior segment of cultured corneal endothelial cells after detachment of the posterior elastic membrane and simultaneous transplantation of a laminin 511-E8 fragment are shown in a monkey bullous keratopathy model. In a cynomolgus monkey model with mechanically dissected corneal endothelial cells, cultured cynomolgus monkey corneal endothelial cells were injected into the anterior chamber after detachment of the posterior elastic membrane and the monkey was held face down for 3 hours. The top left image shows the results on day 1, the top right image on day 3, the bottom left image on day 7, and the bottom right image on day 14.

[0059] [ Figure 11 ] Figure 11The figures show corneal thickness after endothelial transplantation cultured in a monkey bullous keratopathy model using the laminin 511-E8 fragment, and corneal thickness after in vivo-coated cultured endothelial transplantation co-treated with E8 in a monkey bullous keratopathy model with detached posterior elastic membrane. Corneal thickness (μm) is shown for individuals with and without posterior elastic membrane detachment. The horizontal axis represents the number of days after treatment, and the vertical axis represents corneal thickness (μm). Solid lines represent instances without posterior elastic membrane detachment, and dashed lines represent individuals with posterior elastic membrane detachment. Solid circles and triangles indicate individual differences. No thinning of corneal thickness was observed in either of the two instances with posterior elastic membrane detachment.

[0060] [ Figure 12 ] Figure 12 Images of the anterior segment of the cornea are shown in a monkey bullous keratopathy model. After detachment of the posterior elastic membrane, a laminin 511-E8 fragment was injected into the anterior chamber at a concentration of 21 nM. The model was then left to stand for 1 hour to coat the corneal stroma exposed in vivo due to posterior elastic membrane detachment. Images of the anterior segment following transplantation of cultured corneal endothelial cells with the laminin 511-E8 fragment are also shown. The top left image shows the result on day 1, the top right image on day 3, the bottom left image on day 7, and the bottom right image on day 14.

[0061] [ Figure 13 ] Figure 13 The effect of integrin on corneal endothelial cell adhesion is illustrated. A laminin 511-E8 fragment was added to achieve a final concentration of 2.1 nM, and corneal endothelial cells were inoculated. The number of adherent cells 24 hours after inoculation with (from left to right) mouse IgG, antibody against integrin α3, antibody against integrin α6, antibody against integrin α2, antibody against integrin β1, antibody against integrin α3β1, and antibody against integrin α6β1 (shown as a percentage relative to mouse IgG) is shown. The right side shows a control group in which only mouse IgG was added without the addition of the laminin 511-E8 fragment.

[0062] [ Figure 14 ] Figure 14The activation of cell adhesion-related proteins is shown to be mediated by integrin. A group without the laminin 511-E8 fragment was prepared as a control on the left. From the second column onwards, groups with the laminin 511-E8 fragment added to achieve a final concentration of 2.1 nM were prepared. From the second column onwards, the results of Western blots are shown for the following cases, in order of inoculation with mouse IgG, anti-integrin α3 antibody, anti-integrin α6 antibody, anti-integrin α2 antibody, anti-integrin β1 antibody, anti-integrin α3β1 antibody, and anti-integrin α6β1 antibody. p-FAK, FAK, p-pile protein, and background GAPDH are shown from the top. The numerical value of each band represents the relative value of the band intensity quantified when the absence of laminin 511-E8 on the left is assumed to be 1. Detailed Implementation

[0063] The present invention is described below. Unless otherwise expressly stated, throughout this specification, singular expressions should be understood as including the plural form of the described concept. Therefore, unless otherwise expressly stated, articles in the singular form (e.g., "a," "an," "the," etc. in English) should be understood as including the plural form of the described concept. Furthermore, unless otherwise expressly stated, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all specialized terms and scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0064] (definition) As used herein, "corneal endothelial cells" refers to the meaning commonly used in the art. The cornea is one of the thin layers of tissue that make up the eye. The cornea is transparent and located in the part closest to the external environment. In humans, it should be understood that the cornea consists of five layers, starting from the outermost (body surface): corneal epithelium, anterior elastic lamina, lamina propria, posterior elastic lamina (corneal endothelial basement membrane), and corneal endothelium. Unless otherwise specifically stated, the portion other than the epithelium and endothelium may be collectively referred to as the "corneal stroma" and is so named herein. As used herein, "HCEC" is an abbreviation for human corneal endothelial cells. Rabbit corneal endothelial cells are abbreviated as "RCEC," and monkey corneal endothelial cells are abbreviated as "MCEC." It should be understood that naturally occurring cells and cells differentiated from stem cells (e.g., differentiated cells induced by iPS, etc.) can be used as corneal endothelial cells in this invention.

[0065] As used herein, "isolated" refers to a state in which the substances naturally associated with an entity in a normal environment are at least reduced, preferably a state in which said entity is substantially devoid of such substances. Thus, isolated cells, tissues, etc., refer to cells, tissues, etc., that are substantially devoid of other substances (e.g., other cells, proteins, or nucleic acids, etc.) that are naturally associated with them in the environment.

[0066] Laminin The term "lamin" as used in this article refers to the constituent proteins of the basement membrane of the extracellular matrix. Laminins promote multicellularity / tissue construction and maintenance, cell adhesion, cell migration, and cell growth, and are closely related to cancer cells. Laminins are thought to be expressed early in blastogenesis (2-cell stage). Laminins are heterotrimers composed of one α chain, one β chain, and one γ chain. For the nomenclature of laminins, a known nomenclature is based on the order of discovery (laminin-1, laminin-2, etc.). However, this does not consider the correspondence with subunits; therefore, this article uses a newer nomenclature in which the name of the subclass α, β, or γ is commonly described (a three-digit number, with the hundreds digit for α, the tens digit for β, and the units digit for γ). In the case of α1, β1, and γ1, such a laminin is referred to as laminin 111. Five types of α chains, three types of β chains, and three types of γ chains have been identified for laminins. Therefore, the theoretical maximum number of combinations is 5 × 3 × 3 = 45, making it possible for 45 different laminin molecules to exist. However, it is recognized that not all combinations are naturally occurring. For example, each subunit of the α chain is referred to as LAMA1, LAMA2, LAMA3, LAMA4, or LAMA5; each subunit of the β chain is referred to as LAMB1, LAMB2, or LAMB3; and each subunit of the γ chain is referred to as LAMC1, LAMC2, or LAMC3. The laminin used in this invention can be a naturally occurring laminin or a modified form of protein having one or more amino acid residues modified to maintain its biological activity (particularly its activity of promoting cell adhesion). Furthermore, the source of the laminin of this invention, its production method, etc., is not limited, as long as the laminin has the characteristics described herein. Therefore, the laminin used in this invention can be any naturally occurring protein, a protein expressed from recombinant DNA by genetic engineering methods, or a chemically synthesized protein. The source of the laminin used in this invention is not particularly limited, but is preferably derived from humans. When culturing human cells for the purpose of obtaining medical materials, laminin derived from humans is preferred, but not limited to, to avoid the use of materials derived from other animals.

[0067] The binding molecules of laminin are known. α1β1, α2β1, α2β2, α3β1, α6β1, α6β4, α7β1, α9β1, ανβ3, ανβ5, and ανβ8 are integrins known as laminin receptors.

[0068] The table below describes representative laminin proteins and provides an explanation of them.

[0069] [Table 1] The “α1 chain” (LAMA1) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, and is also known as LAMA1, LAMA, S-LAM-α, etc. For human LAMA1, the gene and protein sequences are registered under NCBI accession numbers NM_005559 and NP_005550, respectively. OMIM was identified using accession number 150320. When used for the purposes of this article, it should be understood that “α1 chain” or “LAMA1” means not only a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0070] The “α2 chain” (LAMA2) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMA2, LAMM, etc. For human LAMA2, the gene and protein sequences are registered under NCBI accession numbers NM_000426 and NP_000417, respectively. OMIM was identified using accession number 156225. When used for the purposes of this article, it should be understood that “α2 chain” or “LAMA2” refers not only to a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0071] The “α3 chain” (LAMA3) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, and is also known as LAMA3, BM600, E170, LAMNA, LOCS, lama3a, etc. For human LAMA3, the gene and protein sequences are registered under NCBI accession numbers NM_000227 and NP_000218, respectively. OMIM was identified using accession number 600805. When used for the purposes of this article, it should be understood that “α3 chain” or “LAMA3” refers not only to a protein (or the nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0072] The “α4 chain” (LAMA4) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, and is also known as LAMA4, LAMA3, LAMA4*-1, CMD1JJ, etc. For human LAMA4, the gene and protein sequences are registered under NCBI accession numbers NM_001105206 and NP_001098676, respectively. OMIM was identified using accession number 600133. When used for the purposes of this article, it should be understood that “α4 chain” or “LAMA4” means not only a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0073] The “α5 chain” (LAMA5) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMA5, KIAA1907, etc. For human LAMA5, the gene and protein sequences are registered under NCBI accession numbers NM_005560 and NP_005551, respectively. OMIM was identified using accession number 601033. When used for the purposes of this article, it should be understood that “α5 chain” or “LAMA5” means not only a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0074] The “β1 chain” (LAMB1) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMB1, CLM, LIS5, etc. For human LAMB1, the gene and protein sequences are registered under NCBI accession numbers NM_002291 and NP_002282, respectively. OMIM was identified using accession number 150240. When used for the purposes of this article, it should be understood that “β1 chain” or “LAMB1” refers not only to a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0075] The “β2 chain” (LAMB2) (lamin S) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMB2, LAMS, NPHS5, etc. For human LAMB2, the gene and protein sequences are registered under NCBI accession numbers NM_002292 and NP_002283, respectively. OMIM was identified using accession number 150325. When used for the purposes of this article, it should be understood that “β2 chain” or “LAMB2” refers not only to a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0076] The “β3 chain” (LAMB3) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMB3, BM600-125KDA, LAM5, LAMNB1, etc. For human LAMB3, the gene and protein sequences are registered under NCBI accession numbers NM_000228 and NP_000219, respectively. OMIM was identified using accession number 150310. When used for the purposes of this article, it should be understood that “β3 chain” or “LAMB3” refers not only to a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0077] The “γ1 chain” (LAMC1) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMC1, LAMB2, etc. For human LAMC1, the gene and protein sequences are registered under NCBI accession numbers NM_002293 and NP_002284, respectively. OMIM was identified using accession number 150290. When used for the purposes of this article, it should be understood that “γ1 chain” or “LAMC1” refers not only to a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0078] The “γ2 chain” (LAMC2) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, and is also known as LAMC2, B2T, BM600, CSF, EBR2, EBR2A, LAMB2T, LAMNB2, etc. For human LAMC2, the gene and protein sequences are registered under NCBI accession numbers NM_005562 and NP_005553, respectively. OMIM was identified using accession number 150292. When used for the purposes of this article, it should be understood that “γ2 chain” or “LAMC2” refers not only to a protein (or the nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0079] The “γ3 chain” (LAMC3) used in this article refers to the subunit of laminin, a cell adhesion molecule in the extracellular matrix, also known as LAMC3, OCCM, etc. For human LAMC3, the gene and protein sequences are registered under NCBI accession numbers NM_006059 and NP_006050, respectively. OMIM was identified using accession number 604349. When used for the purposes of this article, it should be understood that “γ3 chain” or “LAMC3” refers not only to a protein (or nucleic acid encoding the protein) having the amino acid sequence described by the specific sequence number or accession number, but also to its functionally active derivatives, functionally active fragments or homologs, or mutants encoded by nucleic acids that hybridize with the nucleic acid encoding the protein under high or low stringency conditions.

[0080] The term "laminar adhesion proteins expressed in corneal endothelial cells" as used in this article refers to laminar adhesion protein types that are expressed in corneal endothelial cells in a normal state or, preferably, significantly expressed at the protein level. The analysis in this article confirmed the expression of α5, β1, β2, and γ1 (as described in WO 2015 / 080297). Figure 2Therefore, at least laminin 511 and laminin 521 were confirmed to be expressed. Laminin 511 is described in detail in Dev. Dyn. 218, 213-234, 2000 and J. Biol. Chem. 277(15), 12741-12748, 2002. Therefore, the contents disclosed in these documents are incorporated herein by reference. For laminin 511, commercially available proteins may be used. For example, recombinant proteins of laminin 511 and laminin 521 are commercially available and available from BioLamina AB.

