Choroidal neovascularization inhibitor or vitreous verruca inhibitor, and method for evaluating or screening same

Abstract

Therapeutic agents for CNV only use symptomatic therapies, and have been strongly seeked to be used for radical treatment. In addition, there is no therapeutic agent for dry AMD, and a therapeutic agent for radically treating dry AMD is strongly sought. The present invention provides a prophylactic and / or therapeutic agent for choroidal neovascularization, which contains, as an active ingredient, a compound having an activity of inhibiting epithelial-mesenchymal transition of retinal pigment epithelial cells. Furthermore, the present invention provides a glassy wart inhibitor containing, as an active ingredient, a compound having an activity of inhibiting epithelial-mesenchymal transition of retinal pigment epithelial cells.

Description

[0001] This application is a divisional application of the invention patent application with Chinese application number 201680074156.1 (the invention name of the original application is "Choroidal neovascularization inhibitor or drusen inhibitor and its evaluation or screening method", and the application date of the original application is December 16, 2016). Technical Field

[0002] The present invention relates to a medicine for preventing or treating choroidal neovascularization.

[0003] In addition, the present invention relates to a drusen inhibitor and a method for evaluating or screening the drusen inhibitor. Background Art

[0004] Choroidal neovascularization (CNV) is a disease in which new blood vessels form in the choroid in the macula of the fundus. In addition to age-related macular degeneration (AMD), this disease can also be seen in retinal angioid streaks, pathological myopia, ocular histoplasmosis, and choroidal rupture after trauma.

[0005] Since choroidal neovascularization is fragile, blood components may leak out, causing retinal edema and subretinal fluid retention. In addition, bleeding due to rupture of fragile neovascularization may accelerate the progression of impairment that affects vision loss (Non-Patent Document 1).

[0006] For example, angiogenesis inhibitors such as anti-VEGF drugs that inhibit choroidal neovascularization are used to treat choroidal neovascularization in age-related macular degeneration (Non-Patent Document 2).

[0007] AMD is a disease that causes loss of vision due to age-related factors such as obstruction of the macula of the retina. If AMD progresses, objects or linear objects may appear deformed (metamorphopsia), colors may be lost, and the patient may become sensitive to light, which has a significant impact on the quality of life. In addition, if the disease is serious, a portion of the visual field may be lost (central scotoma, etc.), or even blindness may occur (Non-Patent Document 3).

[0008] AMD is roughly classified into wet AMD (exudative AMD) and dry AMD (atrophic AMD) according to characteristic symptoms. Wet AMD is a type characterized by abnormal new blood vessels and the rupture of the new blood vessels. Wet AMD is from the choroidal new blood vessels under the retinal pigment epithelial cells (Retinal Pigment Epithelium; RPE cells), and blood components ooze out from these fragile blood vessels, and cause retinal edema or retention of subretinal fluid. In addition, bleeding due to fragile blood vessel rupture can accelerate the obstacle affecting decreased vision (non-patent literature 1).

[0009] As a treatment method for AMD, angiogenesis inhibitors such as anti-VEGF (Vascular Endothelial Growth Factor) drugs that inhibit angiogenesis are used for wet AMD (Non-Patent Document 2).

[0010] On the other hand, dry AMD is the disease (non-patent literature 4) that diagnoses according to standards such as vision, fundus examination result, image observation result, exclusion criteria, severe degree classification.Now think that a reason is: due to the structure that is called drusen (drusen), cause visual cell disorder (non-patent literature 5).There is a report that some dry AMD has the example that is transferred to wet AMD.

[0011] [Prior art literature]

[0012] [Non-patent literature]

[0013] Non-patent document 1: Iwata Eiji et al. (2011); Age-related macular degeneration; Modern Medicine, Vol. 59, No. 1, pp. 133 to 138.

[0014] Non-patent document 2: Mariko Hirayama et al. (2011); Anti-VEGF treatment of exudative age-related macular degeneration; Medical Progress Vol. 236 pp. 1165-1167.

[0015] Non-patent document 3: Ishida Susumu et al. (2010); Age-related AMD Pharma Medica Vol. 28; No. 12; pp. 9 to 12.

[0016] Non-patent document 4: Takahashi Kanji et al. (2015); Diagnostic criteria for atrophic age-related macular degeneration; Journal of the Japanese Ophthalmological Society 119; pp. 671-677.

[0017] Non-patent document 5: Yasukawa Tsuyoshi (2011); Seminar 5; Precursor lesions of age-related macular degeneration (AMD) and its symptoms; Geriatric Medicine Vol. 49; No. 4; pp. 409 to 412. Summary of the invention

[0018] An object of the present invention is to provide a preventive and / or therapeutic agent for CNV.

[0019] Another object of the present invention is to provide a drusen inhibitor.

[0020] The use of antibodies targeting VEGF to block new blood vessels is a symptomatic treatment, not to inhibit or improve the behavior of the cells that produce VEGF. In addition, for biological preparations such as antibodies, the longer the treatment period, the higher the medical expenses required. Moreover, for medicines that require intraocular injection, they are highly invasive and impose a heavy burden on patients. Therefore, the industry strongly seeks a low-invasive and economical therapeutic drug for radical treatment of CNV.

[0021] The inventors of the present invention have conducted intensive research to solve the aforementioned problems and have found that by inhibiting the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE cells) using specific nonsteroidal anti-inflammatory agents (NSAIDs), aldose reductase inhibitors, leukotriene receptor antagonists, chemical mediator release inhibitors and thromboxane A2 receptor antagonists, the expression of VEGF in RPE cells can be inhibited, and choroidal neovascularization can be prevented and / or treated, thereby completing the present invention.

[0022] That is, the present invention relates to:

[0023] (1) A preventive and / or therapeutic agent for choroidal neovascularization, comprising as an active ingredient at least one selected from the group consisting of zaltoprofen, oxaprozin, tiaprofenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, seratrodast, and pharmaceutically acceptable salts thereof;

[0024] (2) The agent for preventing and / or treating choroidal neovascularization as described in (1) above, which is an oral agent;

[0025] (3) The agent for preventing and / or treating choroidal neovascularization according to (1) or (2) above, wherein the choroidal neovascularization occurs in age-related macular degeneration, polypoidal choroidal vasculopathy (PCV), or retinal angiomatous proliferation (RAP).

[0026] In addition, in the present invention, it is expected that the visual field distortion and other symptoms of dry AMD can be improved by inhibiting drusen. However, there is no effective therapeutic drug for dry AMD, and the industry is strongly seeking a therapeutic drug for radical cure of dry AMD.

[0027] The inventors of the present invention have made great efforts to solve the aforementioned problems, and the following new insights have been obtained as a result: drusen is a structure caused by the situation that RPE cells cause epithelial-mesenchymal transition. Based on this insight, the inventors of the present invention have found that: EMT can be easily induced in RPE cells; drusen can be reproduced in RPE cells; EMT-inhibiting compounds that can inhibit EMT in RPE cells are evaluated or screened, and these compounds can inhibit drusen in RPE cells; thus completing the present invention.

[0028] That is, the present invention relates to:

[0029] (4) A drusen inhibitor comprising as an active ingredient a compound having an epithelial-mesenchymal transition inhibitory activity on retinal pigment epithelial cells;

[0030] (5) The drusen inhibitor according to (4) above, wherein the compound having the above-mentioned epithelial-mesenchymal transition inhibitory activity is an agent that inhibits the expression of a mesenchymal marker or an extracellular matrix (ECM) in the above-mentioned cells;

[0031] (6) The drusen inhibitor as described in (5) above, wherein the aforementioned interstitial marker is at least one selected from the group consisting of Snail, Slug, cadherin 3, MMP1 (matrix metalloproteinase 1) and MMP7 (matrix metalloproteinase 7); and / or the aforementioned extracellular matrix is ​​at least one selected from the group consisting of COL5A3 (Collagen Type V Alpha 3 Chain), COL6A3 (Collagen Type VI Alpha 3 Chain), LAMC2, HMMR (Hyaluronan Mediated Motility Receptor) and TNC (thymic nurse cell);

[0032] (7) The drusen inhibitor as described in any one of (4) to (6) above, wherein the compound having the aforementioned epithelial-mesenchymal transition inhibitory activity is a nonsteroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor and / or a thromboxane A2 receptor antagonist;

[0033] (8) The drusen inhibitor as described in (7) above, wherein the non-steroidal anti-inflammatory agent is at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, ibuprofen, flurbiprofen and pharmaceutically acceptable salts thereof; the aldose reductase inhibitor is epalrestat and / or pharmaceutically acceptable salts thereof; the leukotriene receptor antagonist is at least one selected from the group consisting of zafirlukast, montelukast, pranlukast and pharmaceutically acceptable salts thereof; the chemical mediator release inhibitor is amlexanox and / or pharmaceutically acceptable salts thereof; and / or the thromboxane A2 receptor antagonist is serotonin and / or pharmaceutically acceptable salts thereof.

[0034] In addition, the present invention relates to:

[0035] (9) A preventive and / or therapeutic agent for age-related macular degeneration, comprising a propionic acid-based nonsteroidal anti-inflammatory agent, an aminoarylcarboxylic acid-based nonsteroidal anti-inflammatory agent, an arylacetic acid-based nonsteroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor, or a thromboxane A2 receptor antagonist as an effective ingredient; the aldose reductase is epalrestat and / or a pharmaceutically acceptable salt thereof;

[0036] (10) The agent for preventing and / or treating age-related macular degeneration as described in (9), wherein the propionic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of zaltoprofen, oxaprozin, teiperfenic acid, ibuprofen, flurbiprofen, and pharmaceutically acceptable salts thereof; and the aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of flufenamic acid, mefenamic acid, and pharmaceutically acceptable salts thereof. The aforementioned non-steroidal anti-inflammatory agent is sulindac and / or its pharmaceutically acceptable salts; the aforementioned leukotriene receptor antagonist is at least one selected from the group consisting of zafirlukast, montelukast, pranlukast and their pharmaceutically acceptable salts; and / or the aforementioned chemical mediator release inhibitor is amlexanox and / or its pharmaceutically acceptable salts; the aforementioned thromboxane A2 receptor antagonist is serotonin and / or its pharmaceutically acceptable salts.

[0037] In addition, the present invention relates to:

[0038] (11) A preventive and / or therapeutic agent for age-related macular degeneration, comprising a combination of an angiogenesis inhibitor and the drusen inhibitor according to any one of (4) to (8).

[0039] In addition, the present invention relates to:

[0040] (12) A method for evaluating or screening a drusen inhibitor, comprising: measuring the inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells in vitro in the presence of a test agent.

[0041] In addition, the present invention relates to:

[0042] (13) A method for evaluating or screening a drusen inhibitor, comprising: measuring the inhibition of retinal pigment epithelial cell focus in vitro in the presence of a test agent.

[0043] In addition, the present invention relates to:

[0044] (14) A method for evaluating or screening a drug for preventing and / or treating age-related macular degeneration, comprising: measuring the inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells in vitro in the presence of a test drug.

[0045] In addition, the present invention relates to:

[0046] (15) A method for evaluating or screening a drug for preventing and / or treating age-related macular degeneration, comprising: measuring the inhibition of retinal pigment epithelial cell aggregation in vitro in the presence of a test drug.

[0047] Since the compound of the present invention can inhibit the expression of VEGF by inhibiting the EMT of RPE cells, it can be used as a preventive and / or therapeutic agent for choroidal neovascularization. In addition, the present invention has sufficient safety as a pharmaceutical product.

[0048] Furthermore, from another viewpoint, according to the present invention, drusen can be effectively inhibited by using an EMT-inhibiting compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a graph showing the results of verifying EMT induction in RPE cells based on changes in expression of EMT marker molecules.

[0050] Figure 2 The graph shows the results of verifying EMT induction in RPE cells based on the changes in EMT marker expression over time.

[0051] Figure 3 This is a graph showing how oxaprozin inhibits EMT of RPE cells.

[0052] Figure 4 This is a graph showing how epalrestat inhibits EMT of RPE cells.

[0053] Figure 5 This is a graph showing how zafirlukast inhibits EMT of RPE cells.

[0054] Figure 6 This is a graph showing how amlexanox inhibits EMT of RPE cells.

[0055] Figure 7 This is a graph showing how zaltoprofen inhibits EMT of RPE cells.

[0056] Figure 8 This is a graph showing how aluminum flufenamate inhibits EMT of RPE cells.

[0057] Fig. 9 This is a graph showing how mefenamic acid inhibits EMT of RPE cells.

[0058] Fig.10 This is a graph showing how sulindac inhibits EMT of RPE cells.

[0059] Fig.11 This is a graph showing how Tepefiic acid inhibits EMT of RPE cells.

[0060] Fig.12 This is a graph showing how serotastat inhibits EMT of RPE cells.

[0061] Fig.13 This is a graph showing the results of verifying the effect of oxaprozin on the expression levels of VEGF and EMT markers in RPE cells.

[0062] Fig.14 This is a graph showing the results of verifying the effect of epalrestat on the expression levels of VEGF and EMT markers in RPE cells.

[0063] Fig.15 This is a graph showing the results of verifying the effect of zaltoprofen on the expression levels of VEGF and EMT markers in RPE cells.

[0064] Fig.16 This is a graph showing the results of verifying the effect of etodolac on the expression levels of VEGF and EMT markers in RPE cells.

[0065] Fig.17 This is a graph showing the results of verifying the effect of indometacin on the expression levels of VEGF and EMT markers in RPE cells.

[0066] Fig.18 This is a graph showing the results of verifying the effect of aluminum flufenamic acid on the expression levels of VEGF and EMT markers in RPE cells.

[0067] Fig.19 This is a graph showing the results of verifying the effect of mefenamic acid on the expression levels of VEGF and EMT markers in RPE cells.

[0068] Fig. 20 This is a graph showing the results of verifying the effect of serolast on the expression levels of VEGF and EMT markers in RPE cells.

[0069] Fig.21 This is a graph showing the results of verifying the CNV inhibitory effect of zaltoprofen by laser-induced fibrosis test.

[0070] Fig. 22 This is a fluorescent fundus photograph showing the results of verifying the CNV inhibitory effect of zaltoprofen by a laser-induced fibrosis test.

[0071] Fig.23This is a graph showing the results of verifying the EMT inhibitory effect of zaltoprofen by laser-induced fibrosis test.

[0072] Fig.24 The fluorescence micrographs show the results of verifying the EMT-inhibiting effect of zaltoprofen by laser-induced fibrosis test.

[0073] Fig.25 The fluorescence micrographs show the results of verifying the EMT-inhibiting effect of zaltoprofen by laser-induced fibrosis test.

[0074] Fig.26 The figure compares the stained photographs showing the morphology of RPE cells in drusen of AMD patients and the stained photographs showing the morphology of RPE cells of healthy subjects.

[0075] Fig. 27 The diagram compares immunostaining photographs showing the staining of epithelial markers in RPE cells in drusen of AMD patients with immunostaining photographs showing the staining of epithelial markers in RPE cells of healthy subjects.

[0076] Fig.28 The figure compares immunostaining photographs showing the staining of mesenchymal markers in RPE cells in drusen of AMD patients with immunostaining photographs showing the staining of mesenchymal markers in RPE cells of healthy subjects.

[0077] Fig.29 The diagram compares immunostaining photographs showing the staining of epithelial markers and mesenchymal markers in RPE cells of AMD model animals with immunostaining photographs showing the staining of epithelial markers and mesenchymal markers in RPE cells of healthy model animals.

[0078] Fig.30 These are graphs and photographs showing the results of verifying the induction of EMT in RPE cells by enhancing motility.

[0079] Fig.31 These are photographs showing the results of verifying the induction of EMT in RPE cells by the formation of drusen-like structures.

[0080] Fig.32 This is a graph showing how the enhancement of motility is suppressed by an EMT-inhibiting compound in an EMT-inducing model of RPE cells.

[0081] Fig.33 This is a graph showing how the enhancement of motility is suppressed by an EMT-inhibiting compound in an EMT-inducing model of RPE cells.

[0082] Fig.34This is a graph showing how the enhancement of motility is suppressed by an EMT-inhibiting compound in an EMT-inducing model of RPE cells.

[0083] Fig.35 These are photographs showing how an EMT-inhibiting compound inhibits the formation of drusen-like structures in an EMT-induced model of RPE cells.

[0084] Fig.36 These are photographs showing how an EMT-inhibiting compound inhibits the formation of drusen-like structures in an EMT-induced model of RPE cells.

[0085] Fig.37 These are photographs showing how an EMT-inhibiting compound inhibits the formation of drusen-like structures in an EMT-induced model of RPE cells.

[0086] Fig.38 These are photographs showing how an EMT-inhibiting compound inhibits the formation of drusen-like structures in an EMT-induced model of RPE cells.

[0087] Fig.39 These are photographs showing how an EMT-inhibiting compound inhibits the formation of drusen-like structures in an EMT-induced model of RPE cells.

[0088] Fig.40 The photographs show the results of induction of drusen-like structures in an in vivo model test for evaluating drusen inhibition.

[0089] Fig.41 The photographs show the staining results of E-cadherin in the induced drusen-like structures.

[0090] Fig.42 The photographs show the results of staining for cytokeratin in the induced drusen-like structures.

[0091] Fig.43 The photographs show the results of staining for fibronectin in the induced drusen-like structures.

[0092] Fig.44 The photographs show the staining results of vimentin in the induced drusen-like structures.

[0093] Fig.45 The photographs show the pharmacological evaluation of oxaprozin on the formation of drusen-like structures by HE staining (hematoxylin-eosin staining).

[0094] Fig.46 These are photographs showing the pharmacological evaluation of oxaprozin on the formation of drusen-like structures by immunostaining of E-cadherin.

[0095] Fig.47 These are photographs showing the pharmacological evaluation of oxaprozin on the formation of drusen-like structures by immunostaining of fibronectin.

[0096] Fig.48 These are photographs showing the pharmacological evaluation of epalrestat on the formation of drusen-like structures by HE staining.

[0097] Fig.49 These are photographs showing the pharmacological evaluation of epalrestat on the formation of drusen-like structures by immunostaining of E-cadherin.

[0098] Fig.50 These are photographs showing the pharmacological evaluation of epalrestat on the formation of drusen-like structures by immunostaining of fibronectin.

[0099] Fig.51 These are photographs showing the pharmacological evaluation of zaltoprofen on the formation of drusen-like structures by HE staining.

[0100] Fig.52 These are photographs showing the pharmacological evaluation of zaltoprofen on the formation of drusen-like structures by immunostaining of E-cadherin.

[0101] Fig.53 These are photographs showing the pharmacological evaluation of zaltoprofen on the formation of drusen-like structures by immunostaining of fibronectin. DETAILED DESCRIPTION

[0102] Hereinafter, the present invention will be described in detail.

[0103] [Choroidal neovascularization prevention and / or treatment agent]

[0104] The preventive and / or therapeutic agent for choroidal neovascularization of the present invention contains as an active ingredient at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof.

[0105] In this specification, "choroidal neovascularization (CNV)" refers to the generation, proliferation and / or expansion of new blood vessels from the choroid of the fundus. Although not limited to, the new blood vessels generated from the choroid extend through the layer where Bruch's membrane and / or retinal pigment epithelial cells exist. CNV can be examined by known methods. Although not limited to, CNV can be examined, for example, by fundus angiography using fluorescent pigments such as fluorescein.

[0106] In the present specification, the "retinal pigment epithelial cells (RPE cells)" refer to single-layer cuboidal epithelial cells present under the retina of the eye.

[0107] The inventor of the present invention etc. speculates that the epithelial-mesenchymal transition (EMT) in RPE cells is the main cause of CNV.In this specification, so-called " epithelial-mesenchymal transition (EMT) " refers to the phenomenon that the properties of epithelial cells are lost and the properties of mesenchymal system are obtained in epithelial cells.By removing the intercellular adhesion in epithelial cells, the morphology of cells changes, and obtains motor ability.As the expression of epithelial system markers expressed by epithelial cells is suppressed, the expression of mesenchymal system markers or the secretion of extracellular matrix (ECM) become hyperactive (in this specification, epithelial system markers, mesenchymal system markers and / or ECM are also referred to as EMT markers).

[0108] The inventor of the present invention etc. establishes the following hypothesis for the generation of CNV, and verifies.The inventor of the present invention etc. finds that if by certain external stimulation and produce EMT in RPE cells, then RPE cells can be degenerated and produce drusen.It is believed that the RPE cells peeled off from Bruch's membrane in the process of forming drusen are blocked due to the supply of oxygen and nutrition from the choriocapillary plate just below Bruch's membrane, therefore due to local hypoxia state, VEGF or a part of MMPs (matrix metalloproteinases; Matrix metalloproteinases) secretion hyperactivity.In addition, it is believed that even if it is not reached in the case of forming drusen, EMT will also be produced in RPE cells, thus making the secretion hyperactivity of the interstitial system marker and the extracellular matrix from RPE cells, and VEGF and a part of MMPs secretion hyperactivity.By these MMPs, decomposition and perforation are produced in Bruch's membrane, by VEGF, induce the angiogenesis from choroid.

[0109] Since the new blood vessels are fragile, blood can easily leak out. If the fragile new blood vessels rupture and cause subretinal hemorrhage, it will rapidly cause vision loss and lead to blindness.

[0110] Diseases associated with CNV (ICD10: International Classification of Diseases, 10th edition (revised in 2003)) include, for example, age-related macular degeneration, exudative age-related macular degeneration, drusen, exudative retinitis, polypoidal choroidal vasculopathy (PCV), exudative retinopathy, central serous chorioretinopathy, central serous choroidopathy, hemorrhagic retinal pigment epithelial detachment, proliferative vitreoretinopathy, proliferative retinopathy, serous retinal pigment epithelial detachment, idiopathic choroidal neovascularization, retinal edema, retinal angiogenesis, retinal angiomatous proliferation (RAP), retinal vasculopathy, atrophic age-related macular degeneration, angiogenic maculopathy, soft drusen, myopic choroidal neovascularization, macular degeneration, maculopathy, macular vascular ectopia, macular lesions, etc.

[0111] As a functional effect on these diseases, for example, age-related macular degeneration accompanied by subfoveal choroidal neovascularization can be mentioned.

[0112] In addition, examples of diseases related to CNV (standard disease name ICD10) include myopic choroidal neovascularization and idiopathic choroidal neovascularization.

[0113] As a functional effect on these diseases, for example, choroidal neovascularization in pathological myopia can be mentioned.

[0114] Examples of diseases associated with CNV (standard disease name ICD10) include diabetic macular edema, type 1 diabetic macular edema, type 1 diabetic maculopathy, type 2 diabetic macular edema, type 2 diabetic maculopathy, and diabetic maculopathy.

[0115] Examples of functional effects against these diseases include diabetic macular edema.

[0116] In addition, diseases related to CNV (standard disease name ICD10) include, for example, macular edema caused by retinal vein occlusion, central retinal vein thrombosis, central retinal vein thrombosis, central retinal vein occlusion, macular edema caused by central retinal vein occlusion, retinal vein thrombosis, retinal vein occlusion, etc.

[0117] As a functional effect on these diseases, for example, macular edema associated with retinal vein occlusion can be mentioned.

[0118] As for the classification of age-related macular degeneration (AMD), in addition to the general classification of dry AMD and wet AMD, there are also reports of classification of early AMD, intermediate AMD or late AMD, classification of non-progressive AMD or progressive AMD, etc. Although there is no limitation, as a special type of wet AMD, there is retinal angiomatous proliferation (RAP).

[0119] Although not limited to this, the new blood vessels in CNV may extend in various directions above Bruch's membrane or RPE cells, or may form polypoid new blood vessels under RPE cells, such as polypoidal choroidal vasculopathy (PCV).

[0120] Exudative AMD is known to be associated with subfoveal CNVs. This type of AMD is sometimes divided into three types: classic AMD, PCV, or RAP, each of which can be treated initially.

[0121] In the present specification, any retinal disease that causes CNV is a target of prevention and / or treatment regardless of the classification of the disease.

[0122] Among the classifications of AMD, wet AMD (exudative AMD) in particular causes CNV under the fovea due to bleeding or wetting, and subretinal hemorrhage is often seen around the CNV, so it has a strong correlation with CNV.

[0123] Based on this speculated mechanism of CNV generation, zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and their pharmaceutically acceptable salts respectively inhibit the production of VEGF or MMPs related to the occurrence or expansion of CNV by strongly inhibiting the EMT of RPE cells, thereby expecting to fundamentally prevent and / or treat CNV.

[0124] Can be measured or evaluated by known methods to produce EMT or EMT is suppressed. Although not limited, for example, the loss of epithelial traits can be confirmed by measuring the expression of the following epithelial markers. In addition, for example, the interstitial traits can be confirmed by measuring the expression of the following interstitial markers or the secretion of the extracellular matrix (ECM). The expression of genes, the expression or secretion of proteins can be measured by known methods such as microarray, real-time PCR (Polymerase chain reaction; Polymerase chain reaction) method, PCR method, protein blotting (western blot) method, ELISA (enzyme-linkedimmunosorbent assay; Enzyme-linked immunosorbent assay) method, immunohistostaining. In addition, for example, the hyperactivity of motor ability can be measured by known methods such as tissue staining such as Giemsa staining (Giemsa staining), invasion assay (Invasion Assay) method. In addition, for example, the morphological changes of cells can be measured by known methods such as tissue staining such as HE staining.

[0125] The term "prevention" as used herein refers to preventing the onset of a disease or symptom, delaying the onset of a disease, or preventing the recurrence of a disease in an individual.

[0126] The term "treatment" as used herein means alleviating the symptoms of a disease, eliminating the symptoms of a disease, or inhibiting the maintenance of the symptoms of a disease, or inhibiting the progression, aggravation, deterioration or aggravation of the symptoms in an individual.

[0127] In this specification, so-called " suppressing the EMT of RPE cells " refers to suppressing the situation of the proterties of the epithelial system losing in RPE cells, suppressing the situation of obtaining the proterties of the mesenchymal system, or making the situation of the proterties of the mesenchymal system obtained lose. Although not limited, for example, in RPE cells, the EMT of RPE cells can be evaluated by detecting the situation of maintaining the expression of epithelial system markers, suppressing the expression of the mesenchymal system markers, suppressing the expression and / or secretion of ECM, or suppressing the hyperactivity of motor ability.

[0128] Compounds with EMT inhibitory activity of RPE cells (also referred to as EMT inhibitory compounds in this specification) include compounds known to have inhibitory activity on EMT in epithelial cells. EMT inhibitory compounds include nonsteroidal anti-inflammatory agents, aldose reductase inhibitors, leukotriene receptor antagonists, chemical mediator release inhibitors and thromboxane A2 receptor antagonists; from the perspective of exerting a significant EMT inhibitory effect, zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotrast or their pharmaceutically acceptable salts can be used. From the perspective of the permeability of the blood-retinal barrier (BRB; Blood retinal barrier), EMT inhibitory compounds are preferably compounds with excellent factors related to BRB permeability such as fat solubility. Although not limited, compounds with high fat solubility are excellent in BRB permeability, so even in the case of oral administration that is not directly delivered to the eye, high prevention and high therapeutic effects on retinal diseases can be expected. In addition, from the viewpoint that long-term administration can be performed according to symptoms, the EMT inhibitory compound is preferably a compound with few side effects. As side effects, although not limited, examples include: loss of appetite, diarrhea, stomatitis, indigestion, gastritis, itching, abnormal liver function, etc. From the viewpoint of these BRB permeability and / or side effects, preferably zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotrast or their pharmaceutically acceptable salts. These components can be synthesized and used by known methods, or commercially available products can be purchased and used.