[0081] As used herein, "expression" of genes, polynucleotides, polypeptides, etc., refers to the transformation of a gene, etc., into another form through some in vivo process. Preferably, expression refers to the transcription and translation of genes, polynucleotides, etc., into polypeptide forms, but transcription to produce mRNA can also be an expression form. More preferably, such polypeptide forms can be those that have undergone post-translational processing (referred to herein as derivatives). For example, the expression level of each laminin chain can be determined by any method. Specifically, the expression level of each laminin chain can be determined by assessing the amount of mRNA, the amount of protein, and the biological activity of the protein. The amount of mRNA or protein of each laminin chain can be determined by the methods described herein.

[0082] As used herein, “functional equivalent” refers to any substance having the same target function as the original entity but with a different structure. Therefore, it should be understood that “lamin or each laminin chain or its functional equivalent” or “the group consisting of laminins, each laminin chain and their functional equivalents” encompasses: laminins or each laminin chain themselves, and fragments, mutants, or variants (e.g., amino acid sequence variants, etc.) of said laminins or each laminin chain having one or more cell adhesion, differentiation regulation, and / or growth-promoting effects on ocular cells, etc.; and substances that, upon action, can be converted into laminins or each laminin chain themselves, or fragments, mutants, or variants of said laminins or each laminin chain (including, for example, nucleic acids encoding laminins or each laminin chain themselves or fragments, mutants, or variants of said laminins or each laminin chain, and vectors, cells, etc., containing said nucleic acids). Typical examples of "laminin or each laminin chain or its functional equivalent" or "the group consisting of laminins, each laminin chain and its functional equivalents" include at least one reagent selected from laminins and fragments thereof. In this invention, it should be understood that the functional equivalents of laminins or each laminin chain can be used interchangeably with laminins or each laminin chain without any particular description.

[0083] As used herein, a “fragment” refers to a polypeptide or polynucleotide sequence of length 1 to n-1 relative to the full-length polypeptide or polynucleotide (of length n). The length of the fragment may be appropriately varied depending on the purpose. Examples of lower limits for the length of a polypeptide include 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, and more amino acids. Furthermore, lengths represented by integers not specifically listed herein (e.g., 11, etc.) are also suitable as lower limits. Examples of lower limits for the length of a polynucleotide include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides. Lengths represented by integers not specifically listed herein (e.g., 11, etc.) are also suitable as lower limits. It should be understood that, herein, fragments of the laminin chain are within the scope of the invention when they themselves function as factors contributing to their activity (e.g., growth promotion or maintenance). According to the present invention, the term "activity" as used herein refers to molecular function in the broadest sense. Activity generally includes, but is not intended to be limited to, the biological, biochemical, physical, or chemical functions of a molecule. Examples of activity include, enzymatic activity, the ability to interact with another molecule, the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of another molecule, stability, and the ability to localize to a specific location within a cell. Where applicable, the term also refers to the function of protein complexes in the broadest sense. When referring to genes or related nucleic acids or peptides, "biological function" as used herein refers to the specific function that the gene, nucleic acid, or peptide may possess in living organisms. Examples include, but are not limited to, the production of specific antibodies, enzyme activity, and the administration of resistance. As used herein, a biological function can be exerted through "biological activity." "Biological activity" as used herein refers to the activity that a reagent (e.g., polynucleotides and proteins, etc.) may possess in living organisms, including activities that perform multiple functions (e.g., transcription-promoting activity), such as activating molecules to interact with another molecule or inactivating molecules to prevent them from interacting with another molecule. When two reagents interact, their biological activity can be considered as the bond between the two molecules and the resulting biological alteration; for example, when the precipitation of one molecule with an antibody leads to the co-precipitation of another molecule, the two molecules are bound together. Therefore, an assay includes observing the co-precipitation. For example, when a reagent is an enzyme, its biological activity includes its enzymatic activity. One example includes, when a reagent is a ligand, causing the ligand to bind to a corresponding receptor. This biological activity can be measured using techniques known in the art. Therefore, "activity" refers to a variety of measurable indicators that indicate or show the binding (directly or indirectly) or influence on the response (i.e., having a measurable effect in response to some exposure or stimulus).Examples include affinity for compounds that directly bind to the polypeptides or polynucleotides of the present invention, the amount of upstream or downstream proteins after some exposure or stimulation, or a measure of another similar function.

[0084] As used herein, “functionally active” means a polypeptide, fragment, or derivative that has a structural, regulatory, or biochemical function (e.g., biological activity) of a protein, according to embodiments relating to the polypeptide, fragment, or derivative of the present invention.

[0085] The term "fragment" of laminin as used herein refers to any fragment of laminin. As the reagents used in this invention, it should be understood that not only the full-length laminin but also fragments of laminin can be used, provided that the fragment possesses the functions of the full-length laminin, particularly the cell adhesion ability of endothelial cells. Therefore, the laminin fragments used in this invention typically possess at least one function of laminin. Specifically, this function may include the cell adhesion ability of endothelial cells.

[0086] The sequences of laminins found to be expressed in corneal endothelial cells in this invention will be explained below. It should be understood that these laminins are preferred representative examples of this invention, and the invention is not limited to these specific laminin subtypes.

[0087] The typical nucleotide sequence of the laminin α5 chain can be: (a) A polynucleotide having the base sequence or a fragment thereof as described in SEQ ID NO: 1; (b) A polynucleotide encoding a polypeptide or a fragment thereof consisting of the amino acid sequence described in SEQ ID NO: 2; (c) A polynucleotide encoding a variant polypeptide or a fragment thereof, wherein one or more amino acids in the polypeptide or fragment thereof have mutations in substitution, addition, and deletion selected from the amino acid sequence described in SEQ ID NO: 2, wherein the variant polypeptide is biologically active; (d) Polynucleotide, which is an allele or splice mutant of the base sequence described in SEQ ID NO: 1 or a fragment thereof; (e) A species homolog of or a fragment thereof encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 2; (f) A polynucleotide encoding a biologically active polypeptide and hybridizing with one of (a)-(e) polynucleotides under stringent conditions; or (g) is a polynucleotide consisting of a base sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with one of (a)-(e) or its complementary sequence, and encoding a biologically active polypeptide. In this respect, biological activity generally refers to the activity of the laminin α5 chain. For the α5 chain, see Doi M et al., J. Biol. Chem. 277(15), 12741-12748, 2002 and U.S. Patent No. 6,933,273.

[0088] The amino acid sequence of the laminin α5 chain can be: (a) A polypeptide consisting of the amino acid sequence or fragments thereof described in SEQ ID NO: 2; (b) A polypeptide having biological activity and having one or more mutated amino acids, wherein the mutation is selected from substitutions, additions and deletions in the amino acid sequence described in SEQ ID NO: 2; (c) A polypeptide encoded by an allele or splice mutant of the base sequence described in SEQ ID NO: 1; (d) A polypeptide that is a species homolog of the amino acid sequence described in SEQ ID NO: 2; or (e) A polypeptide having an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of (a)-(d), and having biological activity. In this respect, biological activity generally refers to the activity of the laminin α5 chain. For the α5 chain, see Doi M et al., J. Biol. Chem. 277(15), 12741-12748, 2002 and U.S. Patent No. 6,933,273.

[0089] The typical nucleotide sequence of the laminin β1 chain can be: (a) A polynucleotide having the base sequence or a fragment thereof as described in SEQ ID NO: 3; (b) A polynucleotide encoding a polypeptide or a fragment thereof consisting of the amino acid sequence described in SEQ ID NO: 4; (c) A polynucleotide encoding a variant polypeptide or a fragment thereof, wherein one or more amino acids in the polypeptide or fragment thereof have mutations selected from the amino acid sequence described in SEQ ID NO: 4, wherein the variant polypeptide is biologically active; (d) A polynucleotide, which is an allele or splice mutant of the base sequence described in SEQ ID NO: 3 or a fragment thereof; (e) A species homolog of or a fragment thereof encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 4; (f) A polynucleotide encoding a biologically active polypeptide and hybridizing with one of (a)-(e) polynucleotides under stringent conditions; or (g) is a polynucleotide consisting of a base sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with one of (a)-(e) or its complementary sequence, and encoding a biologically active polypeptide. In this respect, biological activity generally refers to the activity of the laminin β1 chain. For the β1 chain, see Pillarainen et al., J. Biol. Chem. 262(22),10454-10462, 1987 and U.S. Patent No. 6,933,273.

[0090] The amino acid sequence of the laminin β1 chain can be: (a) A polypeptide consisting of the amino acid sequence described in SEQ ID NO: 4 or a fragment thereof; (b) A polypeptide having biological activity and having one or more mutated amino acids, wherein the mutation is selected from substitutions, additions and deletions in the amino acid sequence described in SEQ ID NO: 4; (c) A polypeptide encoded by an allele or splice mutant of the base sequence described in SEQ ID NO: 3; (d) A polypeptide that is a species homolog of the amino acid sequence described in SEQ ID NO: 4; or (e) A polypeptide having an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of (a)-(d), and having biological activity. In this respect, biological activity generally refers to the activity of the laminin β1 chain. For the β1 chain, see Pillarainen et al., J. Biol. Chem. 262(22), 10454-10462, 1987 and U.S. Patent No. 6,933,273.

[0091] The typical nucleotide sequence of the laminin β2 chain can be: (a) A polynucleotide having the base sequence or a fragment thereof as described in SEQ ID NO: 5; (b) A polynucleotide encoding a polypeptide or a fragment thereof consisting of the amino acid sequence described in SEQ ID NO: 6; (c) A polynucleotide encoding a variant polypeptide or a fragment thereof, wherein one or more amino acids in the polypeptide or fragment thereof have mutations in substitution, addition, and deletion selected from the amino acid sequence described in SEQ ID NO: 6, wherein the variant polypeptide is biologically active; (d) A polynucleotide, which is an allele or splice mutant of the base sequence described in SEQ ID NO: 5 or a fragment thereof; (e) A species homolog of or a fragment thereof encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 6; (f) A polynucleotide encoding a biologically active polypeptide and hybridizing with one of (a)-(e) polynucleotides under stringent conditions; or (g) is a polynucleotide consisting of a base sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with one of (a)-(e) or its complementary sequence, and encoding a biologically active polypeptide. In this respect, biological activity generally refers to the activity of the laminin β2 chain. For the β2 chain, see Wewer UM et al., Genomics. Nov 15, 1994; 24(2):243-52., 1987 and U.S. Patent No. 6,933,273.

[0092] The amino acid sequence of the laminin β2 chain can be: (a) A polypeptide consisting of the amino acid sequence described in SEQ ID NO: 6 or a fragment thereof; (b) A polypeptide having biological activity and having one or more mutated amino acids, said mutation being selected from substitutions, additions and deletions in the amino acid sequence described in SEQ ID NO: 6; (c) A polypeptide encoded by an allele or splice mutant of the base sequence described in SEQ ID NO: 5; (d) A polypeptide that is a species homolog of the amino acid sequence described in SEQ ID NO: 6; or (e) A polypeptide having an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of (a)-(d), and having biological activity. In this respect, biological activity generally refers to the activity of the laminin β2 chain. For the β2 chain, see Wewer UM et al., Genomics. Nov 15, 1994; 24(2): 243-52., 1987 and U.S. Patent No. 6,933,273.