[0129] In the present specification, "pharmaceutically acceptable salts" are not particularly limited, and specifically, examples include organic acid salts, inorganic acid salts, organic bases, or inorganic bases. Examples of organic acid salts include monocarboxylates such as acetate, trifluoroacetate, butyrate, palmitate, and stearate; polycarboxylates such as fumarate, maleate, succinate, and malonate; hydroxycarboxylates such as lactate, tartrate, and citrate; and organic sulfonates such as methanesulfonate, toluenesulfonate, and p-toluenesulfonate. Examples of inorganic acid salts include hydrochlorides, sulfates, nitrates, hydrobromides, and phosphates. Examples of salts with organic bases include salts with organic amines such as methylamine, triethylamine, triethanolamine, diethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, picoline, and ethylenediamine. Examples of salts with inorganic bases include ammonium salts, salts with alkali metals such as sodium or potassium, alkaline earth metals such as calcium or magnesium, and various salts such as aluminum. These salts may be used alone or in any combination of two or more. "Pharmaceutically acceptable salts" may also include solvates or hydrates of salts.

[0130] The inventors of the present invention have found that at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof significantly inhibits the expression of mesenchymal markers or extracellular matrix (ECM).

[0131] In the present specification, the mesenchymal markers are not limited, and known mesenchymal markers can be used, for example, Snail (SNAI1), Slug (SNAI2), MMP1 (matrix metalloproteinase 1), MMP7 (matrix metalloproteinase 7), MMP2 (matrix metalloproteinase 2), MMP3 (matrix metalloproteinase 3), MMP9 (matrix metalloproteinase 9), N-cadherin (CDH2), CDH3, ZEB1, ZEB2 (SIP1), α-SMA (Alpha-Smooth Muscle Actin; α-smooth muscle actin), vimentin (VIM), FSP-1 (Fibroblast-Specific Protein-1; fibroblast-specific protein-1), β-catenin (β-catenin), OB-cadherin, α5β1 integrin (α5β1integrin), syndecan-1 (Syndecan-1), ETS (E-twenty six), Twist1, Goosecoid, LEF-1 (lymphoid enhancer factor-1 (lymphoid enhancer factor-1), FOXC2 (Forkhead box protein C2), miR10b, miR21, RDX (Radixin), RHOA (ras homolog gene family, member A), TJP1 (Tight Junction Protein 1), CTNNB1, HAS2 (hyaluronan synthase 2), SERPINE1 (Plasminogen activator inhibitor 1), MSN (Moesin), TCF3 (transcription factor 3), ITGAV (Integrin Subunit Alpha V), etc. In order to evaluate EMT inhibition, these mesenchymal markers can be used alone or in combination.

[0132] Since EMT inhibition in RPE cells can be effectively evaluated, the mesenchymal marker is preferably at least one selected from the group consisting of Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2 and VIM, and more preferably at least one selected from the group consisting of Snail, Slug, CDH3, MMP1 and MMP7.

[0133] In the present specification, ECM is not limited, and known ECM can be used as a marker, for example: 5α3 collagen (COL5A3), COL6A3, laminin γ2 (LAMC2), hyaluronan-mediated motility receptor (HMMR), tenascin C (TNC), fibronectin 1 (FN1), COL1A1, COL1A2, COL5A1, COL5A2, COL11A2, COL13A1, COL16A1, COL27A1, chondroitin sulfate proteoglycan 4 (CSPG4; Chondroitin Sulfate Proteoglycan 4), laminin β3 (LAMB3), serglycin (SRGN; serglycin), SPARC (Secreted Protein Acidic and Rich in Cysteine; cysteine-rich acidic secretory glycoprotein), etc. In order to evaluate EMT inhibition, these ECMs may be used alone or in combination as a marker.

[0134] Since EMT inhibition in RPE cells can be effectively evaluated, ECM is preferably at least one selected from the group consisting of COL5A3, COL6A3, laminin γ2 (LAMC2), hyaluronan-mediated motility factor receptor (HMMR), tenascin C (TNC), COL1A1, COL1A2, SRGN, FN1, COL5A2, COL13A1 and LAMB3; more preferably at least one selected from the group consisting of COL5A3, COL6A3, laminin γ2 (LAMC2), hyaluronan-mediated motility factor receptor (HMMR) and tenascin C (TNC).

[0135] In addition, in the present specification, epithelial markers are not limited, and known epithelial markers can be used, for example, ID1, ID2, MUC1, cytokeratin 18 (KRT18; Keratin 18), THBS1 (thrombospondin 1; thrombospondin 1), VIL2 (villin 2), E-cadherin (CDH1), etc. These epithelial markers can be used alone or in combination.

[0136] In this specification, as an EMT inhibitory compound, in addition to zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, seroquel or their pharmaceutically acceptable salts, compounds or components known to have inhibitory activity on EMT in epithelial cells may also be included. Although not limited, for example, can be exemplified: Alisertib, MK-0457 (Tozasertib), PHA-739358 (danusertib), AMG-900, Barasertib, CYC116, MLN8054, Baicalin, Baicalein, Lupeol, Istanbulin A (9a-hydroxy-3,4a,5-trimethyl-4,5,6,7,8a,9-hexahydrobenzo[f][1]benzofuran-2,8-dione), Phytol, EF24, Crucmin, Phloroglucinol, Plumbagin, Rapamycin, FK506 (Tacrolimus), Thalidomide, LY550410, SB-505124, SD-208, TAPI-0, TAPI-1, JNJ-38877605, PF-04217903, AG1478 (Tyrphostin), Erlotinib, Gefitinib, Lapatinib, PD153035, PD158780, WHI-P154, BMS-536924, A83-01, D4476, LY-364947, SB-431542, SD-208, AZD6244 (Selumetinib), CI-1040, PD0325901, GDC-0941 (Pictilisib (4-[2-(1H-indazole-4- 1-[(4-methylsulfonylpiperazin-1-yl)methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine), PI-103, PIK-90, ZSTK474, API-2, AZD0530 (Saracatinib), PP1, 2-Hydroxycinnamaldehyde, 5-aza-dC, BI 5700, Celecoxib, CX-4945 (Silmitasertib (5-[(3-chlorophenyl)amino]benzo[c][2,6] naphthyridine-8-carboxylic acid), disulfiram, eribulin mesyate, evodiamine, EW-7203, fasudil, nintedanib, fuzheng huayu recipe, grape seed proanthocyanidins, vorinostat, herbimycin A, entinostat, honokiol, NPI-0052, methacycline, dasatinib, Ki26894, NSC 74859, NVP-LDE-225 (Erismodegib), Palbociclib, Pinocembrin, Salvianolic Acid B, Sorafenib, Resveratrol, S-Allylcysteine, Silibinin meglumine, Simvastatin, Centchroman, ML327, GN-25, Trichostatin A, Sarasinoside A1, Panobinostat, Danusertib, Cystatin C, Thymoquinone, Ulinastatin, Dendrofalconerol A (DF-A), ginsenoside (ginseng saponin), Celastrus orbiculatus seed extract, salicin (Willow extract), salicylic acid, scutellaria extract, osthole, muscadine grape skin extract, Tongxinluo, procyanidin C1 (cinnamon), Withania somnifera root extract, Qingyihuaji, roselle extract, epigallocatechin gallate, proanthocyanidins (grape seed extract), salvianolic acid B, etc.

[0137] [Exudative age-related macular degeneration prevention and / or treatment agent]

[0138] The existing angiogenesis inhibitor used in wet AMD is centered on the medicine using antibodies, so there is a problem that treatment costs become expensive. Especially in the treatment of wet AMD, if administration is stopped, angiogenesis will occur again, and the risk of blindness will increase. By using the present invention, the dosage, dosing interval, and administration number of angiogenesis inhibitors can be suppressed, and the effect on medical economy can be expected. By the present invention, for the situation of suppressing the EMT in RPE cells, it can be expected to prevent dry AMD from shifting to wet AMD, or it can also be expected that wet AMD is cured. By the present invention, owing to being able to suppress the EMT in RPE cells, the present invention can also be used as VEGF production inhibitor or VEGF expression inhibitor.

[0139] [EMT induction model]

[0140] The inventors of the present invention have developed a method for inducing an EMT model in vitro by including a process for imparting stimulation to epithelial cells. By using an EMT induction model, the effects produced by EMT in various epithelial cells can be evaluated. In addition, it is possible to evaluate and / or screen whether a medicament inhibits EMT in various epithelial cells.

[0141] Epithelial cells are not particularly limited as long as they are known epithelial cells, and with regard to the viewpoint of the impact produced by EMT, epithelial cells preferably use RPE cells, more preferably use human RPE cells, and further preferably use ARPE-19 as RPE cell strain. The EMT induction model using this RPE cell can evaluate EMT in aspects such as gene expression, secretion of protein, motor ability, agglomeration formation.

[0142] The stimulation given to epithelial cells can be exemplified by stimulation produced by drugs, stimulation produced by mechanical operation, stimulation produced by electrical operation, etc., and there is no particular limitation. From the perspective of significantly inducing EMT, it is preferably a stimulation that makes the drug contact cells. As the aforementioned drug, there is no limitation, and it is preferably at least one selected from the group consisting of cytokines, growth factors and chemokines.

[0143] As the cytokine used for stimulation, known substances can be used, for example, interleukin 1β (IL-1β), IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-16, tumor necrosis factor-α (TNF-α), interferon γ (INF-γ), etc. From the viewpoint that EMT can be significantly induced, the cytokine is preferably selected from at least one of the group consisting of IL-1β, IL-6, INF-γ and TNF-α, more preferably selected from at least one of the group consisting of IL-1β, IL-6 and TNF-α, and further preferably IL-1β and / or TNF-α. The cytokine can be used alone or in combination.

[0144] As the growth factor used for stimulation, known substances can be used, for example, transforming growth factor (TGF-β; Transforming growth factor-β), fibroblast growth factor (fibroblast growth factors; FGFs), hepatocyte growth factor (hepatocyte growth factor; HGF), epidermal growth factor (epidermal growth factor; EGF), platelet-derived growth factor (platelet-derived growth factor; PDGF), etc. From the viewpoint of being able to significantly induce EMT, the growth factor is preferably at least one selected from the group consisting of TGF-β, EGF and FGFs, and more preferably TGF-β and / or EGF. The growth factor can be used alone or in combination.

[0145] As the chemokine used for stimulation, known substances can be used, for example, IL-8, macrophage inflammatory protein 1α (MIP-1α; macrophage inflammatory protein 1-alpha), monocyte chemoattractant protein-1 (MCP-1; monocyte chemoattractant protein 1), RANTES (regulated on activationnormal T cell expressed and secreted; regulating activation of normal T cell expression secretion factor), eotaxin, etc. From the viewpoint of significantly inducing EMT, chemokine is preferably selected from at least one of the group consisting of MIP-1α, MCP-1 and RANTES, more preferably MIP-1α and / or MCP-1. Chemokine can be used alone or in combination.

[0146] The aforementioned stimulation may be used in combination with, for example, at least one cytokine selected from the group consisting of IL-1β, IL-6, INF-γ and TNF-α, at least one growth factor selected from the group consisting of TGF-β, EGF and FGFs, and at least one chemokine selected from the group consisting of MIP-1α, MCP-1 and RANTES.

[0147] The concentration of cytokines, growth factors, or chemokines during stimulation can be appropriately adjusted by the type of epithelial cells, the state of the cells, the type of cytokines, etc., for example, preferably 1 ng / mL or more, more preferably 5 ng / mL or more; more preferably 10 ng / mL or more, particularly preferably 15 ng / mL or more, most preferably 20 ng / mL or more. In addition, the concentration of cytokines, growth factors, or chemokines is preferably 1 μg / mL or less, more preferably 700 ng / mL or less, further preferably 500 ng / mL or less, particularly preferably 200 ng / mL or less, and most preferably 100 ng / mL or less. In addition, the concentration of cytokines, growth factors, or chemokines is preferably 1 ng / mL to 1 μg / mL, more preferably 5 ng / mL to 700 ng / mL, further preferably 10 ng / mL to 500 ng / mL, particularly preferably 15 ng / mL to 200 ng / mL, and most preferably 20 ng / mL to 100 ng / mL.

[0148] Although not limited, when the cytokine is IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, or IL-16, the concentration of the cytokine is preferably 1 ng / mL to 1 μg / mL, more preferably 5 ng / mL to 700 ng / mL, further preferably 10 ng / mL to 500 ng / mL, particularly preferably 15 ng / mL to 200 ng / mL, and most preferably 20 ng / mL to 100 ng / mL.

[0149] In addition, although not limited, when the cytokine or chemokine is INF-γ, TNF-α, or MIP-1α, its concentration is preferably 10 ng / mL to 1 μg / mL, more preferably 20 ng / mL to 800 ng / mL, further preferably 30 ng / mL to 700 ng / mL, particularly preferably 40 ng / mL to 600 ng / mL, and most preferably 50 ng / mL to 500 ng / mL.

[0150] In addition, although not limited, when the growth factor is TGF-β or EGF, the concentration of the growth factor is preferably 1 ng / mL to 200 ng / mL, more preferably 2 ng / mL to 100 ng / mL, further preferably 3 ng / mL to 50 ng / mL, particularly preferably 4 ng / mL to 600 ng / mL, and most preferably 5 ng / mL to 25 ng / mL.