[0093] The typical nucleotide sequence of the laminin γ1 chain can be: (a) A polynucleotide having the base sequence or a fragment thereof as described in SEQ ID NO: 7; (b) A polynucleotide encoding a polypeptide or a fragment thereof consisting of the amino acid sequence described in SEQ ID NO: 8; (c) A polynucleotide encoding a variant polypeptide or a fragment thereof, wherein one or more amino acids in the polypeptide or fragment thereof have mutations in substitution, addition, and deletion selected from the amino acid sequence described in SEQ ID NO: 8, wherein the variant polypeptide is biologically active; (d) Polynucleotide, which is an allele or splice mutant of the base sequence described in SEQ ID NO: 7 or a fragment thereof; (e) A species homolog of or a fragment thereof encoding a polypeptide consisting of the amino acid sequence described in SEQ ID NO: 8; (f) A polynucleotide encoding a biologically active polypeptide and hybridizing with one of (a)-(e) polynucleotides under stringent conditions; or (g) is a polynucleotide consisting of a base sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with one of (a)-(e) or its complementary sequence, and encoding a biologically active polypeptide. In this respect, biological activity generally refers to the activity of the laminin γ1 chain. For the γ1 chain, see Pillarainen et al., J. Biol. Chem. 263(14),6751-6758, 1988 and U.S. Patent No. 6,933,273.

[0094] The amino acid sequence of the laminin γ1 chain can be: (a) A polypeptide consisting of the amino acid sequence described in SEQ ID NO: 8 or a fragment thereof; (b) A polypeptide having biological activity and having one or more mutated amino acids, wherein the mutation is selected from substitutions, additions and deletions in the amino acid sequence described in SEQ ID NO: 8; (c) A polypeptide encoded by an allele or splice mutant of the base sequence described in SEQ ID NO: 7; (d) A polypeptide that is a species homolog of the amino acid sequence described in SEQ ID NO: 8; or (e) A polypeptide having an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of (a)-(d), and having biological activity. In this respect, biological activity generally refers to the activity of the laminin γ1 chain. For the γ1 chain, see Pillarainen et al., J. Biol. Chem. 263(14), 6751-6758, 1988 and U.S. Patent No. 6,933,273.

[0095] As used herein, the terms “protein,” “polypeptide,” “oligopeptide,” and “peptide” are used interchangeably with the same meaning and refer to polymers of amino acids of any length. The polymer can be linear, branched, or cyclic. Amino acids can be naturally occurring or non-naturally occurring, or can be modified amino acids. The term may also include those assembled into complexes having multiple polypeptide chains. The term also includes polymers of naturally occurring or artificially modified amino acids. Examples of such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or alteration (e.g., conjugation with a labeled component). The definition also includes, for example, polypeptides containing one or more amino acid analogs (e.g., containing non-naturally occurring amino acids, etc.), peptide-like compounds (e.g., peptides), and other modifications known in the art. For the proteins of the present invention (e.g., each laminin chain), DNA encoding the gene for each target chain is integrated into a suitable vector and introduced into eukaryotic or prokaryotic cells using expression vectors capable of expression in various hosts, allowing expression of the individual chains to obtain the desired protein. There are no particular limitations on the host cells that can be used to express laminins. Examples include prokaryotic host cells, such as *Escherichia coli* and *Bacillus subtilis*; and eukaryotic host cells, such as yeast, fungi, insect cells, plants and plant cells, and mammalian cells. Vectors for expressing target laminin chains, etc., can be introduced into these host cells using transformation, transfection, conjugation, protoplast fusion, electroporation, gene gun technology, calcium phosphate precipitation, *Agrobacterium* method, direct microinjection, etc. Cells containing the vector are grown in a suitable culture medium to produce laminin chains used in this invention, and the laminin chains are purified from the cells or culture medium to obtain the laminin chains, etc. Purification is performed using molecular sieve chromatography, HPLC, ion exchange chromatography, immunoaffinity chromatography, etc.

[0096] The "amino acids" used in this article may be naturally occurring or non-natural, as long as they can achieve the purpose of this invention.

[0097] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably and refer to polymers of nucleotides of any length. These terms also include "oligonucleotide derivatives" or "polynucleotide derivatives." The terms "oligonucleotide derivative" or "polynucleotide derivative" are used interchangeably and refer to derivatives of nucleotides, oligonucleotides, or polynucleotides in which the bonds between nucleotides are different from normal bonds. Specific examples of such oligonucleotides include: 2'-O-methyl-ribonucleotides; oligonucleotide derivatives wherein the phosphodiester bond in the oligonucleotide is converted to a thiophosphate bond; oligonucleotide derivatives wherein the phosphodiester bond in the oligonucleotide is converted to an N3'-P5' aminophosphate bond; oligonucleotide derivatives wherein the ribose and phosphodiester bond in the oligonucleotide is converted to a peptide-nucleic acid bond; oligonucleotide derivatives wherein uracil in the oligonucleotide is replaced by C-5-propynyluracil; oligonucleotide derivatives wherein uracil in the oligonucleotide is replaced by C-5-thiazouracil; oligonucleotide derivatives wherein cytosine in the oligonucleotide is replaced by C-5-propynylcytosine; an oligonucleotide derivative wherein cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine; oligonucleotide derivatives wherein the ribose in the DNA is replaced by 2'-O-propylribose; and oligonucleotide derivatives wherein the ribose in the oligonucleotide is replaced by 2'-methoxyethoxyribose. Unless otherwise stated, a specific nucleic acid sequence is intended to include explicitly stated sequences, as well as variants with conserved alterations (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term “nucleic acid” as used herein may be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. The term “nucleotide” as used herein may be naturally occurring or non-natural.

[0098] In this article, "gene" refers to an agent that defines a hereditary trait. Genes are typically arranged in a given order on chromosomes. Genes that define the primary structure of a protein are called structural genes, and genes that influence its expression are called regulatory genes. In this article, "gene" may also refer to "polynucleotide," "oligonucleotide," and "nucleic acid."

[0099] Amino acids are referred to in this paper using either well-known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides are referred to using recognized single-letter codes. In this paper, the similarity, identity, and homology of amino acid and base sequences are compared using default parameters and calculations with the sequence analysis tool BLAST. Identity was retrieved using NCBI's BLAST 2.2.26 (released October 30, 2011). In this paper, identity values ​​generally refer to those obtained using the above BLAST under default conditions. However, when higher values ​​are obtained by changing parameters, the highest value is considered the identity value. When identity is assessed across multiple regions, the highest value among them is considered the identity value. Similarity is a numerical value calculated considering similar amino acids in addition to identity.

[0100] As used herein, "polynucleotides hybridized under stringent conditions" refers to conditions commonly known in the art. It should be understood that laminins encoded by the "polynucleotides hybridized under stringent conditions" corresponding to the nucleic acid sequence of each specifically disclosed laminin can also be used as laminins in this invention. Such polynucleotides can be obtained using colony hybridization, plaque hybridization, or DNA blotting, and a polynucleotide selected from the polynucleotides of this invention can be used as a probe. Specifically, it refers to polynucleotides that can be determined by hybridizing at 65°C in the presence of 0.7 to 1.0 M NaCl using a filter immobilized with DNA from colonies or plaques, followed by rinsing the filter at 65°C with a 0.1 to 2-fold concentration of SSC (sodium citrate saline) solution (wherein a 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 mM sodium citrate). The hybridization can be performed according to the methods described in experimental literature (e.g., Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, 2nd Edition, Oxford University Press (1995)). In this regard, sequences containing only A or T sequences are preferably excluded from the sequences hybridized under stringent conditions. Therefore, the polypeptides used in this invention (e.g., laminins) also include polypeptides encoded by nucleic acid molecules, so that the nucleic acid molecules hybridize under stringent conditions with nucleic acid molecules encoding the polypeptides specifically described in this invention. These low-toughness conditions include: hybridization at 40°C for 18 to 20 hours in a buffer containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% BSA, 100 μg / ml denatured salmon sperm DNA, and 10% (w / v) dextran sulfate; washing at 55°C for 1 to 5 hours in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS; and washing at 60°C for 1.5 hours in a buffer consisting of 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS.

[0101] Functional equivalents of the present invention may be those having one or more amino acid insertions, substitutions, deletions, or additions at one or both ends of an amino acid sequence. In this document, “one or more amino acid insertions, substitutions, deletions, or additions at one or both ends of an amino acid sequence” refers to changes made by known technical methods (e.g., site-directed mutagenesis) or naturally occurring mutations, having multiple naturally occurring amino acid substitutions, etc.

[0102] The modified amino acid sequence of each laminin chain, etc., used in this invention can be a sequence having, for example, about 1 to 30, preferably about 1 to 20, more preferably about 1 to 9, still more preferably about 1 to 5, and particularly preferably about 1 to 2 amino acid insertions, substitutions, deletions, or additions at one or both ends. The modified amino acid sequence can be an amino acid sequence having one or more (preferably one or more, or 1, 2, 3, or 4) conserved substitutions in the amino acid sequence of each laminin chain, etc. Hereinafter, "conserved substitution" means the substitution of one or more amino acid residues with other chemically similar amino acid residues, said substitution not substantially altering the protein function. Examples include: the substitution of a given hydrophobic residue with another hydrophobic residue, or the substitution of a given polar residue with another polar residue having the same charge. For each amino acid, functionally similar amino acids that can be used for said substitution are known in the art. Specific examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Specific examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Specific examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Furthermore, specific examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0103] The term "reagent" as used herein can be broadly defined as any substance or other element (e.g., light, radiation, heat, electricity, and other forms of energy), provided that the substance is interchangeable and achieves the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA, genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling molecules, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)) and their complex molecules. Typical examples of reagents specifically targeting polynucleotides include, but are not limited to, polynucleotides having a sequence homology (e.g., 70% or more sequence identity) with the sequence of the polynucleotide, polypeptides such as transcription factors that bind to promoter regions, etc. Typical examples of reagents specifically targeting polypeptides include, but are not limited to, antibodies (e.g., single-chain antibodies) specifically targeting the polypeptide or its derivatives or analogs, specific ligands or receptors when the polypeptide is a receptor or ligand, substrates when the polypeptide is an enzyme, etc.

[0104] As used herein, “normal cellular function” refers to the function that a cell inherently possesses when referring to a specific cell type (e.g., corneal endothelial cells). For corneal endothelial cells, examples of such functions include, but are not limited to, the ability to adapt to corneal transplantation, ZO-1, and Na+. + / K +-ATPase (Matsubara M, Tanishima T: Wound-healing of the corneal endothelium in the monkey: a morphometric study, Jpn J Ophthalmol 1982, 26: 264-273; Matsubara M, Tanishima T: Wound-healing of corneal endothelium in the monkey: an autoradiographic study, Jpn J Ophthalmol 1983, 27: 444-450; Van Horn DL, Hyndiuk RA: Endothelial wound repair inprimate cornea, Exp Eye Res 1975, 21: 113-124 and Van Horn DL, Sendele DD, Seideman S, Buco PJ: Regenerative capability of the corneal endothelium inrabbit and cat, Invest Ophthalmol Vis Sci 1977, 16: 597-613) etc.

[0105] ZO-1 and Na + / K + -ATPases can be assessed by observing gene expression at the nucleic acid level, such as through RT-PCR or immunological methods. + / K + Confirmation of the expression and / or function of ATPase and ZO-1 at the same level as in normal cells makes it possible to confirm whether the test cells have normal function.

[0106] The ability to adapt to corneal transplantation can be tested by mechanically scraping away the corneal endothelium as a bullous keratopathy model in experimental animals (e.g., rabbits) and transplanting cultured cells. However, because rabbit corneal endothelial cells grow in vivo, the possibility of spontaneous healing due to the growth of host corneal endothelial cells cannot be ruled out (Matsubara M et al., Jpn J Ophthalmol 1982, 26: 264-273; Matsubara M et al., Jpn J Ophthalmol 1983, 27: 444-450; Van Horn DL et al., Exp Eye Res 1975, 21: 113-124 and Van Horn DL et al., Invest Ophthalmol Vis Sci 1977, 16: 597-613). Therefore, to more accurately assess the ability to adapt to transplantation, it is preferable to evaluate graft implantation in primates. When assessing the ability to adapt for transplantation in humans, for example, adaptability is evaluated in primates (e.g., cynomolgus monkeys) after at least one month, preferably at least two months, more preferably at least three months, still more preferably at least six months, and still more preferably at least 12 months. Confirmation of the ability to adapt for transplantation in primates (e.g., monkeys) is important, especially when applied to humans.

[0107] General Technology The molecular biology, biochemical, and microbiological methods used in this paper are well-known and commonly used in the field, and are described in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its third edition (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular 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 ofNucleic Acids, Weinheim;Blackburn, G. M. et al. (1996). Nucleic Acids inChemistry and Biology, Oxford University Press;Hermanson, G.T. (1996).Bioconjugate Techniques, Academic Press, Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume], Idenshi Donyu Oyobi Hatsugen Kaiseki JikkenHo [Experimental Methods for Transgenesis & Expression Analysis], Yodosha, 1997, etc., are well-known. As mentioned above, long-term culture and subculturing lead to fibroblast-like transformation, research on efficient culture methods is still ongoing. Relevant portions of the aforementioned literature (which may be complete works) are incorporated herein by reference.

[0108] Preferred implementation scheme Preferred embodiments will be described below. It should be understood that these embodiments are examples of the present invention, and the scope of the invention should not be limited to these preferred embodiments. It should also be understood that those skilled in the art can easily make changes and modifications within the scope of the invention by referring to the following preferred embodiments. It should also be understood that any embodiments can be combined.

[0109] Prevention or treatment On one hand, the present invention provides a treatment or preventive agent for diseases, disorders, or conditions of the cornea (e.g., corneal endothelium), said agent comprising at least one agent selected from laminin and fragments thereof. In this respect, the present invention also provides at least one agent selected from laminin and fragments thereof for treating or preventing diseases, disorders, or conditions of the corneal endothelium. Alternatively, the present invention provides a method for treating or preventing diseases, disorders, or conditions of the corneal endothelium, said method comprising administering an effective amount of at least one agent selected from laminin and fragments thereof to a subject requiring such treatment or prevention. In this respect, it should be understood that similar therapeutic or preventive effects on the cornea can be achieved for the corneal endothelium and epithelium, etc.

[0110] In one specific embodiment, the present invention provides a treatment or preventive agent for diseases, disorders, or conditions of the corneal endothelium, said agent comprising at least one agent selected from laminin and fragments thereof.

[0111] In one embodiment, the reagents or laminins used in this invention comprise RGD sequences. While not wishing to be bound by any theory, RGD sequences are considered to be associated with cell adhesion. It should be understood that laminins with outstanding cell adhesion capabilities can be used to treat or prevent, or improve, diseases, disorders, or conditions of the corneal endothelium.

[0112] In another embodiment, the reagents or laminins used in this invention comprise an α5 chain. While not wishing to be bound by any theory, this is because the results shown in the examples, etc., demonstrate that types of laminins comprising an α5 chain can treat or prevent or improve diseases, disorders, or conditions of the corneal endothelium, and that the β and γ chains are considered to have a degree of flexibility as long as the α5 chain is present.

[0113] In another embodiment, the reagents or laminins used in this invention comprise a γ1 chain. While not wishing to be bound by any theory, this is because the results shown in the examples, etc., demonstrate that types of laminins containing a γ1 chain can treat or prevent or improve diseases, disorders, or conditions of the corneal endothelium, and that the α and β chains are considered to have a degree of flexibility as long as the γ1 chain is present.

[0114] In another embodiment, the reagents or laminins used in this invention comprise α5 chains and / or γ1 chains. While not wishing to be bound by any theory, this is because the results shown in the examples, etc., demonstrate that types of laminins comprising α5 chains and / or γ1 chains can treat or prevent or improve diseases, disorders, or conditions of the corneal endothelium, and demonstrate the role of laminins 511 and 521, thus proving that the β chains possess a certain degree of flexibility once the α5 and / or γ1 chains are identified.

[0115] In a preferred embodiment, the laminin comprises laminin 511 and laminin 521. Therefore, in this embodiment, the reagent of the present invention can be laminin 511, laminin 521, or a fragment thereof. Any fragment can be used as a fragment of laminin 511 or laminin 521 of the present invention, provided that the fragment is capable of treating or preventing, or improving, diseases, disorders, or conditions of the corneal endothelium. Examples of such fragments include, but are not limited to, the laminin 511-E8 fragment and the laminin 521 fragment (SEQ ID NO: 9 and 10 (nucleic acid and amino acid sequences) and SEQ ID NO: 11 and 12 (nucleic acid and amino acid sequences), respectively) (see Taniguchi Y, Ido H, Sanzen N, Hayashi M, Sato-Nishiuchi R, Futaki S, Sekiguchi K. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins. J Biol Chem. 284: 7820-7831, 2009; available from Nippi. Inc.). The laminin 511-E8 fragment and the laminin 521 fragment are fragments obtained by elastase treatment and consist of a portion of the coiled-coil domain of the heterotrimer and three LG domains (LG1 to LG3) in the C-terminal region of the α-chain. The E8 fragment is believed to correspond to the integrin binding site of a heterotrimeric molecule in which the α, β, and γ chains of laminin are assembled together via coiled-coil domains. Therefore, a full-length laminin fragment in which the integrin binding site is substantially preserved can be used as a preferred fragment. It should be understood that such fragments can be prepared by appropriate modifications based on information from the laminin 511-E8 and laminin 521 fragments.

[0116] Here, the E8 fragment of human laminin α5β1γ1 (also referred to herein as "human laminin 511-E8") refers to a fragment of human laminin α5β1γ1 (hereinafter referred to as "human laminin 511") corresponding to the E8 fragment of mouse laminin α1β1γ1 (hereinafter referred to as "mouse laminin 111-E8"). As used herein, the term "laminin 511-E8 fragment" is also referred to as "laminin 511-E8 fragment," "laminin 511 E8," or "laminin 511-E8." The E8 fragment of laminin has been identified as a fragment with strong cell adhesion activity that can be obtained by digesting mouse laminin α1β1γ1 (hereinafter referred to as "mouse laminin 111") with elastase (Edgar D., Timpl R., Thoenen H. The heparin-binding domain of lamininis responsible for its effects on neurite outgrowth and neuronal survival. EMBOJ., 3: 1463-1468, 1984., Goodman SL., Deutzmann R., von der Mark K. Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading. J. Cell Biol., 105: 589-598, 1987). For human laminin 511 and human laminin 332, it is assumed that a fragment corresponding to mouse laminin 111-E8 exists during elastase digestion. The human laminin 511-E8 fragment used in this invention only needs to be a fragment of human laminin 511 having the same cell adhesion activity, structure, and similar molecular weight as mouse laminin 111-E8, and does not need to be an elastase digestion product of human laminin 511. The method for preparing the human laminin 511-E8 fragment is not particularly limited. Examples of such methods include methods for digesting full-length human laminin 511 with a proteolytic enzyme (such as elastase) to fractionate and purify the target fragment, and methods for preparing recombinant proteins. From the perspective of yield, quality consistency, and preparation cost, methods for preparing recombinant proteins are preferred. The recombinant human laminin 511-E8 fragment can be prepared by appropriately using known genetic engineering techniques.The recombinant human laminin 511-E8 fragment can be prepared by, for example, obtaining DNA encoding each α, β, and γ chain of the human laminin 511-E8 fragment, inserting each obtained DNA into an expression vector, expressing the three resulting expression vectors by co-transfection into suitable host cells, and purifying the trimeric proteins using known methods (e.g., see Hiroyuki Ido et al., "The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin γ chains in integrin binding by laminins," The Journal of Biological Chemistry, 282, 11144-11154, 2007). For specific production methods, refer to Japanese Patent Application 2011-78370. A similar fragment can also be produced using human laminin 521. This is referred to as the laminin 521-E8 fragment. It should be understood that such fragments can be prepared in the same manner as the laminin 511-E8 fragments and retain the same activity. In this invention, it should be understood that E8 fragments can be prepared in a similar manner for any laminin containing an α5 chain and / or a γ1 chain. It should also be understood that such E8 fragments can be similarly used for the full-length laminins of this invention.

[0117] In a preferred embodiment, the laminin comprises laminin 511 (α5β1γ1) and laminin 521 (α5β2γ1), or the reagent is laminin 511, laminin 521, laminin 511-E8 fragment or laminin 521-E8 fragment.

[0118] In another embodiment, the fragment used in this invention has the ability to adhere to corneal cells (corneal endothelial cells).

[0119] In one implementation, the concentration of the reagent used (e.g., laminin or fragments thereof) can be any concentration, as long as it has a therapeutic or preventive effect (also referred to as an effective concentration, or a therapeutically effective concentration for treatment, or a preventively effective concentration for prevention). Examples include, but are not limited to, approximately 0.1 nM or above, approximately 0.2 nM or above, approximately 0.3 nM or above, approximately 0.4 nM or above, approximately 0.5 nM or above, approximately 0.6 nM or above, approximately 0.7 nM or above, approximately 0.8 nM or above, approximately 0.9 nM or above, approximately 1 nM or above, approximately 2 nM or above, approximately 2.1 nM or above, approximately 3 nM or above, approximately 4 nM or above, approximately 5 nM or above, approximately 6 nM or above, approximately 7 nM or above, approximately 8 nM or above, approximately 9 nM or above, approximately 10 nM or above, approximately 15 nM or above, approximately 20 nM or above, approximately 21 nM or above, approximately 25 nm or above, approximately 30 nM or above, approximately 40 nM or above, approximately 50 nM or above, approximately 60 nM or above, approximately 70 nM or above, approximately 80 nM or above, approximately 90 nm. nM or above, approximately 100 nM or above, etc.

[0120] In one embodiment, the target site of the present invention includes the corneal endothelium. Therefore, the diseases, disorders, or conditions targeted by the present invention include, but are not limited to, diseases, disorders, or conditions of the corneal endothelium targeted by the present invention.

[0121] In one embodiment, the ophthalmic site is derived from a primate. In another embodiment, the ophthalmic site is derived from a human.

[0122] In one embodiment, the eye cells are derived from primates. In another embodiment, the eye cells are derived from humans.

[0123] In one embodiment, the corneal endothelium is derived from a primate. In another embodiment, the corneal endothelium is derived from a human.

[0124] In one embodiment, the corneal endothelial cells are derived from primates. In another embodiment, the corneal endothelial cells are derived from humans. While not wishing to be bound by any theory, those skilled in the art will understand that similar therapeutic or preventative effects can be achieved in any mammal, given that the therapeutic or preventative effects of laminin in the corneal endothelial models described in this specification have been demonstrated not only in rabbits but also in primates.

[0125] Examples of corneal endothelial diseases, disorders, or conditions targeted by this invention include diseases requiring corneal endothelial transplantation, such as bullous keratopathy, corneal edema, and corneal leukoma, particularly bullous keratopathy resulting from corneal endothelial disorders caused by corneal dystrophy, trauma, or internal ophthalmic surgery. Grafts can be used for their treatment. Examples of the causes of such bullous keratopathy, corneal endothelial disorders, etc., include surgery, as well as Fuchs keratinocyte dystrophy, trauma, pseudoepidermal detachment syndrome, corneal endotheliitis, etc.

[0126] In another implementation, examples of diseases, disorders, or conditions of the corneal endothelium include photophobia, blurred vision, visual impairment, eye pain, epiphora, congestion, pain, bullous keratosis, eye discomfort, decreased contrast, glare, corneal stromal edema, bullous keratosis, and corneal opacity.

[0127] Examples of subjects who treat or prevent diseases, disorders or conditions of the corneal endothelium of the present invention include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, etc.), and preferably primates (e.g., humans).

[0128] In one embodiment, the corneal endothelium targeted by the present invention comprises the corneal endothelial layer, the posterior elastic membrane, or both.

[0129] In a preferred embodiment, the corneal endothelium targeted by the present invention comprises the posterior elastic membrane. The corneal endothelium targeted by the present invention includes corneal endothelium having a detached posterior elastic membrane. It has been found that the technology of the present invention can treat detached posterior elastic membranes that are difficult to fully recover using conventional techniques. The technology of the present invention can also be understood as a qualitative improvement in this respect.