[0151] The reaction time during stimulation can be appropriately adjusted by the type of epithelial cells, the state of cells, the type of cytokines, etc., for example, it can be set to at least 5 minutes or more, and with respect to the viewpoint of losing epithelial properties in EMT and obtaining interstitial properties, it is preferably set to at least 10 minutes or more, more preferably set to at least 1 hour or more, further preferably set to at least 10 hours or more, particularly preferably set to at least 24 hours or more, and most preferably set to at least 48 hours or more. In addition, the reaction time during stimulation can be set to, for example, within 120 hours, and with respect to the viewpoint of losing epithelial properties in EMT and obtaining interstitial properties, it is preferably set to within 108 hours, more preferably set to within 96 hours, further preferably set to within 84 hours, particularly preferably set to within 72 hours, and most preferably set to within 60 hours. In addition, the reaction time during stimulation can be set, for example, to 5 minutes to 120 hours. From the perspective of losing epithelial properties and acquiring mesenchymal properties in EMT, it is preferably set to 10 minutes to 108 hours, more preferably to 1 hour to 96 hours, further preferably to 10 hours to 84 hours, particularly preferably to 24 hours to 72 hours, and most preferably to 48 hours to 60 hours.

[0152] The reaction temperature during stimulation can be appropriately adjusted according to the type of epithelial cells, the state of the cells, the type of cytokines, etc., and can be set, for example, to 4°C to 50°C, preferably 10°C to 45°C, more preferably 20°C to 40°C, and even more preferably 30°C to 37°C.

[0153] Evaluation whether the method for EMT is induced to cells is not limited, and can be exemplified: measure the expression (secretion) of at least one selected from the group consisting of epithelial markers, mesenchymal markers and ECM before and / or after epithelial cell stimulation; Observe the morphological changes of cells; Or measure the change of the motility of cells. Although not limited, if RPE cells are induced to EMT, then epithelial markers that are suppressed can be confirmed to be hypertrophic, hypermotility and / or cell morphology are deformed into spindles, and cells condense each other and form phenomena such as drusen-like structures (Focus).

[0154] Gene expression or protein expression or secretion can be measured by known methods such as microarray, real-time PCR, PCR, Western blotting, ELISA, and immunohistochemical staining. Changes in motility can be measured by known methods such as tissue staining such as Giemsa staining, invasion assays, etc. In addition, for example, morphological changes in cells can be measured by known methods such as tissue staining such as HE staining.

[0155] [Evaluation of CNV inhibition]

[0156] Can use EMT induction model, evaluate the inhibition of CNV in vitro.That is, after inducing EMT to RPE cells, compare the expression of VEGF in the presence of medicament and non-presence, thus can evaluate the CNV inhibition produced by medicament suppressing the expression of VEGF in RPE cells.About the determination of the state of EMT, for example, can exemplify the expression (secretion) of at least one selected from the group consisting of epithelial marker, mesenchymal marker and ECM.If the EMT in RPE cells is suppressed, then mesenchymal marker and / or ECM are suppressed.EMT marker or VEGF expression, for example, can be evaluated by the expression of mRNA (messengerribonucleic acid;Messenger RNA).

[0157] The step of extracting RNA from cells can use a known RNA extraction method, and preferably use a commercially available RNA extraction kit or the like.

[0158] The step of quantitative analysis of mRNA expression is not limited, preferably using real-time PCR method.As the EMT marker measured by real-time PCR method, the aforementioned epithelial marker, mesenchymal marker, ECM and other EMT markers can be used;With regard to the viewpoint of the EMT marker measured by real-time PCR method using good reproducibility, as epithelial marker, preferably ID1, ID2, MUC1, cytokeratin 18 (KRT18), THBS1, VIL2, E-cadherin (CDH1), more preferably MUC1, cytokeratin 18 (KRT18), E-cadherin (CDH1).As mesenchymal marker, preferably Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2, vimentin, more preferably Snail, Slug, CDH3, MMP1, MMP7, vimentin. In addition, as ECM, from the viewpoint of using an EMT marker that can be measured with good reproducibility by a real-time PCR method, preferably COL5A3, COL6A3, LAMC2, HMMR, TNC, COL1A1, COL1A2, CSPG4, SRGN, FN1, VIM, COL5A2, COL13A1, and LAMB3, and more preferably COL5A3, COL6A3, LAMC2, HMMR, TNC, and FN1.

[0159] [Drusen Inhibitors]

[0160] The drusen inhibitor of the present invention contains a compound having an epithelial-mesenchymal transition (EMT) inhibitory activity of retinal pigment epithelial cells (RPE cells) as an active ingredient.

[0161] In this manual, so-called " drusen " refers to the deposit that is present in RPE cell below or around RPE cell that also visible increases change along with age for healthy elderly.Histologically, it is the deposition of the polymorphic material in the collagen fiber layer between the basement membrane of RPE cell and Bruch's membrane, and constituent components are the secretion of cell components such as organelles, cell membrane sample residues, non-esterified cholesterol, complement, RPE cell etc.The deposit that forms the macula of drusen has the basement membrane deposit (basal laminar deposit) that is present in the basement membrane inboard of RPE cell and the basal line deposit (basal linear deposit) that is present in the outside under the electron microscope.

[0162] One theory holds that, for humans, drusen can be classified into hard drusen (less than 63 μm) and soft drusen (more than 63 μm) based on the results of funduscopy. If soft drusen fuse, it is called confluent drusen. When drusen fuse and expand to cause retinal pigment epithelial detachment (PED), it is called drusenoid PED. In addition, in recent years, it has been reported that pseudodrusen, which is classified as another type of drusen, can affect prognosis, and is therefore clinically important. In this specification, "drusen" includes not only hard drusen and soft drusen, but also pseudodrusen and other deposits or structures similar to drusen.

[0163] The diagnostic criteria of AMD in Japan include soft drusen (more than 63 μm) and RPE cell abnormalities as precursor lesions. In the past, the formation mechanism of drusen was not known, and it was difficult to prevent or treat fundamentally. When completing the present invention, the inventors of the present invention etc. obtained the following new insights: drusen is a structure produced by RPE cells causing EMT.

[0164] The inventors of the present invention predict that epithelial cells headed by RPE cells will change their properties due to external stimuli such as aging, UV (Ultra Violet; ultraviolet) light, and obtain properties (referred to as interstitial properties) such as the acquisition of hypersecretion and motor ability of the extracellular matrix. It is strongly inferred that drusen is generated by the property conversion of the retinal pigment epithelial cells produced in the macula.

[0165] Therefore, the inventors used a retinal pigment epithelial cell line (ARPE-19) and confirmed that the cells acquired traits such as hypersecretion of the extracellular matrix and acquisition of motility based on the transformation of their traits, and ultimately formed aggregates (also called "Focus") equivalent to drusen composed of cellular components and their secretions. While verifying the mechanism of its occurrence, they obtained confirmation that the drusen-like structure (aggregate (Focus)) generated in the same process is equivalent to the drusen produced in a living body.

[0166] In the present specification, the so-called "drusen inhibition" refers to reducing the number of drusen formed or decomposing the drusen formed, or reducing the size of the drusen formed (which can be appropriately specified according to the diameter, area, volume, etc.) or preventing the increase, and also includes preventing the formation of new drusen. Although not limited, in the case of reducing the number of drusen formed, for example, compared with a control not treated with a drug, the number of drusen is reduced by at least 5%, preferably by more than 10%, more preferably by more than 20%, particularly preferably by more than 30%, and most preferably by more than 40%. The number of drusen can be evaluated by a known method, for example, using a fundus photograph. In addition, although not limited, in the case of reducing the size of the drusen formed or preventing the increase, for example, compared with a control not treated with a drug, at least the size of the drusen does not change, preferably by more than 5%, more preferably by more than 10%, more preferably by more than 20%, particularly preferably by more than 30%, and most preferably by more than 40%. The size of drusen can be evaluated by known methods, for example, fundus photographs, spectral domain optical coherence tomography (SD-OCT) examination results, fluorescent fundus angiography using fluorescein or indocyanine green, etc. In addition, although not limited, in the case of preventing the formation of new drusen, for example, compared with a control not treated with a drug, the formation of at least 5% or more of new drusen is prevented, preferably 10% or more, more preferably 20% or more, particularly preferably 30% or more, and most preferably 40% or more. The formation of new drusen can be evaluated by known methods, for example, fundus photographs, fluorescent fundus angiography, etc. The evaluation method for drusen inhibition is not particularly limited as long as it can be appropriately evaluated, and preferably a comprehensive evaluation is performed based on the number, thickness, and volume of drusen.

[0167] The formation of drusen is related to the progression of AMD, that is, the reduction of visual field or vision. One theory reports that the increase in the number of drusen on the retina and the progression and size of each drusen lead to the progression of dry AMD, and symptoms such as map-like atrophy are observed. In addition, with the formation of drusen, new blood vessels are generated in the choroid under the drusen, and retinal edema and subretinal fluid retention characteristic of wet AMD can be seen. There are reports that the new blood vessels are fragile, and if they rupture, there are cases where AMD is transferred from dry AMD to wet AMD.

[0168] In addition, it is known that VEGF (vascular endothelial growth factor), which is an inducing factor for neovascularization, and VEGFR (vascular endothelial growth factor receptor), which is a VEGF receptor, are expressed more frequently when cells undergo EMT. However, based on the new insight that drusen formation in dry AMD is caused by EMT, it is speculated that the formation of drusen in dry AMD will also induce neovascularization.

[0169] Therefore, it is believed that the inhibition of drusen is related to the prevention and treatment of wet AMD in addition to dry AMD, and is also related to the prevention and treatment of angiogenesis in dry AMD, which is the pre-stage of transition to wet AMD.

[0170] In addition, the inventors of the present invention have established the following hypothesis: one of the reasons for the very slow development of AMD is the balance between the newly generated drusen and the decomposed (inhibited) drusen. Below, it will be specifically described. Even if it is a healthy person, drusen will be generated due to age increase change, and since decomposition (inhibition) is dominant, during the period of maintaining this equilibrium relationship, the development of AMD is not easy. However, if this equilibrium relationship is unbalanced, the generation of drusen is dominant than decomposition (inhibition), and drusen slowly remains, the development of AMD. The inventors of the present invention speculate that the main factor that strongly affects this equilibrium relationship is EMT. That is, for healthy people, since EMT is suppressed, RPE cells maintain the original epithelial properties, but if the hyperactivity of EMT is dominant due to age increase or external stimulation, RPE cells lose the epithelial properties and become the properties of the interstitial system. As shown in the embodiment described later, in the RPE cells that have obtained the properties of the interstitial system, the generation of ECM such as collagen as the constituent elements of drusen is hyperactive. On the other hand, in organisms, although these ECMs are decomposed by degrading enzymes MMP (matrix metalloproteinases), the expression of TIMP (tissue inhibitors of metalloproteinases; tissue inhibitors of metalloproteinases) that inhibit the activity of MMP due to the hyperactivity of EMT is also hyperactive. By suppressing the activity of MMP, ECM is not decomposed, and the decomposition of drusen is suppressed. As a result, drusen is hyperactive due to the hyperactivity of EMT, causing AMD to develop.

[0171] In addition, by inhibiting EMT, the expression of ECM such as collagen, which is a component of drusen, is inhibited, and the expression of TIMP is inhibited, so that the activity of MMP and the like is significantly expressed, and the decomposition of ECM is promoted. As a result, the generation of drusen is inhibited, and the development of AMD is inhibited, and the cure of AMD caused by the significant decomposition of drusen can be expected. The inventors of the present invention speculate that in the organism, due to the hyperactivity of EMT, the inhibitory mechanism caused by TIMP plays a role, so that the generation of drusen becomes dominant, and the condition of AMD slowly develops.

[0172] Based on this inferred mechanism of AMD development, the overexpression of factors related to the traits of the mesenchymal system can be inhibited by using external factors to inhibit EMT, and / or the mechanism of decomposing drusen in the organism can be fully exerted to improve the balance between the generation and decomposition of drusen. The inventors of the present invention have established a cell model that can reproduce drusen in an organism, and used the cell model to evaluate and screen compounds that inhibit EMT, and also verified whether the compound can inhibit drusen.

[0173] As the classification of AMD, in addition to the general division of dry AMD and wet AMD, the classification of early AMD, intermediate AMD or late AMD, the classification of non-progressive AMD or progressive AMD, etc. are also reported. In this specification, as long as it is AMD related to the onset and aggravation of drusen, it becomes the object of prevention and / or treatment regardless of which classification it is.

[0174] The classification of early AMD, intermediate AMD or late AMD is advocated by the NIH (National Institutes of Health) (refer to NIH Eligibility Categories: A Randomized, Placebo-Controlled, Clinical Trial of High-Dose Supplementation With Vitamins C and E, Beta Carotene, and Zinc for Age-Related Macular Degeneration and Vision Loss Arch Ophthalmol. 2001 October; 119(10): 1417-1436). This classification classifies AMD into no AMD (category 1), early AMD (category 2), intermediate AMD (category 3a, category 3b), and late AMD (category 4a, category 4b) based on the size of drusen in one eye, the area of ​​drusen, and pigment abnormalities, and the symptoms of the other eye.

[0175] No AMD (Class 1) means that there are no drusen in one eye or the size of drusen is small (less than 63 μm), the area of ​​drusen is a circle with a diameter of less than 125 μm (5 to 15 drusen), there is no pigment abnormality, and the same symptoms are present in the other eye. According to this classification, since drusen are produced even in No AMD (Class 1), they can be the subject of prevention and / or treatment using the drusen inhibitor of the present invention.

[0176] For early AMD or intermediate AMD, which is classified as a symptom in which the size and area of ​​drusen further increase, it is estimated that the drusen inhibitor of the present invention will be more effectively used.

[0177] Based on the new understanding that drusen are structures generated by RPE cells inducing EMT, it is understood that drusen can be suppressed by inhibiting EMT in RPE cells.