[0130] Combination therapy In another aspect, the present invention provides a treatment or preventative agent for diseases, disorders, or conditions of the corneal endothelium, said agent using at least one agent selected from laminin and fragments thereof, and corneal endothelial cells. In this respect, the agent and corneal endothelial cells of the present invention can be used as a mixture or administered independently. Therefore, in this respect, the present invention provides a method for treating or preventing diseases, disorders, or conditions of the corneal endothelium, said method comprising administering an effective amount of at least one agent selected from laminin and fragments thereof to a subject requiring such treatment or prevention, and administering corneal endothelial cells and / or a ROCK inhibitor to said subject. It should be understood that the agents (laminin, fragments thereof, etc.), corneal endothelial cells, ROCK inhibitors, etc., used in the methods of the present invention in this aspect may be used in any form as explained herein.

[0131] While not wishing to be bound by any theory, the use of corneal endothelial cells and at least one agent selected from laminin and its fragments in the treatment itself, as demonstrated in the examples, resulted in the clarification of corneal opacities, a reduction in corneal thickness, and a return to normalcy of functional markers. Furthermore, previously unattainable treatment outcomes were achieved. Moreover, the treatment time is characterized by a significantly shorter duration, as evidenced by cases where significant effects were observed within two to three days and near-complete recovery was achieved within a week.

[0132] On the other hand, the present invention provides a treatment or preventative agent for diseases, disorders, or conditions of the corneal endothelium, said agent using at least one agent selected from laminin and fragments thereof, and a ROCK inhibitor (the term is synonymous with "Rho kinase inhibitor"). In this respect, the agent and ROCK inhibitor of the present invention can be used as a mixture or administered independently. It should be understood that the agents (laminin, fragments thereof, etc.) used in the methods of the present invention can be used in any form as explained herein.

[0133] In this invention, "Rho kinase" refers to a serine / threonine kinase that is activated by activating Rho. Examples include ROKα (ROCK-II: Leung, T. et al., J. Biol. Chem., 270, 29051-29054, 1995), p160ROCK (ROKβ, ROCK-I: Ishizaki, T. et al., The EMBO J., 15(8), 1885-1893, 1996) and other proteins with serine / threonine kinase activity.

[0134] Examples of ROCK inhibitors include compounds disclosed in the following publications: US Patent 4,678,783, Japanese Patent 3,421,217, International Publication WO 95 / 28387, International Publication WO 99 / 20620, International Publication WO 99 / 61403, International Publication WO 02 / 076976, International Publication WO 02 / 076977, International Publication WO 2002 / 083175, International Publication WO 02 / 100833, International Publication WO 03 / 059913, International Publication WO 03 / 062227, International Publication WO 2004 / 009555, International Publication WO 2004 / 022541, International Publication WO 2004 / 108724, International Publication WO 2005 / 003101, International Publication WO 2005 / 039564, and International Publication WO International Publication WO 2005 / 034866, WO 2005 / 037197, WO 2005 / 037198, WO 2005 / 035501, WO 2005 / 035503, WO 2005 / 035506, WO 2005 / 080394, WO 2005 / 103050, WO 2006 / 057270, WO 2007 / 026664, etc. These compounds can be prepared by the methods described in the respective publications that disclose them. Specific examples include 1-(5-isoquinolinesulfonyl)piperazine or its salts (e.g., fasudil (1-(5-isoquinolinesulfonyl)piperazine)), (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) or its salts (e.g., Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) etc.). Commercially available products (Wako Pure Chemical Industries, Ltd, Asahi Kasei Pharma Corporation, etc.) may also be used appropriately for these compounds.

[0135] In a preferred embodiment, examples of ROCK inhibitors (Rho kinase inhibitors) used in the present invention include, but are not limited to, Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate), etc.

[0136] Any corneal endothelial cell may be used as “corneal endothelial cell” as used herein. The corneal endothelial cell may be isolated or cultured. The corneal endothelial cell may be those cultured using normal culture methods developed by the inventors, or those cultured by other methods. For example, corneal endothelial cells cultured using the methods described in WO 2013 / 100208 may be used. For example, during the culture of said corneal endothelial cells, fibrosis inhibitors may be present continuously, while adhesion promoters may be present for a period of time (e.g., 24 to 72 hours, or 48 hours, etc.), then temporarily removed, and then present again for a period of time (e.g., 24 to 72 hours, or 48 hours, etc.; this period may vary each time or remain constant). Alternatively, these culture methods may optionally be methods that do not use adhesion promoters. For example, the following three types are examples.

[0137] Cultivation Method 1 During primary and subculture, Y-27632 (e.g., available from WAKO, catalog number: 253-00513), a ROCK inhibitor with adhesion-promoting effects, was added for 48 hours to achieve a final concentration of 10 μmol / L.

[0138] Cultivation Method 2 During the culture process, the ROCK inhibitor Y-27632 was continuously added to achieve a final concentration of 10 μmol / l.

[0139] Cultivation Method 3 Cells were cultured in basal medium supplemented with SB431542 (e.g., available from Merck Millipore, Billerica, MA) (1 μmol / l) and SB203580 (1 μmol / l) without the addition of Y-27632.

[0140] The culture medium used can be a commercially available and used culture medium component, or a component developed specifically for corneal endothelium. Examples of such culture medium components include, but are not limited to, OptiMEM, DMEM, M199, MEM, etc. (these are available from INVITROGEN, etc.). Typical examples include: for humans, Opti-MEM I reduced serum medium, liquid (INVITROGEN catalog number: 31985-070) + 8% FBS (BIOWEST, catalog number: S1820-500) + 200 mg / ml CaCl2·2H2O (SIGMA catalog number: C7902-500G) + 0.08% chondroitin sulfate (SIGMA catalog number: C9819-5G) + 20 μg / ml ascorbic acid (SIGMA catalog number: A4544-25G) + 50 μg / ml gentamicin (INVITROGEN catalog number: 15710-064) + 5 ng / ml EGF (INVITROGEN catalog number: PHG0311) adapted for use as basal medium for 3T3 feeder cells, as well as SB431542 (1 μmol / l) and SB203580 (1 μmol / l).

[0141] <1> Corneal endothelial cells were collected and cultured in test tubes. Corneal endothelial cells are collected from the cornea of ​​the recipient or a suitable donor using conventional methods. Considering the transplantation conditions of this invention, corneal endothelial cells from the same species can be prepared. For example, the posterior elastic membrane and endothelial cell layer of the corneal tissue are detached from the corneal stroma, then transferred to a culture dish and treated with a neutral protease, etc. Thus, the corneal endothelial cells detach from the posterior elastic membrane. Corneal endothelial cells remaining on the posterior elastic membrane can be detached by pipetting, etc. After removal of the posterior elastic membrane, the corneal endothelial cells are cultured in a culture medium (e.g., as described in WO2013 / 100208). The following reagents can be used as cultures or culture solutions, for example, FBS (fetal bovine serum) (e.g., BIOWEST, catalog number: S1820-500), β-FGF (basic fibroblast growth factor) (e.g., INVITROGEN, catalog number: 13256-029), and antibiotic substances (e.g., penicillin and streptomycin) can be appropriately added to commercially available DMEM (Dalbeco modified Eagle medium) (e.g., INVITROGEN, catalog number: 12320, etc.), and then the components of the culture normalizer shown in WO2013 / 100208 can be added. Coating with the reagents of the present invention for culturing promotes corneal endothelial cell adhesion to the surface of the culture vessel, achieving excellent growth. When culturing by adding laminin to the culture solution, culture dishes whose surfaces are coated with type I collagen, type IV collagen, fibronectin, laminin, or the extracellular matrix of bovine corneal endothelial cells are preferably used. Alternatively, commercially available coating reagents (e.g., FNC coating mixtures) can be used. ® (50 ml (AES-0407), ATHENA, catalog number: 0407)) standard culture container. There are no particular restrictions on the temperature conditions for culturing corneal endothelial cells, as long as the corneal endothelial cells grow. For example, the temperature is about 25°C to about 45°C, preferably about 30°C to about 40°C, and more preferably about 37°C, considering growth efficiency. The culture method is carried out in a standard cell culture incubator under humidified conditions at a CO2 concentration of about 5-10%.

[0142] <2> Transgeneration After the cultured corneal endothelial cells have grown, they can be passaged. Preferably, passage is performed at subconfluence or confluence. Passage can be performed as follows: First, the cells are treated with trypsin-EDTA or the like to detach them from the surface of the culture vessel. Then, the cells are collected. The culture standard of the present invention or culture medium is added to the collected cells to obtain a cell suspension. Preferably, the cells are centrifuged at or after collection. This centrifugation allows for the preparation of a high-density cell suspension. A preferred cell density is about 1-2 × 10⁻⁶ cells / year. 6 Cells / mL. Examples of centrifugation conditions include, but are not limited to, 500 rpm (30 g) to 1000 rpm (70 g) for 1 to 10 minutes.

[0143] The cell suspension is seeded into a culture vessel and cultured in the same manner as the primary culture described above. Although the dilution ratio for subculture varies depending on the cell state, it is approximately 1:2 to 1:4, preferably 1:3. Subculture can be performed under culture conditions similar to those for primary culture described above. The incubation time varies depending on the state of the cells to be used, etc. Examples include 7 to 30 days. The above subculture can be performed multiple times as needed. When using ROCK inhibitors, cell adhesion in the early stages of culture can be enhanced, resulting in a shorter culture period.

[0144] High-density corneal endothelial cells were purified using density gradient centrifugation. In one embodiment, the high-density corneal endothelial cells can be used in this invention after purification using density gradient centrifugation. The method is generally as follows. Suitable methods (e.g., OptiPrep) can be used. TM Cultured human corneal endothelial cells (a mixture of low-density and high-density cells) were centrifuged at 800×g for 15 minutes using a density gradient. Cells contained in the pellet and supernatant can be collected, and an appropriate number of each type of cell (e.g., 420 cells / mm³) can be extracted. 2 Cells were seeded and cultured as the precipitate group and the supernatant group, respectively. After 30 days, morphology was observed using phase-contrast microscopy to analyze the expression of corneal endothelial function-related markers by immunostaining and to measure cell density / cell area. After centrifugation, the cultured cells showed a monolayer polygonal cell morphology in both the precipitate and supernatant groups. Cells exhibiting Na+ expression were obtained. + / K + - Cells expressing ATPase and ZO-1. Furthermore, the precipitate group typically has a significantly higher cell density. The median cell area (interquartile range) in the precipitate group is generally lower, indicating less dispersion. Therefore, it should be understood that high-density cells can be purified and used in this invention using density gradient centrifugation.

[0145] Wrapped In one embodiment, the present invention provides a treatment or preventative agent for diseases, disorders, or ailments of the corneal endothelium, comprising at least one agent selected from laminin and fragments thereof, wherein the agent is injected into the eye to contact the tissues within the eye. Accordingly, the present invention also provides a method for treating or preventing diseases, disorders, or ailments of the corneal endothelium, the method comprising administering an effective amount of at least one agent selected from laminin and fragments thereof to a subject requiring such treatment or prevention, wherein the agent is injected into the subject's eye to contact the tissues within the eye. It should be understood that the agents (laminin, fragments thereof, etc.) used in the methods of the present invention may be used in any form as explained herein. In this respect, it should be understood that injecting the agent into the eye to contact the tissues within the eye results in the formation of a coating of at least one agent selected from laminin and fragments thereof (also referred to herein as a laminin coating) in the eye to promote corneal healing.