[0178] As previously mentioned, the compound with the EMT inhibitory activity of RPE cells (also referred to as EMT inhibitory compound in this specification) includes a compound known to have the inhibitory activity to EMT in epithelial cells. As the compound for suppressing EMT in RPE cells, the compound for observing EMT inhibition is also included in the EMT induction model newly found in the present invention, using RPE cell strain.

[0179] Although not limited, the EMT-inhibiting compound may be an agent that inhibits the expression of a mesenchymal lineage marker or an extracellular matrix (ECM).

[0180] Regarding the drusen inhibitor of the present invention, the EMT inhibitory compound is not limited and may be a non-steroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor and / or a thromboxane A2 receptor antagonist.

[0181] The nonsteroidal anti-inflammatory agents (NSAIDs) are not limited, and examples thereof include oxaprozin, zaltoprofen, pranoprofen, alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, teprofenic acid, naproxen, flunoxaprofen, flurbiprofen, flurbiprofen axetil, fenoprofen calcium, naproxen, piketoprofen, pirprofen, protizinic acid, and tadalafil. Propionic acid NSAIDs such as suprofen, tiaprofenic acid, ximoprofen, and loxoprofen sodium;Aceclofenac, Acemetacin, Alclofenac, Amfenacsodium, Antolmetinguacil, Bufexamac, Cinmetacin, Clopirac, Felbinac, Fenclozic acid, Fentiazac, Glucametacin, Ibufenac, Indometacin, Indomethacin farnesil, Isofezolac, Isoxepac, Lonazolac, Metiazinic acid acid, oxametacin, pirazolac, proglumetacin, tiaramide, tolmetin, tropecin, zomepirac, etodolac, diclofenac sodium, sulindac, nabumetone, fenbufen, proglumetacin maleate, mofezolac and other arylacetic acid-based NSAIDs; enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, flufenamate aluminum, mefenamic acid, niflumic acid aminoaryl carboxylic acid-based NSAIDs such as talniflumate, terofenamate, and tolfenamic acid; aryl butyric acid-based NSAIDs such as bumadizon, butibufen, fenbufen, and xenbucin; aryl carboxylic acid-based NSAIDs such as clidanac, ketorolac, and tinoridine;Acetaminosalol, benoylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, arginine acetylsalicylate, mesalazine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide O-acetic acid, salicylsulfuric acid Salicylic acid NSAIDs such as salsalate, sulfasalazine, etc.; fenamic acid NSAIDs such as mefenamic acid, flufenamic acid, tolfenamic acid, etc.; pyrimidine NSAIDs such as bucolome; oxicams NSAIDs such as ampiroxicam, tenoxicam, piroxicam, meloxicam, lornoxicam, droxicam, isoxicam, etc.; pyrazole NSAIDs such as epirizole, difenamizole, etc.; azapropazone, etc. e), pyrazolone-based NSAIDs such as benzpiperylone, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, and thiazolinobutazone; basic NSAIDs such as thiazolamide hydrochloride and emorfazone, or pharmaceutically acceptable salts thereof. These NSAIDs may be used alone or in any combination of two or more. These NSAIDs may be synthesized by a known method or may be purchased as commercial products. ;

[0182] Among these NSAIDs, from the viewpoint of exerting a significant EMT inhibitory effect, propionic acid NSAIDs, aminoarylcarboxylic acid NSAIDs or arylacetic acid NSAIDs are preferred, propionic acid NSAIDs, fenamic acid NSAIDs or arylacetic acid NSAIDs are more preferred, at least one selected from the group consisting of zaltoprofen, oxaprozin, teipfenic acid, ibuprofen, flurbiprofen, flufenamic acid, mefenamic acid, sulindac and pharmaceutically acceptable salts thereof is more preferred, and at least one selected from the group consisting of zaltoprofen, oxaprozin, flufenamic acid, mefenamic acid and pharmaceutically acceptable salts thereof is further preferred.

[0183] The aldose reductase inhibitor is not limited, and examples thereof include epalrestat, zenarestat, statiel, sorbinil, etc. These aldose reductase inhibitors may be used alone or in any combination of two or more. These aldose reductase inhibitors may be synthesized by a known method and used, or may be purchased as a commercial product and used.

[0184] Among these aldose reductase inhibitors, epalrestat and / or a pharmaceutically acceptable salt thereof is preferred from the viewpoint of exhibiting a significant EMT inhibitory effect.

[0185] Leukotriene receptor antagonists are not limited, and examples thereof include antagonists of cysteinyl leukotriene type 1 (CysLT1; Cysteinyl leukotriene 1) receptors such as zafirlukast, montelukast, and pranlukast, and 5-lipoxygenase inhibitors such as zileuton. These leukotriene receptor antagonists may be used alone or in any combination of two or more. These leukotriene receptor antagonists may be synthesized and used by known methods, or may be purchased as commercial products and used.

[0186] Among these leukotriene receptor antagonists, from the viewpoint of exerting a significant EMT inhibitory effect, preferred are antagonists of cysteinyl leukotriene type 1 (CysLT1) receptors such as zafirlukast, montelukast, and pranlukast, more preferably at least one selected from the group consisting of zafirlukast, montelukast, pranlukast, and pharmaceutically acceptable salts thereof, and further preferably zafirlukast and / or its pharmaceutically acceptable salts.

[0187] The chemical mediator release inhibitor is not limited, and for example, can be exemplified by: amlexanox, sodium cromoglycate, pemirolast potassium, ibudilast, etc. These chemical mediator release inhibitors can be used alone or in combination of two or more. These chemical mediator release inhibitors can be synthesized by a known method and used, or commercial products can be purchased and used.

[0188] Among these chemical mediator release inhibitors, amlexanox and / or a pharmaceutically acceptable salt thereof is preferred from the viewpoint of exhibiting a significant EMT inhibitory effect.

[0189] Thromboxane A2 antagonists are not limited, for example, thromboxane A2 receptor antagonists or thromboxane A2 synthetase inhibitors can be cited. As thromboxane A2 receptor antagonists, for example, serostat or Ramatroban can be cited, and as thromboxane A2 synthetase inhibitors, for example, Ozagrel hydrochloride can be cited. These thromboxane A2 antagonists can be used alone or in combination of two or more. These thromboxane A2 antagonists can be synthesized and used by known methods, or they can be purchased and used as commercial products.

[0190] Among these thromboxane A2 antagonists, seratrolast and / or a pharmaceutically acceptable salt thereof is preferred from the viewpoint of exhibiting a significant EMT inhibitory effect.

[0191] [Agent for preventing and / or treating age-related macular degeneration]

[0192] The drusen inhibitor of the present invention is mainly used as a preventive and / or therapeutic agent for age-related macular degeneration. As such an agent for preventing and / or treating age-related macular degeneration, it contains a propionic acid-based non-steroidal anti-inflammatory agent, an aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent, an arylacetic acid-based non-steroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor, or a thromboxane A2 receptor antagonist as an active ingredient, and the aldose reductase inhibitor can use epalrestat and / or its pharmaceutically acceptable salt. Such an active ingredient refers to the ingredients described in the item [drusen inhibitor].

[0193] [Combination use with angiogenesis inhibitors]

[0194] The present invention provides a preventive and / or therapeutic agent for AMD comprising a combination of an angiogenesis inhibitor and the drusen inhibitor of the present invention.

[0195] In this specification, the so-called "angiogenesis inhibitor" refers to an agent having an effect of preventing angiogenesis, an agent having an effect of preventing angiogenesis from worsening, or an agent having an effect of reducing or eliminating the formed new blood vessels. As angiogenesis inhibitor, there is no limitation, and examples thereof include: anti-VEGF antibody or a fragment of the VEGF antibody (Fab fragment, single-chain variable region fragment (scFv), etc.), a fusion protein of a fragment of the anti-VEGF antibody and the Fc region of the IgG molecule, and an anti-angiogenesis aptamer (RNA aptamer, etc.). Angiogenesis inhibitors can also use known angiogenesis inhibitors that are already on the market, and there is no limitation, for example, examples thereof include: bevacizumab (Avastin (registered trademark)), ranibizumab (Lucentis (registered trademark)), aflibercept (Eylea (registered trademark)), pegaptanib sodium (Macugen (registered trademark)), ESBA-1008, Lampalizumab, MC-1101, doxycycline hydrochloride (Doxycycline Hyclate), and amitraz hydrochloride. Specifically, the aforementioned EMT-inhibiting compound and angiogenesis inhibitor can be used in appropriate combination in consideration of the patient's symptoms and condition, the expected therapeutic effect, and the like.

[0196] About AMD, it is known that angiogenesis occurs in the choroid under the formed drusen, and it is inferred that the angiogenesis under the drusen is inhibited and interacts with the inhibition of drusen, and obtains the prevention and treatment effect of excellent AMD. As the classification of angiogenesis in AMD, wet AMD, early stage and / or mid-term AMD can be exemplified. About early stage and / or mid-term AMD, it is inferred that except the quantity or size increase of drusen, fragile new blood vessels are also generated. In addition, about dry AMD, it is inferred that angiogenesis can also be induced when drusen is produced. Due to the exudation of blood components from this fragile new blood vessel, or the rupture of fragile blood vessels, the transfer to wet AMD can be rapidly carried out. In addition, it is believed that it is important to preventively treat from the early stage AMD that will produce tiny fragile blood vessels. Therefore, in the present invention, drusen inhibitor is by using with angiogenesis inhibitor, is preferably used for the prevention and / or treatment of wet AMD, more preferably used for the prevention and / or treatment of mid-term AMD, further preferably used for the prevention and / or treatment of early stage and / or mid-term AMD.

[0197] By using the drusen inhibitor of the present invention as a combined drug, the dosage, dosing interval, and dosing frequency of the angiogenesis inhibitor can be suppressed, and medical economic effects can be expected.

[0198] In addition, in one embodiment, the drusen inhibitor of the present invention can be used in combination with photodynamic therapy (PDT) for treatment. In photodynamic therapy, drugs such as Verteporfin that selectively transfer to new blood vessels are used, but it is known that laser irradiation will more or less cause damage to the normal choroid. Therefore, by reducing the dosage of drugs such as Verteporfin in photodynamic therapy, or reducing the irradiation time and irradiation amount of the laser, and using the drusen inhibitor of the present invention, effective prevention and / or treatment of AMD can be performed.

[0199] In addition, in one embodiment, the drusen inhibitor of the present invention may be used for the prevention and / or treatment of AMD by appropriately combining an angiogenesis inhibitor with photodynamic therapy.

[0200] The EMT induction model using RPE cells described in the item [EMT induction model] shows the same characteristics as drusen of AMD in terms of gene expression, protein secretion, motility, aggregate formation, etc., and can therefore be effectively used as a cell model of AMD.

[0201] [Evaluation or screening methods for test substances]

[0202] The aforementioned EMT induction model can be used to evaluate drug sensitivity in vitro. In addition, the EMT induction model of the present invention is useful for evaluating or screening drusen inhibitors.

[0203] Although not limited, for example, the EMT induction model of RPE cells can be used, drusen is reproduced in vitro, this drusen-like structure is contacted in the presence or absence of the tested drug of different concentrations, the state of the drusen-like structure before and after the addition of the drug is measured and compared, thus evaluating or screening the drug. In addition, the evaluation or screening of the drug can also be carried out in the following manner: in RPE cells, EMT is induced in the presence or absence of the drug, and the state of the drusen-like structure in the presence or absence of the drug is measured and compared. The determination of the state of the drusen-like structure can be exemplified, for example: the expression (secretion) of at least one selected from the group consisting of epithelial markers, mesenchymal markers and ECM is measured; the morphological changes of the cells are observed; or the changes in the motility of the cells are measured. If the EMT in the RPE cells is suppressed, it is confirmed that the mesenchymal markers and / or ECM are suppressed, the motility is suppressed and / or the cell morphology is deformed from the spindle-like to the cubic shape and suppresses the formation of the drusen-like structure (Focus) and other phenomena.

[0204] In the case of evaluation and / or screening of medicament according to the formation degree of drusen-like structure (Focus), it is not limited, and can be carried out by the method comprising the steps of: using EMT induction model to induce EMT to RPE cells, making RPE cells contact with tested agent and reacting, measuring the formation of drusen-like structure (Focus) of cells. In one embodiment, using EMT induction model to induce EMT to RPE cells and making RPE cells contact with tested agent and reacting can also be carried out simultaneously. In one embodiment, the aforementioned method may also be included in the step of fixing cells after contact reaction.

[0205] In the step of making RPE cell contact with the tested agent and reacting it, comprising reacting the concentration of the tested agent in stages from low concentration to high concentration. The concentration of the tested agent can be appropriately adjusted according to the type of the agent, for example, can be set to 0 μM (non-existence, non-addition) to 500 μM, can also be 0.1 μM to 400 μM, can also be 0.5 μM to 300 μM.

[0206] The contact reaction time between RPE cells and the tested drug can be set, for example, to 5 minutes to 120 hours. From the viewpoint of fully suppressing interstitial properties by inhibiting EMT, it is preferably set to 10 minutes to 108 hours, more preferably to 1 hour to 96 hours, further preferably to 10 hours to 84 hours, particularly preferably to 24 hours to 72 hours, and most preferably to 48 hours to 60 hours.

[0207] The contact reaction temperature between RPE cells and the test agent can be appropriately adjusted according to the type of the agent, and can be set to, for example, 4 to 50°C, preferably 10 to 45°C, more preferably 20 to 40°C, and even more preferably 30 to 37°C.

[0208] In the step of fixing the cells after the contact reaction, a known method can be used as a method for fixing the cells. For example, although not limited to, the cells can be fixed using polyformaldehyde.

[0209] The step of measuring the formation of drusen-like structures (Focus) in cells may further include staining the cells and / or analyzing the formation rate or formation inhibition rate of drusen-like structures (Focus).

[0210] The staining of cells is not limited, and known staining methods can be used, preferably fluorescent staining, more preferably nuclear staining and / or actin staining using fluorescent substances. As nuclear staining, for example, 7-AAD (7-aminoactinomycin D), acridine orange, DAPI (4', 6-diamidino-2-phenylindole; 4', 6-diamidino-2-phenylindole), DMAO, ethidium bromide, Hoechst staining, propidium iodide (PI; Propidium Iodide), etc. can be cited. From the perspective of staining, Hoechst staining is preferred. As actin staining, for example, phalloidin is preferably used.

[0211] The formation rate or formation inhibition rate analysis of drusen-like structures (Focus) includes, for example, imaging the formation state of drusen-like structures (Focus) when no candidate agent is added and the formation state of drusen-like structures (Focus) when a candidate agent with a specific concentration is added, and digitizing. And, including calculating the formation rate or formation inhibition rate of drusen-like structures (Focus) by comparing the numerical data of the two. The formation state of drusen-like structures (Focus) is imaged and digitized by known imaging techniques and digitization techniques. For example, the diameter, area, volume, etc. of drusen-like structures (Focus) can be set as an index. From the perspective of more accurately performing a high reproducibility analysis, it is preferred to measure the volume of drusen-like structures (Focus) and digitize it, and it is also preferred that the number, thickness, and volume of drusen-like structures (Focus) are comprehensively evaluated and digitized.

[0212] In the case of evaluating and / or screening of medicament by the inhibition of mesenchymal system markers and / or ECM, it is not limited and can be carried out by the method comprising the following steps: using EMT induction model to induce EMT in RPE cells; making RPE cells contact with the tested agent and reacting; extracting RNA from cells after contact reaction; quantitative analysis of mRNA expression. In one embodiment, using EMT induction model to induce EMT in RPE cells and making RPE cells contact with the tested agent and reacting can also be carried out simultaneously.

[0213] The step of inducing EMT in RPE cells using the EMT induction model and the step of contacting RPE cells with a test drug to allow them to react refer to the item of the degree of formation of drusen-like structures (Focus).

[0214] The step of extracting RNA from cells after the contact reaction can use a known RNA extraction method, and preferably uses a commercially available RNA extraction kit or the like.

[0215] The step of quantitative analysis of mRNA expression is not limited, and real-time PCR is preferably used. As an EMT marker measured by real-time PCR, the interstitial markers, ECM and other EMT markers described in the project of the aforementioned drusen inhibitor can be used, and with respect to the viewpoint of the EMT marker measured by real-time PCR with good reproducibility, as an interstitial marker, preferably Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2, more preferably Snail, Slug, CDH3, MMP1, MMP7. In addition, as ECM, from the viewpoint of using an EMT marker that can be measured with good reproducibility by a real-time PCR method, preferably COL5A3, COL6A3, LAMC2, HMMR, TNC, COL1A1, COL1A2, CSPG4, SRGN, FN1, VIM, COL5A2, COL13A1, and LAMB3, and more preferably COL5A3, COL6A3, LAMC2, HMMR, and TNC.

[0216] In the case of evaluating and / or screening the agent by suppressing the motor ability, it is not limited and can be carried out by the method comprising the following steps: using the EMT induction model to induce EMT to RPE cells; making RPE cells contact with the tested agent to react; recovering cells after the contact reaction; using the recovered cells to carry out invasion experiment. In one embodiment, using the EMT induction model to induce EMT to RPE cells and making RPE cells contact with the tested agent to react can also be carried out simultaneously.

[0217] The step of inducing EMT in RPE cells using the EMT induction model and the step of contacting RPE cells with a test drug to allow them to react refer to the item of the degree of formation of drusen-like structures (Focus).

[0218] After the contact reaction, the cells can be recovered by a known method and their motility can be evaluated using a commercially available invasion assay kit, etc. The motility can be evaluated by image analysis of cell migration (infiltration) and / or counting the number of infiltrating cells.

[0219] Here, the test agent to be evaluated may be a compound known to have an epithelial-mesenchymal transition inhibitory activity, or a compound not yet known to have an epithelial-mesenchymal transition inhibitory activity, or may be a newly synthesized compound.

[0220] [Preparations, etc.]

[0221] The present invention can be formulated with additives as needed into a dosage form suitable for administration. For example, it can be provided in the form of tablets, capsules, syrups, fine granules, granules, powders, or pills suitable for oral administration. The present invention can also be provided in the form of injections, ophthalmic preparations, or patches suitable for parenteral administration. In addition to these preparations, the present invention can also be provided in the form of a preparation that has been systematized for drug delivery.

[0222] The dosage of the choroidal neovascularization prevention and / or treatment agent or drusen inhibitor of the present invention varies depending on the type of target disease, the patient's age, weight, indications, and dosage form. For example, in the case of an injection, an adult can be administered about 1 mg to about 1000 mg once or several times a day. In the case of oral administration, an adult can usually be administered 0.01 mg to 10000 mg, preferably 0.1 mg to 5000 mg, and more preferably 0.5 mg to 2500 mg once or several times a day. In the case of an injection, an adult can usually be administered 0.0001 mg to 2000 mg once or several times a day. In the case of eye drops or inserts, a drug having an active ingredient concentration of 0.000001% (w / v) to 10% (w / v), preferably 0.00001% (w / v) to 1% (w / v), more preferably 0.0001% (w / v) to 0.1% (w / v) can be administered once or several times a day. In the case of a patch, a patch containing 0.0001 mg to 2000 mg can be applied to an adult.

[0223] In this specification, "Cmax" refers to the peak plasma concentration of the drug obtained within the dosing interval. In the case of existing drugs, it can be obtained based on the interview form or accompanying documents of the drug. Cmax occurs from about 1 hour to about 48 hours, about 1 hour to about 20 hours, about 1 hour to about 18 hours, about 1 hour to about 16 hours, about 1 hour to about 12 hours, about 1 hour to about 10 hours, about 1 hour to about 8 hours, about 1 hour to about 6 hours, or about 1 hour to about 4 hours after administration.

[0224] Although not limited, when using zaltoprofen as an active ingredient, the daily dose for adults is preferably 25 mg to 360 mg, more preferably 50 mg to 300 mg, further preferably 70 mg to 260 mg, particularly preferably 80 mg to 240 mg, and most preferably 80 mg. In addition, in the case of single use, the daily dose of zaltoprofen can be 80 mg to 160 mg. Regarding the dosage of zaltoprofen, the maximum daily dose can be 240 mg. In the case of zaltoprofen, Cmax is 20.4 μM ("Japanese Pharmacopoeia Zaltoprofen Tablets Non-steroidal Analgesic and Anti-inflammatory Agent Soluirubin (Registered Trademark) Tablets 80" Pharmaceutical Interview Form, April 2012 (Revised 4th Edition), single administration). The dosage of zaltoprofen is preferably a dosage that produces a Cmax of about 20.4 μM or more. As long as the effect of the present invention is exerted, it may be a dosage that produces a blood concentration of 1 / 2 or more of Cmax or 1 / 4 or more of Cmax. In addition, the Cmax of zaltoprofen may be 16.8 μM depending on the type of additives used ("Non-steroidal analgesic and anti-inflammatory agent Soleton tablets (registered trademark) 80 Japanese Pharmacopoeia Zaltoprofen Tablets" pharmaceutical interview form, revised in August 2015 (6th edition), when administered as a single dose). In this case, the dosage of zaltoprofen is preferably a dosage that produces a Cmax of about 16.8 μM or more. As long as the effect of the present invention is exerted, it may be a dosage that produces a blood concentration of 1 / 2 or more of Cmax or 1 / 4 or more of Cmax. From the viewpoint of producing the aforementioned Cmax, the daily dose of zaltoprofen can be set to 240 mg to 360 mg, 240 mg to 300 mg, or 240 mg to 260 mg. From the viewpoint of producing a blood concentration of 1 / 2 or more of the aforementioned Cmax, the daily dose of zaltoprofen can be set to 120 mg to 360 mg, 120 mg to 300 mg, 120 mg to 260 mg, 120 mg to 240 mg, 120 mg to 220 mg, 120 mg to 200 mg, or 120 mg to 180 mg. From the viewpoint of producing a blood concentration of 1 / 4 or more of the aforementioned Cmax, the daily dose of zaltoprofen can be set to 60 mg to 360 mg, 60 mg to 300 mg, 60 mg to 260 mg, 60 mg to 240 mg, 60 mg to 200 mg, 60 mg to 160 mg, 60 mg to 120 mg, 60 mg to 80 mg. The aforementioned administration is preferably an injection or oral administration. By producing the aforementioned blood concentration, it can be expected that zaltoprofen penetrates the BRB and effectively inhibits CNV or drusen.

[0225] Although not limited, the dosage when using oxaprozin as an active ingredient is preferably set to 100 mg to 700 mg per day for adults, more preferably 200 mg to 600 mg, further preferably 300 mg to 500 mg, and particularly preferably 400 mg. The peak plasma concentration (Cmax) of the drug is calculated by a known method. In the case of oxaprozin, Cmax is 340.9 μM ("Sustained anti-inflammatory and analgesic Alvo (registered trademark) tablets 100 mg Alvo (registered trademark) tablets 200 mg oxaprozin preparation" pharmaceutical product interview form, November 2011 (new revised 3rd edition), when repeatedly administered). The dosage of oxaprozin is preferably a dosage that produces a Cmax of about 340.9 μM or more. As long as the effect of the present invention is exerted, it may also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax. From the perspective of producing the aforementioned Cmax, the daily dose of oxaprozin can be set to 400mg to 700mg, 400mg to 600mg. From the perspective of producing a blood concentration of 1 / 2 of the aforementioned Cmax or more, the daily dose of oxaprozin can be set to 200mg to 700mg, 200mg to 600mg, 200mg to 500mg, 200mg to 400mg, 200mg to 300mg. From the perspective of producing a blood concentration of 1 / 4 of the aforementioned Cmax or more, the daily dose of oxaprozin can be set to 100mg to 700mg, 100mg to 600mg, 100mg to 500mg, 100mg to 400mg, 100mg to 300mg, 100mg to 150mg. The aforementioned administration is preferably administered by injection or oral administration. By producing the aforementioned blood concentration, it is expected that oxaprozin penetrates the BRB and effectively inhibits CNV or drusen.

[0226] Although not limited, regarding the dosage when using tepufil acid as an active ingredient, it is preferred that the daily dose for adults is set to 60 mg to 900 mg, more preferably to 100 mg to 800 mg, further preferably to 150 mg to 700 mg, particularly preferably to 200 mg to 600 mg, and most preferably to 600 mg. In addition, in the case of single use, regarding the dosage of tepufil acid, the daily dose can be set to 200 mg. Regarding the dosage of tepufil acid, the maximum daily dose can be set to 600 mg. In the case of tepufil acid, Cmax is 69.15 μM ("Analgesic and Anti-inflammatory Agent, Surgam (Registered Trademark) Tablets 100 mg, 200 mg" Pharmaceutical Interview Form, Revised in August 2014 (Revised 5th Edition), Single Administration). The dosage of teipfic acid is preferably a dosage that produces a Cmax of about 69.15 μM or more. As long as the effect of the present invention is exerted, it can also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax. From the perspective of producing the aforementioned Cmax, the daily dosage of teipfic acid can be set to 600 mg to 900 mg, 600 mg to 800 mg, 600 mg to 700 mg. From the perspective of producing a blood concentration of more than 1 / 2 of the aforementioned Cmax, the daily dosage of teipfic acid can be set to 300 mg to 900 mg, 300 mg to 800 mg, 300 mg to 700 mg, 300 mg to 600 mg, 300 mg to 500 mg, 300 mg to 400 mg. With regard to the viewpoint of producing a blood concentration of 1 / 4 amount or more of the aforementioned Cmax, the daily amount of teipfic acid can be set to 150mg to 900mg, 150mg to 800mg, 150mg to 700mg, 150mg to 600mg, 150mg to 500mg, 150mg to 400mg, 150mg to 300mg, 150mg to 250mg, 150mg to 200mg. The aforementioned administration is preferably carried out by injection or oral administration. By producing the aforementioned blood concentration, it can be expected that teipfic acid penetrates BRB and effectively inhibits CNV or drusen.

[0227] Although not limited, the dosage when using aluminum flufenamic acid as an active ingredient is preferably set to 75 mg to 1250 mg per day for adults, more preferably 150 mg to 1000 mg, further preferably 200 mg to 800 mg, particularly preferably 250 mg to 750 mg, and most preferably 375 mg to 750 mg. In addition, in the case of single use, the dosage of aluminum flufenamic acid can be set to 250 mg per day. Regarding the dosage of aluminum flufenamic acid, the maximum daily dosage can be set to 750 mg. In the case of aluminum flufenamic acid, Cmax is 27.91 μM ("Non-steroidal anti-inflammatory, analgesic, antipyretic Opyrin (registered trademark) tablets 125 mg" pharmaceutical interview form, produced in December 2011 (new version 4), when administered once). The dosage of aluminum flufenamic acid is preferably a dosage that produces a Cmax of about 27.91 μM or more. As long as the effect of the present invention is exerted, it can also be a dosage that produces a blood concentration of 1 / 2 or more of Cmax or 1 / 4 or more of Cmax. From the perspective of producing the aforementioned Cmax, the daily dosage of aluminum flufenamic acid can be set to 750 mg to 1250 mg, 750 mg to 1000 mg, or 750 mg to 800 mg. From the perspective of producing a blood concentration of 1 / 2 or more of the aforementioned Cmax, the daily dosage of aluminum flufenamic acid can be set to 400 mg to 1250 mg, 400 mg to 1000 mg, 400 mg to 800 mg, 400 mg to 750 mg, or 400 mg to 500 mg. From the viewpoint of producing a blood concentration of 1 / 4 or more of the aforementioned Cmax, the daily amount of flufenamic acid aluminum can be set to 200 mg to 1250 mg, 200 mg to 1000 mg, 200 mg to 800 mg, 200 mg to 500 mg, 200 mg to 400 mg, 200 mg to 300 mg, 200 mg to 250 mg. The aforementioned administration is preferably performed by injection or oral administration. By producing the aforementioned blood concentration, it can be expected that flufenamic acid aluminum penetrates the BRB and effectively inhibits CNV or drusen.