[0146] In one embodiment, the concentration of the reagent used during coating can be any concentration as long as it has a therapeutic or preventative effect (also known as the effective concentration; also known as the effective coating concentration for coating). Examples include, but are not limited to, approximately 0.1 nM or more, approximately 0.2 nM or more, approximately 0.3 nM or more, approximately 0.4 nM or more, approximately 0.5 nM or more, approximately 0.6 nM or more, approximately 0.7 nM or more, approximately 0.8 nM or more, approximately 0.9 nM or more, approximately 1 nM or more, approximately 2 nM or more, approximately 2.1 nM or more, approximately 3 nM or more, approximately 4 nM or more, approximately 5 nM or more, approximately 6 nM or more, approximately 7 nM or more, approximately 8 nM or more, approximately 9 nM or more, approximately 10 nM or more, approximately 15 nM or more, approximately 20 nM or more, approximately 21 nM or more, approximately 25 nM or more, approximately 30 nM or more, approximately 40 nM or more, approximately 50 nM or more, approximately 60 nM or more, approximately 70 nM or more, approximately 80 nM or above, approximately 90 nM or above, approximately 100 nM or above, etc.

[0147] In a preferred embodiment, corneal cells (such as corneal endothelial cells) may be further administered before, simultaneously with, or after the reagent is injected near the corneal endothelium to contact the cells or tissues constituting the corneal endothelium. Therefore, in this invention, the reagent and corneal endothelial cells can be administered separately. The timing of administering corneal cells (such as corneal endothelial cells) is preferably after or simultaneously with the injection of the reagent into the eye to contact (coating) intraocular tissues, more preferably after the injection of the reagent into the eye to contact intraocular tissues. It has been revealed that the presence of coating promotes graft implantation of corneal cells (such as corneal endothelial cells) administered to the corneal endothelial tissue in this manner, thereby significantly enhancing therapeutic efficacy.

[0148] On the other hand, the present invention relates to a preventive or therapeutic agent for diseases, disorders, or ailments of the corneal endothelium, comprising a mixture of at least one agent selected from laminin and fragments thereof with corneal cells (such as corneal endothelial cells), wherein at least one agent selected from laminin and fragments thereof (which is different from the at least one agent selected from laminin and fragments thereof) is injected into the eye to contact the tissues within the eye, preferably to a portion of the tissue receiving treatment or prevention (e.g., the corneal endothelium). Therefore, in this respect, the present invention provides a method for treating or preventing diseases, disorders, or ailments of the corneal endothelium, the method comprising administering an effective amount of at least one agent selected from laminin and fragments thereof to a subject requiring such treatment or prevention, wherein the agent is provided in a mixture with corneal endothelial cells, and at least one agent selected from laminin and fragments thereof is injected into the eye to contact the tissues within the eye. In this respect, the mixture may be administered before, simultaneously with, or after the injection of at least one agent selected from laminin and fragments thereof into the eye to contact (coat) the tissues within the eye. The timing of administration of the mixture is preferably after or simultaneously with the injection of the reagent into the eye, thereby bringing it into contact with the intraocular tissues; more preferably, it is after the injection of the reagent into the eye, thereby bringing it into contact with the intraocular tissues. While not wishing to be bound by any theory, it should be understood that this coating provides an environment conducive to the formation of the mixture of the aforementioned reagent and corneal cells (such as corneal endothelial cells), thereby promoting corneal healing. It should be understood that corneal cells (such as corneal endothelial cells) may be used in any form as explained herein or in any known form.

[0149] In a preferred embodiment, the therapeutic or preventative agent of the present invention (in a coated form) further comprises a ROCK inhibitor. The ROCK inhibitor and the agent may be administered simultaneously, sequentially, or independently.

[0150] ROCK inhibitors can be any form as explained herein, preferably Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate), etc.

[0151] In one embodiment of the invention, the reagent mixed with corneal cells (such as corneal endothelial cells) is about 2.1 nM or more, and the reagent to be injected is about 21 nM or more.

[0152] use On the other hand, the present invention provides the use of at least one reagent selected from laminin and fragments thereof for the preparation of a medicament for treating or preventing diseases, disorders, or conditions of the corneal endothelium. Alternatively, in this respect, the present invention provides the use of at least one reagent selected from laminin and fragments thereof for the treatment or prevention of diseases, disorders, or conditions of the corneal endothelium. It should be understood that the reagents (laminin, fragments thereof, etc.) used in the uses of the present invention may be used in any form as explained herein.

[0153] References cited herein (e.g., scientific literature, patents, and patent applications) are incorporated herein by reference to the same extent that each reference is fully cited. As used herein, "or" is used when referring to "at least one or more" listed in a sentence. When explicitly stated herein as "within a range of two values," those two values ​​are themselves included within that range. Unless otherwise specified, as used herein, "about" means a numerical value rounded to significant figures, or, for a specific value, ±10% of that value.

[0154] As described above, the present invention has been explained, and preferred embodiments have been provided to aid understanding. Hereinafter, the invention will be explained with reference to embodiments. However, the above description and the following embodiments are not intended to limit the invention, but are merely illustrative. Therefore, the scope of the invention is not limited to the embodiments and examples specifically described herein, but is defined only by the claims.

[0155] Example Embodiments of the present invention will be disclosed below. When applicable, biological samples and the like are processed in accordance with the standards prescribed by the Ministry of Health, Labour and Welfare, the Ministry of Education, Culture, Sports, Science and Technology, etc.

[0156] Experimental methods: Preparation of cultured corneal endothelial cells (Method) * (Cultivation) Rabbit corneal endothelial cells (RCEC, cell source and culture method): For the rabbit corneal endothelial cells used in the following experiments, the posterior elastic membrane containing the endothelial cell layer was detached from the corneal tissue and placed in 1.2 U / ml Dispase I [(Sanko Pure Chemical) catalog number: GD81060] dissolved in DMEM (Gibco-Invitrogen) and seeded at 37°C. One hour later, the corneal endothelial cells were detached from the posterior elastic membrane by pipetting and recovered, and centrifuged at 1000 rpm for 5 minutes to remove the supernatant. Culture medium was added and mixed with the precipitated corneal endothelial cells. The total amount was seeded into 6-well plates coated with FNC Coating Mix. The culture medium used was DMEM (catalog number: 12320; Gibco-Invitrogen) supplemented with 10% FBS, 50 μg / ml gentamicin (catalog number: 15710-064; Invitrogen), 10 μg / ml Y-27632 (catalog number: 6880005, Calbiochem, LaJolla, CA) and 2 ng / ml basic fibroblast growth factor (catalog number: 13256-029; bFGF; Invitrogen). Similar to monkeys, previously reported lines [Koizumi N et al., Exp Eye Res., 2012; 95: 60-67; Koizumi N et al., Invest Ophthalmol VisSci. 2007; 48: 4519-4526; Okumura N et al., Am J Pathol. 2012; 181: 268-277] were used to culture rabbit corneal endothelial cells (CECs).

[0157] The culture medium should be changed every two days. Subculture is carried out when the confluence reaches 50-80%. Subculture methods include using calcium-free... 2+ Mg 2+ Cells were washed with PBS (PBS-; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and TrypLE was added. TM Select (catalog number: 12563; Invitrogen) and incubate at 37°C for 5 minutes. After detaching and collecting the cells from the plate, centrifuge at 1000 rpm for 5 minutes, add culture medium to prepare a cell suspension. Seed the cells at a density of 1:2 onto plates coated with FNC Coating Mix.

[0158] This was used to culture corneal endothelial cells.

[0159] Statistical analysis The Student's t-test was used to determine statistically significant differences (P-values) in the means when comparing two samples. Dunnett's multiple comparison test was used to analyze statistically significant differences when comparing multiple sample groups. The values ​​shown in the figures represent the mean ± SE.

[0160] Example 1: Corneal endothelial transplantation experiment using a rabbit bullous keratopathy model induced by laminin 511-E8 fragment. In this embodiment, the laminin 511-E8 fragment was used as the laminin, and a rabbit bullous keratopathy model was used as the pathological model for cultured corneal endothelial transplantation.

[0161] Materials and methods Reagents used, etc. The following reagents are used in this embodiment.

[0162] *Cultured rabbit corneal endothelial cells (also referred to as RCEC; prepared as disclosed above). *Laminin 511 E8 fragment (Nippi. Inc., 382-02413) * Rabbit bullous keratopathy model (prepared as described in "Transplantation Methods" below) *Other methods mentioned in the experimental methods transplantation method Figure 1 The experiment shown is conducted as follows.

[0163] A bullous keratopathy model was created by mechanically detaching rabbit corneal endothelium using a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan). The control group consisted of a model without cell injection. For the RCEC group, cultured rabbit corneal endothelial cells were injected into the anterior chamber of the model and held face down for 3 hours. For the RCEC+E8 group, cultured rabbit corneal endothelial cells were injected into the anterior chamber of the model along with DMEM containing a laminin 511-E8 fragment adjusted to a concentration of 2.1 nM, and held face down for 3 hours.

[0164] Measurement of corneal thickness Figure 2 The measurement experiment shown was conducted as follows.

[0165] Measured sequentially using an ultrasonic thickness gauge (SP-2000; Tomey, Nagoya, Japan). Figure 1The corneal thickness of the individuals prepared in the study was measured. When measurement was not possible, the upper limit of the measurable value of 1200 μm was used.

[0166] Histological examination Figure 3 The histological examination was performed as follows. This examination was conducted using Na... + / K + Immunostaining with ATPase and ZO-1 is used to confirm normal function. This is used to examine the function of corneal endothelial cells, namely pump function and barrier function. + / K + -ATPase and ZO-1 represent the normal functioning of corneal endothelial cells, namely pump function and barrier function, respectively. The method is as follows.

[0167] Cell observation methods (historical examination) such as staining Cells were observed using a phase-contrast microscope. After cell fixation, ZnO- and Na+ were used. + / K + -ATPase was used as a functionally relevant marker and immunostained for observation under a fluorescence microscope. For histological examination, corneal tissue extracted from rabbits was fixed with 4% formaldehyde at room temperature (RT) for 10 minutes, followed by incubation with 1% bovine serum albumin (BSA) for 30 minutes. To identify the phenotype of regenerated corneal endothelial tissue, adhesion-binding-related protein ZO-1 and pump function-related protein Na+ were analyzed. + / K + Immunohistochemical analysis was performed using ATPase. ZO-1 and Na... + / K + -ATPases are used as markers related to cell function. ZO-1 and Na... + / K + -ATPase was prepared using a 1:200 dilution of ZO-1 polyclonal antibody (Zymed Laboratories, Inc., South San Francisco, CA) and Na + / K +- Staining was performed with an ATPase monoclonal antibody (Upstate Biotec, Inc., Lake Placid, NY). A 1:2000 dilution of Alexa Fluor® 488 (Life Technologies Corp., Carlsbad, CA) was used as a secondary antibody. Cell nuclei were then stained with DAPI (Vector Laboratories, Inc., Burlingame, CA). Cell morphology was further stained with a 1:400 dilution of Alexa Fluor® 488-conjugated phalloidin (Life Technologies Corp., Carlsbad, CA). Slides were then observed under a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Welzlar, Germany).

[0168] result The results are shown in Figures 1 to 3 middle. Figure 1 Images of the anterior segment of corneal endothelial cells cultured in a rabbit bullous keratopathy model using the laminin 511-E8 fragment are shown below. From left to right: Control: mechanically scraped rabbit corneal endothelial cells were used as a control; RCEC: In the prepared model, cultured rabbit corneal endothelial cells were injected into the anterior chamber and held face down for 3 hours; RCEC+E8: In the prepared model, cultured rabbit corneal endothelial cells were injected into the anterior chamber along with DMEM containing the laminin 511-E8 fragment at a concentration adjusted to 2.1 nM and held face down for 3 hours. The top row shows images after one week, and the bottom row shows images after two weeks. Corneal opacity was observed in the control and RCEC groups, while the cornea in the RCEC+E8 group was clear and healed, indicating that corneal clarity and healing occur when cells and laminin are injected.