[0228] Although not limited, regarding the dosage when using mefenamic acid as an active ingredient, it is preferred to set the daily dose for adults to 150 mg to 2250 mg, more preferably to 250 mg to 1800 mg, further preferably to 400 mg to 1600 mg, particularly preferably to 500 mg to 1500 mg, and most preferably to 1000 mg to 1500 mg. In addition, in the case of single use, regarding the dosage of mefenamic acid, the daily dose can be set to 500 mg. Regarding the dosage of mefenamic acid, the maximum daily dose can be set to 1500 mg. In the case of mefenamic acid, Cmax is 38.54 μM ("Analgesic, Anti-inflammatory, Antipyretic Pontal (Registered Trademark) Tablets 250 mg" Pharmaceutical Interview Form, Revised in July 2015 (9th Edition), Single Administration). The dosage of mefenamic acid is preferably a dosage that produces a Cmax of about 38.54 μM or more. As long as the effect of the present invention is exerted, it can also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax. From the perspective of producing the aforementioned Cmax, the daily dosage of mefenamic acid can be set to 1000 mg to 2250 mg, 1000 mg to 1800 mg, 1000 mg to 1600 mg, 1000 mg to 1500 mg, 1000 mg to 1200 mg. From the perspective of producing a blood concentration of more than 1 / 2 of the aforementioned Cmax, the daily dosage of mefenamic acid can be set to 500 mg to 2250 mg, 500 mg to 1800 mg, 500 mg to 1600 mg, 500 mg to 1500 mg, 500 mg to 1200 mg, 500 mg to 1000 mg, 500 mg to 800 mg. From the viewpoint of producing a blood concentration of 1 / 4 of the aforementioned Cmax or more, the daily amount of mefenamic acid can be set to 150mg to 2250mg, 250mg to 1800mg, 250mg to 1600mg, 250mg to 1500mg, 250mg to 1200mg, 250mg to 1000mg, 250mg to 800mg, 250mg to 500mg, 250mg to 300mg. The aforementioned administration is preferably performed by injection or oral administration. By producing the aforementioned blood concentration, mefenamic acid can be expected to penetrate BRB and effectively inhibit CNV or drusen.

[0229] Although not limited, about the dosage when using sulindac as active ingredient, preferably the daily amount of adult is set to 30mg to 500mg, more preferably to 100mg to 450mg, further preferably to 200mg to 400mg, particularly preferably to 250mg to 350mg, most preferably to 300mg. In the case of sulindac, Cmax is 10.10μM (" non-steroidal anti-inflammatory analgesic Clinoril (registered trademark) tablets 50, 100" pharmaceutical product interview form, revised in March 2011 (revised the 3rd edition), single administration). The dosage of sulindac is preferably the dosage that produces the Cmax above about 10.10μM, as long as the effect of the present application invention is brought into play, then it can also be the dosage that produces the blood concentration of more than 1 / 2 amount of Cmax or more than 1 / 4 amount of Cmax. From the perspective of producing the aforementioned Cmax, the daily amount of sulindac can be set to 300mg to 500mg, 300mg to 450mg, 300mg to 400mg, 300mg to 350mg. From the perspective of producing a blood concentration of 1 / 2 of the aforementioned Cmax, the daily amount of sulindac can be set to 200mg to 500mg, 200mg to 450mg, 200mg to 400mg, 200mg to 350mg, 200mg to 300mg, 200mg to 250mg. From the perspective of producing a blood concentration of 1 / 4 of the aforementioned Cmax, the daily amount of sulindac can be set to 100mg to 500mg, 100mg to 450mg, 100mg to 400mg, 100mg to 350mg, 100mg to 300mg, 100mg to 250mg, 100mg to 200mg. The above-mentioned administration is preferably performed by injection or oral administration. By generating the above-mentioned blood concentration, it is expected that sulindac will penetrate the BRB and effectively inhibit CNV or drusen.

[0230] Although not limited, regarding the dosage when using epalrestat as an active ingredient, it is preferred that the daily dose for adults be set to 10 mg to 400 mg, more preferably 50 mg to 300 mg, further preferably 100 mg to 200 mg, and particularly preferably 150 mg. In the case of epalrestat, Cmax is 12.2 μM ("Aldose reductase inhibitor Japanese Pharmacopoeia epalrestat tablets Kinedak (registered trademark) tablets 50 mg" pharmaceutical interview form, revised in November 2013 (6th edition), single administration). The dosage of epalrestat is preferably a dosage that produces a Cmax of about 12.2 μM or more. As long as the effect of the present invention is exerted, it can also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax. From the perspective of producing the aforementioned Cmax, the daily dose of epalrestat can be set to 150 mg to 400 mg, 150 mg to 300 mg, or 150 mg to 200 mg. From the perspective of producing a blood concentration of 1 / 2 of the aforementioned Cmax, the daily dose of epalrestat can be set to 75mg to 400mg, 75mg to 300mg, 75mg to 200mg, 75mg to 150mg. From the perspective of producing a blood concentration of 1 / 4 of the aforementioned Cmax, the daily dose of epalrestat can be set to 37mg to 400mg, 37mg to 300mg, 37mg to 200mg, 37mg to 150mg. The aforementioned administration is preferably performed by injection or oral administration. By producing the aforementioned blood concentration, it can be expected that epalrestat penetrates the BRB and effectively inhibits CNV or drusen.

[0231] Although not limited, when using zafirlukast as an active ingredient, the daily dose for adults is preferably set to 10 mg to 200 mg, more preferably 20 mg to 150 mg, further preferably 30 mg to 100 mg, and particularly preferably 40 mg to 80 mg. In the case of zafirlukast, Cmax is 0.8 μM ("Leukotriene Receptor Antagonist / Bronchial Asthma Treatment Agent Accolate (Registered Trademark) Tablets 20 mg Zafirlukast Tablets" Pharmaceutical Interview Form, produced in January 2015 (revised 9th edition), single administration). The dosage of zafirlukast is preferably a dosage that produces a Cmax of about 0.8 μM or more. As long as the effect of the present invention is exerted, it can also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax. From the perspective of producing the aforementioned Cmax, the daily dose of zafirlukast can be set to 40mg to 200mg, 40mg to 150mg, 40mg to 100mg, 40mg to 80mg. From the perspective of producing a blood concentration of 1 / 2 of the aforementioned Cmax or more, the daily dose of zafirlukast can be set to 20mg to 200mg, 20mg to 150mg, 20mg to 100mg, 20mg to 80mg, 20mg to 40mg. From the perspective of producing a blood concentration of 1 / 4 of the aforementioned Cmax or more, the daily dose of zafirlukast can be set to 10mg to 200mg, 10mg to 150mg, 10mg to 100mg, 10mg to 80mg, 10mg to 40mg, 10mg to 20mg. The aforementioned administration is preferably performed by injection or oral administration. By producing the aforementioned blood concentration, it can be expected that zafirlukast penetrates the BRB and effectively inhibits CNV or drusen.

[0232] Although not limited, the dosage of amlexanox when used as an active ingredient is preferably set to 10 mg to 150 mg per day for adults, more preferably 15 mg to 100 mg, further preferably 20 mg to 80 mg, and particularly preferably 25 mg to 50 mg. In the case of amlexanox, Cmax is 16.0 μM ("Asthma and Allergic Rhinitis Treatment Agent Japanese Pharmacopoeia Amlexanox Tablets Solfa (Registered Trademark) 25 mg Tablets Solfa (Registered Trademark) 50 mg Tablets" Pharmaceutical Interview Form, Revised in October 2012 (Revised 2nd Edition), when administered as a single dose). The dosage of amlexanox is preferably a dosage that produces a Cmax of about 16.0 μM or more, but may also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax as long as the effect of the present invention is exerted. From the perspective of producing the aforementioned Cmax, the daily dose of amlexanox can be set to 75 mg to 150 mg. From the perspective of producing a blood concentration of more than 1 / 2 of the aforementioned Cmax, the daily dose of amlexanox can be set to 37 mg to 150 mg, 37 mg to 100 mg, or 37 mg to 75 mg. From the perspective of producing a blood concentration of more than 1 / 4 of the aforementioned Cmax, the daily dose of amlexanox can be set to 18 mg to 150 mg, 18 mg to 100 mg, 18 mg to 75 mg, or 18 mg to 37 mg. The aforementioned administration is preferably performed by injection or oral administration. By producing the aforementioned blood concentration, it can be expected that amlexanox will penetrate the BRB and effectively inhibit CNV or drusen.

[0233] Although not limited, the dosage when using seroquel as an active ingredient is preferably set to 8 mg to 120 mg per day for adults, more preferably 25 mg to 110 mg, further preferably 50 mg to 100 mg, particularly preferably 70 mg to 90 mg, and most preferably 80 mg. In the case of seroquel, Cmax is 31.03 μM ("Thromboxane A2 Receptor Antagonist Bronica (Registered Trademark) Tablets 40, 80" Pharmaceutical Interview Form, Revised in October 2016 (4th Edition), Single Administration). The dosage of seroquel is preferably a dosage that produces a Cmax of about 31.03 μM or more, and as long as the effect of the present invention is exerted, it may also be a dosage that produces a blood concentration of more than 1 / 2 of Cmax or more than 1 / 4 of Cmax. From the perspective of producing the aforementioned Cmax, the daily dose of serostat can be set to 80mg to 120mg, 80mg to 110mg, 80mg to 100mg, 80mg to 90mg. From the perspective of producing a blood concentration of 1 / 2 of the aforementioned Cmax or more, the daily dose of serostat can be set to 50mg to 120mg, 50mg to 110mg, 50mg to 100mg, 50mg to 90mg, 50mg to 80mg, 50mg to 70mg. From the perspective of producing a blood concentration of 1 / 4 of the aforementioned Cmax or more, the daily dose of serostat can be set to 25mg to 120mg, 25mg to 110mg, 25mg to 100mg, 25mg to 90mg, 25mg to 80mg, 25mg to 60mg, 25mg to 40mg. The aforementioned administration is preferably performed by injection or oral administration. By producing the above-mentioned blood concentration, serotonin can be expected to penetrate the BRB and effectively inhibit CNV or drusen.

[0234] In the present specification, the dosage form of the preparation is not particularly limited, and may be a liquid preparation (suspension, emulsion, capsule, syrup, injection, etc.) or a solid preparation (powder, granules, granules, pills, tablets, etc.).

[0235] The preparation provided in this specification is not limited and may have a pH value of 4.0 to 8.5. From the viewpoint of significantly exerting the effect of the present invention, the pH value of the preparation is preferably 4.5 or more, more preferably 5.0 or more, further preferably 5.5 or more, particularly preferably 6.0 or more, and most preferably 6.5 or more. In addition, from the viewpoint of significantly exerting the effect of the present invention, the pH value of the preparation is preferably 8.0 or less, more preferably 7.8 or less, further preferably 7.7 or less, particularly preferably 7.6 or less, and most preferably 7.5 or less. In addition, from the viewpoint of significantly exerting the effect of the present invention, the pH value of the preparation is preferably 4.5 to 8.0, more preferably 5.0 to 7.8, further preferably 5.5 to 7.7, particularly preferably 6.0 to 7.6, and most preferably 6.5 to 7.6.

[0236] [Solid preparations]

[0237] As long as the preparation provided in this specification does not damage stability, etc., the conventional carrier components can be added according to the form of the preparation and prepared by conventional methods. For example, if it is a tablet, it can be prepared by mixing the powdered active ingredient with the pharmaceutically acceptable carrier components (excipients, etc.) and compressing them. In addition, powders such as granules in solid preparations can also be prepared by various granulation methods (extrusion granulation, crushing granulation, dry compaction granulation, fluidized bed granulation, rolling granulation, high-speed stirring granulation, etc.), and tablets can be prepared by appropriately combining the aforementioned granulation method, tableting method (wet tableting, direct tableting), etc. For tablets, sugar coating can be implemented to make sugar-coated tablets. In addition, the tablet can be a single-layer tablet or a layered tablet such as a two-layer tablet. The preferred dosage form of the solid preparation is a tablet (such as a chewable tablet in the mouth).

[0238] In solid preparations, carrier components or additives include, for example: excipients (sugar alcohols such as D-sorbitol, D-mannitol, and xylitol, sugars such as glucose, saccharide, lactose, and fructose, crystalline cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, calcium hydrogen phosphate, wheat starch, rice starch, corn starch, potato starch, dextrin, β-cyclodextrin, light silicic anhydride, titanium oxide, magnesium aluminosilicate, talc, kaolin, etc.); disintegrants (low-substituted hydroxypropyl cellulose, calcium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, hydroxypropyl starch, partially alpha-starched starch, etc.); binders (methyl cellulose, Cellulose derivatives such as ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, acrylic polymers, gelatin, gum arabic, polytriglucose, α-starch, agar, tragacanth gum, sodium alginate, propylene glycol alginate, etc.); lubricants (stearic acid, magnesium stearate, calcium stearate, polyoxyl stearate, cetyl alcohol, talc, hydrogenated oil, sucrose fatty acid esters, dimethyl polysiloxane, beeswax, white beeswax, etc.); antioxidants (butylated hydroxytoluene (BHT), propyl gallate, butylated hydroxyanisole (BHA), tocopherol, lemon Acid, etc.); coating agents (hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, ethyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinyl acetal diethylamino acetate, methacrylate aminoalkyl ester copolymer, hydroxypropyl methylcellulose acetate succinate, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, shellac, etc.); coloring agents (turmeric extract, riboflavin, titanium oxide, carotene solution, etc.); flavoring agents (aspartame, ascorbic acid, stevia, menthol, licorice crude extract, simple syrup, etc.); foaming agent (sodium bicarbonate, etc.); fluidizing agent (sodium aluminometasilicate, light silicic anhydride, etc.); surfactant (polyoxyethylene hydrogenated castor oil, glycerol monostearate, sorbitan monostearate, sorbitan monolaurate, polyoxyethylene polyoxypropylene, polysorbate, sodium lauryl sulfate, polyethylene glycol 6000 and other polyethylene glycols, sucrose fatty acid esters, etc.); plasticizer (triethyl citrate, polyethylene glycol, triacetin, cetyl alcohol, etc.); sweetener (sucrose, mannitol, aspartame and other natural or synthetic sweeteners); flavoring agent (menthol, etc.); adsorbent, preservative, wetting agent, antistatic agent, etc.