[0169] [ Figure 2 ] Figure 2 The changes in corneal thickness following transplantation of cultured corneas in a rabbit bullous keratopathy model using the laminin 511-E8 fragment are shown. As illustrated, corneal thickness initially decreased significantly after drug administration. When corneal thickness was measured using an ultrasonic thrombectomy, the cornea was maintained at approximately 1200 μm or greater (the measurement limit) in the control and RCEC groups, but in the RCEC + E8 group, corneal thickness thinned to an average of 637 μm by day 7. This can be understood as the regeneration of the corneal endothelium, as well as pump and barrier functions, due to the transplantation of cells with laminin.

[0170] [ Figure 3 ] Figure 3Histological examination results of corneal endothelial transplantation using a cultured laminin 511-E8 fragment are shown. As illustrated, the gene product expressed in normal corneal endothelial cells is shown. Specifically, Na, an indicator of pump function, is expressed. + / K + -ATPase and ZO-1, which indicates tight junctions (barrier function). Furthermore, it was demonstrated that N-cadherin, which indicates adhesion junctions, is expressed normally. Phalloidin staining also confirmed that the cells possess a monolayer polygonal morphology, identical to normal cells. Based on the above, it has been revealed that the cells have recovered normal function.

[0171] In light of these results, it should be understood that administration of laminin or fragments thereof to corneal endothelial cells can significantly cure corneal endothelial diseases or disorders and restore normal function.

[0172] Example 2: Corneal endothelial transplantation experiment in a rabbit bullous keratopathy model using both laminin and ROCK inhibitors. Previously, it has been reported that injecting cultured corneal endothelial cells with a ROCK inhibitor into the anterior chamber can promote cell adhesion to the stroma. In this regard, the effect of using laminin and a ROCK inhibitor simultaneously was examined.

[0173] Materials and methods Reagents used, etc. The following reagents are used in this embodiment.

[0174] *Cultured rabbit corneal endothelial cells (RCEC; prepared as disclosed above) *Laminin 511 E8 fragment (same as in Example 1; Nippi Inc., 382-02413) * Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) (Catalogue No.: 6880005, Calbiochem, La Jolla, CA) * Rabbit bullous keratopathy model (same as in Example 1; preparation method described in "Transplantation Methods") *Other methods mentioned in the experimental methods transplantation method Figure 4 The experiment shown is conducted as follows.

[0175] A bullous keratopathy model was established by mechanically detaching the rabbit corneal endothelium. The adhesion of injected cells to the stroma was compared 24 hours later between individuals in which cultured rabbit corneal endothelial cells were injected with the ROCK inhibitor Y-27632(+) (100 μM) and individuals in which cells were injected with a laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM). Rabbits were euthanized 24 hours later. Corneal tissue was extracted and stained with phalloidin to evaluate the morphology and number of adherent cells.

[0176] Figures 5 to 8 The measurement experiments shown are conducted as described below.

[0177] transplantation method Four groups (four rabbits per group) were tested: a group in which cultured corneal endothelial cells were injected with Y-27632(+) (100 μM) after the corneal endothelial cells had detached but the elastic membrane had not; a group in which the cells were injected with laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM) after the corneal endothelial cells had detached but the elastic membrane had not; a group in which cells were injected with Y-27632(+) (100 μM) in a bullous keratopathy model with posterior elastic membrane detachment; and a group in which cells were injected with laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM) in a bullous keratopathy model with posterior elastic membrane detachment.

[0178] Measurement of corneal thickness and intraocular pressure Corneal thickness was measured using an ultrasonic pachymeter (SP-2000; Tomey, Nagoya, Japan). When measurement was not possible, the upper limit of measurable thickness of 1200 μm was used. Intraocular pressure was also measured using Tonovet (METechnica, Tokyo).

[0179] Histological examination Performed in the same manner as in Example 1 Figure 8 The histological examination is shown.

[0180] result Figure 4The results show the cell adhesion of corneal endothelial grafts cultured in a rabbit bullous keratopathy model 24 hours after simultaneous administration of laminin and ROCK inhibitors. Phalloidin staining demonstrated greater cell adhesion in individuals injected with both laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM). The density of adherent cells was also higher in individuals injected with both laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM) (average 717.3 cells / mm² in the absence of laminin). 2 Its concentration increased to 1662.8 cells / mm² in the presence of laminin. 2 This should be understood as meaning that when laminin and ROCK inhibitors are used simultaneously, laminin further promotes cell adhesion in vivo.

[0181] Figure 5 The following four groups of anterior segment images, from left to right: Group 1: After corneal endothelial cell detachment but the posterior elastic membrane remained intact, cultured corneal endothelial cells were injected with Y-27632(+) (100 μM); Group 2: After corneal endothelial cell detachment but the posterior elastic membrane remained intact, cells were injected with laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM); Group 3: In a bullous keratopathy model with posterior elastic membrane detachment, cells were injected with Y-27632(+) (100 μM); Group 4: In a bullous keratopathy model with posterior elastic membrane detachment, cells were injected with laminin 511-E8 fragment (2.1 nM) and Y-27632(+) (100 μM). One week after cell injection, regardless of whether the posterior elastic membrane had detached or not, or whether laminin was used, the cornea became transparent.

[0182] Figure 6 and Figure 7 The figures show corneal thickness and intraocular pressure. Corneal thinning was delayed in cases where the posterior elastic membrane was intact, but ultimately thinning occurred in both cases. Intraocular pressure remained within the normal range throughout the observation period.

[0183] Figure 8 Histological examination of corneal endothelial transplantation cultured with laminin 511-E8 fragment d is shown. As illustrated, Na+ expression has been demonstrated in all groups. + / K +The cells contained ATPase (pump function) and ZO-1 (barrier function), and also expressed N-cadherin normally. In the group with the added laminin 511-E8 fragment, phalloidin staining demonstrated that the cells had the same normal monolayer polygonal morphology as normal tissue. Furthermore, in the group with the added laminin 511-E8 fragment, pump function and tight junctions were expressed normally, and adhesion junctions also exhibited normal morphology. Therefore, it was revealed that the cells in the group with the added laminin 511-E8 fragment recovered normal function.

[0184] In light of the results described, it should be understood that the use of laminin or fragments thereof in conjunction with ROCK inhibitors, and administration together with corneal endothelial cells, can significantly cure corneal endothelial diseases or disorders and further improve the function of restoring normal function.

[0185] Example 3: Case study in a monkey bullous keratopathy model Next, a monkey bullous keratopathy model was used as an example in primates to similarly examine the effects of simultaneous use of laminin, ROCK inhibitors, and corneal endothelial cell transplantation.

[0186] Materials and methods Reagents used, etc. The following reagents are used in this embodiment.

[0187] *Cultured monkey corneal endothelial cells (prepared using the same method as rabbit culturing, as described again below) *Laminin 511 E8 fragment (same as in Example 1; Nippi. Inc., 382-02413) *Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) (Same as Example 2; Catalog No.: 6880005, Calbiochem, La Jolla, CA) *Monkey bullous keratopathy model (prepared as described in "Transplantation Methods" below) Cultivation methods Monkey corneal endothelial cells (MCECs) can be obtained and cultured as follows. Specifically, the posterior elastic membrane containing the endothelial cell layer is detached from the corneal tissue and placed in 1.2 U / ml Dispase I [(SankoPure Chemical) catalog number: GD81060] dissolved in DMEM (Gibco-Invitrogen) and seeded at 37°C. After 1 hour, the corneal endothelial cells are detached from the posterior elastic membrane by pipetting and recovered, and centrifuged at 1000 rpm for 5 minutes to remove the supernatant. Culture medium is added and mixed with the precipitated corneal endothelial cells. The total amount is seeded into 6-well plates coated with FNC Coating Mix. The culture medium used was DMEM (catalog number: 12320; Gibco-Invitrogen) supplemented with 10% FBS, 50 μg / ml gentamicin (catalog number: 15710-064; Invitrogen), 10 μg / ml Y-27632 (catalog number: 6880005, Calbiochem, La Jolla, CA) and 2 ng / ml basic fibroblast growth factor (catalog number: 13256-029; bFGF; Invitrogen). Similar to monkeys, previously reported lines [Koizumi N et al., Exp Eye Res., 2012; 95: 60-67; Koizumi N et al., Invest Ophthalmol Vis Sci. 2007; 48: 4519-4526; Okumura N et al., Am J Pathol. 2012; 181: 268-277] were used to culture rabbit corneal endothelial cells (CECs).

[0188] The culture medium should be changed every two days. Subculture is carried out when the confluence reaches 50-80%. Subculture methods include using calcium-free... 2+ Mg 2+ Cells were washed with PBS (PBS-; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and TrypLE was added. TM Select (catalog number: 12563; Invitrogen) and incubate at 37°C for 5 minutes. After detaching and collecting the cells from the plate, centrifuge at 1000 rpm for 5 minutes, add culture medium to prepare a cell suspension. Seed the cells at a density of 1:2 onto plates coated with FNC Coating Mix.

[0189] transplantation method A bullous keratopathy model was created by mechanically detaching the corneal endothelium of cynomolgus monkeys using a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan). Figure 9 In the middle, 5.0×10 5 Cultured monkey corneal endothelial cells, along with DMEM containing a laminin 511-E8 fragment regulated to a concentration of 2.1 nM, were injected into the anterior chamber of a bullous keratopathy model and kept face down for 3 hours. Figure 10 In the prepared bullous keratosis model, the posterior elastic membrane was detached, and similarly, 5.0 × 10 5 Cultured monkey corneal endothelial cells were injected into the anterior chamber of this bullous keratopathy model along with DMEM containing a laminin 511-E8 fragment regulated to a concentration of 2.1 nM, and kept face down for 3 hours.

[0190] Measurement of corneal thickness Corneal thickness was measured using an ultrasonic thimometer (SP-2000; Tomey, Nagoya, Japan). When measurement was not possible, the upper limit of the measurable value of 1200 μm was used.

[0191] result The results are shown in Figures 9 to 10 middle. Figure 9 An anterior segment image of corneal endothelial transplantation after treatment with a monkey bullous keratopathy model simultaneously treated with a laminin 511-E8 fragment is shown. It has been found that, in in vivo primate models where corneal endothelial growth is significantly restricted, the laminin or fragment thereof of the present invention can cure bullous keratopathy by co-administration with corneal endothelial cells and a ROCK inhibitor. On the other hand, Figure 10 This image shows the anterior segment of the cornea after detachment of the posterior elastic membrane and simultaneous transplantation of cultured corneal endothelial cells using a laminin 511-E8 fragment in a monkey bullous keratosis model. In the model of mechanically scraping corneal endothelial cells from cynomolgus monkeys, the cornea was opaque or not healed. While the cornea was transparent and healed in the rabbit bullous keratosis model, no therapeutic effect was observed in the cynomolgus monkey model. This suggests that, depending on the animal species, there is a possibility that corneal endothelial regeneration may not occur upon posterior elastic membrane detachment due to reduced adhesion of transplanted cells to the cornea. Figure 11 The figure shows the corneal thickness changes in individuals who underwent transplantation without posterior elastic membrane detachment and in individuals who underwent transplantation after posterior elastic membrane detachment. In the posterior elastic membrane detachment group, the corneal thickness did not decrease, while in the posterior elastic membrane intact group, the corneal thickness decreased.

[0192] Example 4: A case of treatment with laminin coating in subjects with posterior elastic membrane detachment Next, by coating the inner corneal stroma exposed due to posterior elastic membrane detachment with laminin alone, the improved efficacy of simultaneous use of laminin, ROCK inhibitors, and corneal endothelial cell transplantation was confirmed.

[0193] Materials and methods Reagents used, etc. The following reagents are used in this embodiment.

[0194] *Cultured monkey corneal endothelial cells (prepared in the same manner as in Example 3) *Laminin 511-E8 fragment (same as in Example 1; Nippi. Inc., 382-02413) *Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) (Same as Example 2; Catalog No.: 6880005, Calbiochem, La Jolla, CA) method A bullous keratopathy model was created by mechanically detaching the corneal endothelium of cynomolgus monkeys using a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan). In the created bullous keratopathy model, the posterior elastic membrane detached. A laminin 511-E8 fragment at a concentration of 21 nM was injected into the anterior chamber of the bullous keratopathy model, and the model was incubated for 1 hour. This resulted in in vivo encapsulation of the corneal stroma exposed due to posterior elastic membrane detachment. Then, as in Example 3, 5.0 × 10⁻⁶ mm² of urea was injected into the corneal stroma. 5 Cultured monkey corneal endothelial cells were injected into the anterior chamber of a bullous keratopathy model along with DMEM containing a laminin 511-E8 fragment regulated to a concentration of 2.1 nM, and kept face down for 3 hours.

[0195] result The results are as follows Figure 12 As shown. Figure 12 As shown, after posterior elastic membrane detachment, a laminin 511-E8 fragment at a concentration of 21 nM was injected into the anterior chamber to coat the corneal stroma, resulting in corneal transparency and healing, an effect that could not be achieved without coating. Figure 10 This demonstrates that injecting laminin with cell suspension can not only promote cell adhesion, but also facilitate in vivo graft implantation by using it as a coating agent in vivo.

[0196] Example 5: The effect of integrin on corneal endothelial cell adhesion In this embodiment, the effects of various integrins on corneal endothelial cell adhesion were investigated.

[0197] Materials and methods Reagents used, etc. The following reagents are used in this embodiment.

[0198] *The control group refers to the group that did not receive the laminin 511-E8 fragment.

[0199] *Mouse IgG (DAKO, X0931) *Anti-integrin α3 (Millipore, MAB1952Z-20) *Anti-integrin α6 (Millipore, MAB1378-20) *Anti-integrin α2 (Millipore, MAB1950Z-20) *Anti-integrin β1 (R&D Systems, MAB17781) *For anti-integrin α3β1 and anti-integrin α6β1, use the combination described above.

[0200] *Laminin 511-E8 fragment (same as in the above example) method Completely remove the culture medium from the culture dish containing human corneal endothelial cells. Rinse the cells twice with PBS(-). After rinsing, add phosphate-buffered saline (PBS) and incubate the mixture at 37°C (5% CO2) for 5 minutes. Subsequently, remove the PBS(-) and add TrypLE. TMSelect (10X) (Life Technologies, A12177-01). The mixture was incubated at 37°C (5% CO2) for 10 minutes. Then, Opti-MEMI (Life Technologies, 31985-070) was added to collect cells. After cell collection, the cells were centrifuged at 1200 rpm for 3 minutes to prepare a cell suspension using Opti-MEMI. At this time, a control group without the laminin 511-E8 fragment was prepared, and a group with the laminin 511-E8 fragment added to achieve a final concentration of 2.1 nM was prepared. Meanwhile, mouse IgG and integrin neutralizing antibody were added to the group with the laminin 511-E8 fragment added to adjust the final concentration to 2 μg / ml. After adjustment, cells were seeded at 5000 cells / well in 96-well plates and incubated at 37°C (5% CO2) for 24 hours. After 24 hours of seeding, the culture medium was completely removed, and the cells were washed twice with PBS(-). After rinsing, add culture medium and CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation, Madison, WI) at a 1:1 ratio. Shake the mixture in the dark for 2 minutes, then let it stand for 10 minutes. Measurements were then taken. 24 h, *p < 0.01, Dunnet's test, n = 6.

[0201] result The results are as follows Figure 13 As shown in the figure, compared to the control, adding the laminin 511-E8 fragment to the culture medium at the time of inoculation promoted the adhesion of corneal endothelial cells, but the cell adhesion was inhibited to the same level as the control by the neutralizing antibody against integrin β1.

[0202] Example 6: The relationship between activation of cell adhesion-related proteins and integrins Next, this example demonstrates that the activation of cell adhesion-related proteins is mediated by integrins.

[0203] Materials and methods reagents, etc. In principle, the same conditions as in Example 5 are used.

[0204] *Mouse IgG (same as in Example 5) *Anti-integrin α3 (same as in Example 5) *Anti-integrin α6 (same as in Example 5) *Anti-integrin α2 (same as in Example 5) *Anti-integrin β1 (same as in Example 5) *Anti-integrin α3β1 (same as in Example 5) *Anti-integrin α6β1 (same as in Example 5) method Completely remove the culture medium from the culture dish containing human corneal endothelial cells. Rinse the cells twice with PBS(-). After rinsing, add phosphate-buffered saline (PBS) and incubate the mixture at 37°C (5% CO2) for 5 minutes. Subsequently, remove the PBS(-) and add TrypLE. TM Select (10X) (Life Technologies, A12177-01). The mixture was incubated at 37°C (5% CO2) for 10 minutes. Then, Opti-MEMI (Life Technologies, 31985-070) was added to collect cells. After cell collection, the cells were centrifuged at 1200 rpm for 3 minutes to prepare a cell suspension using Opti-MEMI. At this point, a control group without the laminin 511-E8 fragment was prepared, along with a group with the laminin 511-E8 fragment added to achieve a final concentration of 2.1 nM. Simultaneously, mouse IgG and integrin neutralizing antibody were added to the group with the added laminin 511-E8 fragment to adjust the final concentration to 2 μg / ml. After adjustment, the cells were incubated at 1×10⁻⁶ cells / ml. 5 Cells were seeded onto 12-well plates at 1 / well ratio, and proteins were collected 3 hours after seeding. Phospho-FAK (Cell Signaling Technology, 8556S), FAK (Cell Signaling Technology, 3285S), and p-pilin (Cell Signaling Technology, 2541S) were detected using Western blotting. Each antibody was diluted 1:1000. Density assays were performed using ImageJ.

[0205] result The results are as follows Figure 14 As shown in the figure, 3 hours after inoculation, p-FAK was promoted by the laminin 511-E8 fragment, but was suppressed to the same level as the control group by a neutralizing antibody against integrin β1. p-pole protein was also promoted by the laminin 511-E8 fragment, but was suppressed to the same level as the control group by a neutralizing antibody against integrin β1. Based on these results, it should be understood that E8 promotes cell adhesion by activating adhesion-related proteins via integrin.

[0206] Considering Examples 5 and 6, earlier cell adhesion was observed compared to cells without the fragment after the addition of the laminin 511-E8 fragment. The number of adherent cells significantly increased to 137.3 ± 2.8% after 24 hours (p < 0.01). Furthermore, neutralizing antibodies against integrin α3β1 and α6β1 inhibited the cell adhesion effect of the laminin 511-E8 fragment, resulting in the same level of effect as in cells without the fragment (p < 0.01). FAK phosphorylation was promoted by the laminin 511-E8 fragment but inhibited by the integrin neutralizing antibody. Therefore, it should be understood that laminin 511 binds to integrin and promotes FAK phosphorylation to facilitate stromal adhesion of corneal endothelial cells. Therefore, it should be understood that laminins (such as the laminin 511-E8 fragment) can be used for corneal endothelial cell transplantation.

[0207] Example 7: Formulation Example: Laminin-Cell Mixture In this embodiment, a therapeutic solution containing the reagent of the present invention is prepared as a formulation example, as described below.

[0208] The following solutions were prepared using conventional methods.

[0209] Laminin 511, laminin 521 and / or fragments thereof (0.75 μg / cm) 2 ) The final concentration was 2.1 nM. Cultured corneal endothelial cells An appropriate amount of cells prepared according to Example 1, etc. Suitable buffer solution Appropriate amount Total 100 mL Example 8: Formulation Example: Laminin Coating Composition In this embodiment, the preparation of a coating solution containing the reagent of the present invention is described below as a formulation example.

[0210] The coating solution is prepared using conventional methods as shown below.

[0211] Laminin 511, laminin 521 and / or fragments thereof (0.75 μg / cm) 2 ) The final concentration was 21 nM Suitable buffer solution Appropriate amount Total 100 mL Each component can be obtained as described in Examples 1 to 4.

[0212] As described above, the invention has been illustrated using preferred embodiments. However, it should be understood that the scope of the invention should be interpreted solely by the scope of the claims. It should be understood that patents, patent applications, and documents cited herein are incorporated by reference as if they were specifically described herein. This application claims priority to Japanese Patent Application 2142-222947, filed October 31, 2014, the entire contents of which are incorporated herein by reference.

[0213] Industrial applicability This invention enables novel ophthalmic treatments, particularly those using novel corneal endothelial cells (especially human corneal endothelial cells). In particular, this invention enables near-complete recovery from bullous keratopathy, making it especially useful in the pharmaceutical industry.

[0214] Sequence List Independent Text SEQ ID NO: 1: Laminin α5 chain nucleic acid sequence (NM_005560) SEQ ID NO: 2: Amino acid sequence of laminin α5 chain (NP_005551) SEQ ID NO: 3: Laminin β1 chain nucleic acid sequence (NM_002291) SEQ ID NO: 4: Laminin β1 chain amino acid sequence (NP_002282) SEQ ID NO: 5: Laminin β2 chain nucleic acid sequence (NM_002292) SEQ ID NO: 6: Laminin β2 chain amino acid sequence (NP_002283) SEQ ID NO: 7: Laminin γ1 chain nucleic acid sequence (NM_002293) SEQ ID NO: 8: Laminin γ1 chain amino acid sequence (NP_002284).

Claims

1. The use of at least one agent selected from laminin and fragments thereof for the production of a treatment or preventative agent for diseases, disorders or conditions of the corneal endothelium.

2. The use according to claim 1, wherein the laminin comprises an RGD sequence.

3. The use according to claim 1, wherein the laminin comprises an α5 chain and / or a γ1 chain.

4. The use according to claim 1, wherein the laminin comprises laminin 511 (α5β1γ1) and laminin 521 (α5β2γ1).

5. The use according to claim 1, wherein the fragment has the ability to adhere to corneal endothelial cells.

6. The use according to claim 1, wherein the reagent is laminin 511, laminin 521, or a laminin 511-E8 fragment.

7. The use according to claim 1, wherein the corneal endothelium is derived from primates.

8. The use as claimed in claim 1, wherein the disease, disorder or condition of the corneal endothelium is selected from Fuchs corneal endothelial dystrophy, corneal endotheliitis, and disorders and conditions resulting from trauma and ophthalmic surgery.

9. The use according to claim 1, wherein the disease, disorder or condition of the corneal endothelium is selected from photophobia, blurred vision, visual impairment, eye pain, epiphora, congestion, pain, bullous keratopathy, eye discomfort, decreased contrast, glare, corneal stromal edema, bullous keratopathy and corneal opacity.

10. The use according to claim 1, wherein the corneal endothelium comprises the corneal endothelial layer, the posterior elastic membrane, or both.

11. The use according to claim 1, wherein the corneal endothelium has a detached posterior elastic membrane.

12. The use according to claim 1, wherein the therapeutic or preventive agent further comprises corneal endothelial cells.

13. The use according to claim 1, wherein the therapeutic or preventive agent further comprises a ROCK inhibitor.

14. The use according to claim 1, wherein the therapeutic or preventive agent further comprises corneal endothelial cells and a ROCK inhibitor.

15. The use according to claim 13, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

16. The use according to claim 1, wherein the reagent is injected into the eye and comes into contact with the tissues within the eye.

17. The use according to claim 1, wherein the reagent is present at about 21 nM or more.

18. The use as claimed in claim 1, wherein corneal endothelial cells are further provided.

19. The use according to claim 1, wherein the reagent is provided in combination with corneal endothelial cells, and the at least one reagent selected from laminin and fragments thereof is injected into the eye and comes into contact with the tissues within the eye.

20. The use according to claim 19, wherein the therapeutic or preventive agent further comprises a ROCK inhibitor.

21. The use according to claim 20, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

22. The use according to claim 19, wherein the reagent mixed with the corneal endothelial cells is about 2.1 nM or more, and the reagent to be injected is about 21 nM or more.

23. The use according to claim 14, wherein the ROCK inhibitor is selected from Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate) and pharmaceutically acceptable salts thereof.

24. The use of at least one reagent selected from laminin and fragments thereof for the preparation of a medicament for the treatment or prevention of diseases, disorders or conditions of the corneal endothelium.

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