[0239] In the case where the preparation is a solid preparation, the content of at least one selected from the group consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serostat and their pharmaceutically acceptable salts, or the EMT inhibitory compound is not limited, and can be set to 0.1% to 30% by weight, preferably 0.5% to 25% by weight, and more preferably 0.5% to 20% by weight, relative to the total amount of the preparation. In addition, the content of the present compound can be 0.01% to 3% by weight, 0.02% to 2% by weight, or 0.03% to 1% by weight.

[0240] [Ophthalmic agents]

[0241] When an ophthalmic agent is prepared, the properties are not particularly limited, and the ophthalmic agent may be in any of the following forms, for example, liquid, fluid, gel, semisolid, or solid. The types of ophthalmic agents are not particularly limited. Examples include eye drops, eye ointments (water-soluble eye ointments, oil-soluble eye ointments), and intraocular injections (e.g., intravitreal injections).

[0242] Furthermore, the ophthalmic agent in a liquid, fluid, gel, semisolid, or solid form other than a solid form may be an aqueous composition or an oily composition such as an ointment.

[0243] The preparation method of ophthalmic preparation is well known. It can be prepared by mixing at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and their pharmaceutically acceptable salts with a pharmaceutically acceptable matrix or carrier, and pharmaceutically acceptable ophthalmic preparation additives and other active ingredients (physiologically active ingredients or pharmacologically active ingredients other than the present compound) for preparation.

[0244] Examples of the matrix or carrier include water, aqueous solvents such as polar solvents, polyols, vegetable oils, and oily matrices. Examples of the matrix or carrier for intraocular injections include distilled water for injection and physiological saline. The matrix or carrier may be used alone or in combination of two or more.

[0245] As additives for ophthalmic preparations, for example, surfactants, fragrances or cooling agents, preservatives, bactericides or antibacterial agents, pH adjusters, isotonic agents, chelating agents, buffers, stabilizers, antioxidants, and thickening agents can be cited. Intraocular injections can also contain dissolution aids, suspending agents, isotonic agents, buffers, analgesics, stabilizers, and preservatives. One additive can be used alone, or two or more additives can be used in combination.

[0246] When the ophthalmic agent is a preparation other than a solid preparation, such as a liquid, flowable, gel, or semisolid preparation, the content of at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof, or the EMT inhibitory compound in the ophthalmic composition can be set to 0.00001% by weight or more, preferably 0.0001% by weight or more, and more preferably 0.001% by weight or more. In addition, the content of at least one selected from the group consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serostat and their pharmaceutically acceptable salts in the ophthalmic composition, or the EMT inhibitory compound, relative to the total amount of the composition, may be 0.01% by weight or more, 0.1% by weight or more, or 1% by weight or more. If it is within the above range, the prevention, improvement or treatment effect of retinal diseases is fully obtained.

[0247] In addition, in the case where ophthalmic agents are preparations such as liquid, flowing, gel, or semisolid forms other than solid preparations, the ophthalmic composition is selected from at least one of the groups consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serolast and their pharmaceutically acceptable salts or the content of EMT inhibitory compounds relative to the total amount of the composition, and can be set to 10% by weight or less, preferably 5% by weight or less, more preferably 3% by weight or less. If it is the aforementioned range, it becomes a preparation that fully obtains the prevention, improvement or treatment effect of retinal diseases, and has less foreign body sensation when dropping the eyes.

[0248] When the ophthalmic agent is a preparation other than a solid preparation, for example, a liquid, a flowing, a gel, or a semisolid preparation, the content of the compound in the ophthalmic composition can be 0.00001% to 10% by weight, 0.00001% to 5% by weight, 0.00001% to 3% by weight, 0.0001% to 10% by weight, 0.0001% to 5% by weight, 0.0001% to 5% by weight, 0.0001% to 10% by weight, 0.0001% to 5% by weight, 0.0001% to 3% by weight, 0.0001% to 10% by weight, 0.0001% to 5 ... % to 3 wt%, 0.001 wt% to 10 wt%, 0.001 wt% to 5 wt%, 0.001 wt% to 3 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.01 wt% to 3 wt%, 0.1 wt% to 10 wt%, 0.1 wt% to 5 wt%, 0.1 wt% to 3 wt%, 1 wt% to 10 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%.

[0249] When the ophthalmic agent is a composition containing water, the pH value of the ophthalmic agent is not limited, preferably 4 or more, more preferably 5.5 or more, further preferably 6 or more, and further more preferably 6.5 or more. If it is the aforementioned range, the compound becomes a preparation with good stability to heat and light. In addition, the pH value of the ophthalmic agent is preferably 9 or less, more preferably 8.5 or less, further preferably 8 or less, and further more preferably 7.5 or less. If it is the aforementioned range, the irritation to the eye is suppressed.

[0250] [Injection]

[0251] Injection can make at least one selected from the group consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serolast and their pharmaceutically acceptable salts or EMT inhibitory compounds dissolved or dispersed in distilled water for injection or physiological saline, etc., and prepared by a known method such as that recorded in the Japanese Pharmacopoeia. In addition, the injection may also include pharmaceutically acceptable carriers such as dissolution aids, suspending agents, isotonic agents, buffers, analgesics, stabilizers and preservatives, or may also include pharmaceutically acceptable additives for injection.

[0252] The injection may be formulated with pharmacologically active ingredients or physiologically active ingredients other than the preventive, ameliorative or therapeutic ingredients for retinal diseases. Examples of such pharmacologically active ingredients and physiologically active ingredients include neurotrophic factors, decongestant ingredients, eye muscle regulating drug ingredients, anti-inflammatory drug ingredients or astringent drug ingredients, antihistamine drug ingredients or antiallergic drug ingredients, vitamins, amino acids, antibacterial drug ingredients or bactericidal drug ingredients, etc.

[0253] Injection selected from the group consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotrast and their pharmaceutically acceptable salts at least one or EMT inhibitory compound relative to the total amount of the preparation, preferably 0.001 weight % or more, more preferably 0.01 weight % or more, more preferably 0.1 weight % or more. In addition, preferably 80 weight % or less, more preferably 60 weight % or less, more preferably 50 weight % or less. If the aforementioned range, then can fully obtain retinal disease prevention, improvement or therapeutic effect.

[0254] The content of at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof, or the EMT inhibitory compound in the injection can be set to 0.001% to 80% by weight, 0.001% to 60% by weight, 0.001% to 50% by weight, 0.01% to 80% by weight, 0.01% to 60% by weight, 0.01% to 50% by weight, 0.1% to 80% by weight, 0.1% to 60% by weight, 0.1% to 50% by weight, relative to the total amount of the preparation.

[0255] In the injection, the additive and at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and their pharmaceutically acceptable salts, or a pharmacologically active ingredient or a physiologically active ingredient other than an EMT inhibitory compound may be used alone or in combination of two or more.

[0256] [Capsules]

[0257] Capsules can be prepared by using a conventional method, filling powder and granules (powders, granules, etc.) into capsules (soft or hard capsules). In the case of a capsule, the preparation is selected from the group consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serolast and their pharmaceutically acceptable salts. The content of at least one or EMT inhibitory compound is not limited, relative to the total amount of preparation, can be set to 0.01 weight % to 3 weight %, preferably 0.02 weight % to 2 weight %, more preferably 0.03 weight % to 1 weight %. It is preferred that this liquid preparation is filled into a soft capsule, etc., and soft capsules are made and used.

[0258] In the case of a hard capsule, the content of the hard capsule without capsule coating is not limited to at least one selected from the group consisting of zaltoprofen, oxaprozin, teipfic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serolast and their pharmaceutically acceptable salts, or the content of the EMT inhibitory compound, relative to the total amount of the preparation, can be set to 0.1% to 30% by weight, preferably 0.5% to 25% by weight, more preferably 0.5% to 20% by weight. In addition, the content of the present compound can be 0.01% to 3% by weight, 0.02% to 2% by weight, or 0.03% to 1% by weight.

[0259] Examples of additives in liquid preparations include: oily bases (e.g., vegetable oils such as olive oil, corn oil, soybean oil, sesame oil, and cottonseed oil; medium-chain fatty acid triglycerides, etc.), aqueous bases (e.g., polyethylene glycol 400, water), gel bases (e.g., carboxyvinyl polymers, colloids, etc.), surfactants (e.g., polysorbate 80, hydrogenated castor oil, glycerol fatty acid esters, sorbitan sesquioleate, etc.), suspending agents (e.g., kaolin, sodium carboxymethylcellulose, tragacanth gum, white beeswax or various surfactants, gum arabic, gum arabic powder, xanthan gum, soybean lecithin, etc.), dispersants, emulsifiers, stabilizers, buffers, solubilizing agents, defoaming agents (e.g., dimethylpolysiloxane, silicon defoaming agents, etc.), pH adjusters (e.g., citric acid, malic acid, sodium hydrogen phosphate, dipotassium hydrogen phosphate, etc.), preservatives (preservatives), cooling agents (e.g., l-menthol, mint water, etc.), and the like. Furthermore, antioxidants, sweeteners, acidulants, colorants, flavoring agents, and the like may be appropriately added to these liquid preparations.

[0260] Liquid preparations can be prepared, for example, by dissolving or dispersing each component in an aqueous medium (purified water, purified water containing ethanol, etc.) as a carrier component, filtering or sterilizing as necessary, filling into a predetermined container, and further sterilizing.

[0261] The composition is not particularly limited as long as it exhibits the effects of the present invention, and may be, for example, a pharmaceutical product, a quasi-drug, a functional food, or the like.

[0262] [Functional Foods]

[0263] The present invention can also be provided as a functional food. In this specification, the so-called functional food refers to a food and drink with the efficacy of a pharmaceutical product that is permitted and designated by the state or a public organization, or a food and drink that shows functionality based on the content of the predetermined effect reported by the enterprise to the state, etc. Functional foods can be exemplified by: specific health foods, nutritional functional foods, foods that show functionality, foods for the elderly, health supplements (nutritional balanced foods, supplements), etc. It also includes food and drinks that include packaging or containers, accompanying documents, instructions for use, etc. that indicate the efficacy or functionality of pharmaceuticals. It also includes food and drinks that indicate the efficacy or functionality of pharmaceuticals in applications submitted to the state, etc. This indication can be exemplified by: indications on the prevention and / or treatment of choroidal neovascularization, indications on the prevention and / or treatment of AMD, such as AMD, early AMD, intermediate AMD, dry AMD, wet AMD, etc., indications on the prevention, improvement and / or treatment of symptoms that are characteristic of AMD, such as distortion of visual field (metamorphosis), color tone of visual field, photosensitivity, rapid visual acuity loss, central dark spots, etc.

[0264] [Implementation Method]

[0265] The above-mentioned embodiments are not limited, and the present invention discloses the following embodiments.

[0266] A preventive and / or therapeutic agent for choroidal neovascularization, comprising as an active ingredient at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof.

[0267] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization is an oral agent or an injectable agent.

[0268] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the oral preparation is a solid preparation.

[0269] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the solid preparation is a tablet.

[0270] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization is a liquid preparation.

[0271] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the liquid preparation is an ophthalmic preparation or a capsule preparation.

[0272] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein the choroidal neovascularization occurs in age-related macular degeneration, polypoidal choroidal vasculopathy (PCV), or retinal angiomatous proliferation (RAP).

[0273] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the choroidal neovascularization is caused by EMT of RPE cells.

[0274] The preventive and / or therapeutic agent for choroidal neovascularization as described above, wherein, in order to inhibit the EMT of RPE cells, a sufficient amount of at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof is administered.

[0275] A preventive and / or therapeutic agent for choroidal neovascularization as described above, wherein, in order to inhibit the expression of mesenchymal markers or extracellular matrix in RPE cells, a sufficient amount of at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and their pharmaceutically acceptable salts is administered.

[0276] The preventive and / or therapeutic agent for choroidal neovascularization as described above, wherein the mesenchymal marker is at least one selected from the group consisting of Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2 and VIM.

[0277] The preventive and / or therapeutic agent for choroidal neovascularization as described above, wherein the extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR, TNC, COL1A1, COL1A2, SRGN, FN1, COL5A2, COL13A1 and LAMB3.

[0278] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein zaltoprofen or a salt thereof is administered in an amount that produces a Cmax of 20.4 μM or more.

[0279] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein zaltoprofen or a salt thereof is administered in an amount that produces a Cmax of 16.8 μM or more.

[0280] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein zaltoprofen or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0281] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein zaltoprofen or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0282] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein oxaprozin or a salt thereof is administered in an amount that produces a Cmax of 340.9 μM or more.

[0283] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein oxaprozin or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0284] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein oxaprozin or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0285] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the tetrapfilic acid or a salt thereof is administered in an amount that produces a Cmax of 69.15 μM or more.

[0286] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the tepefiic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0287] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein the tepefiic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0288] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein flufenamic acid or a salt thereof is administered in an amount that produces a Cmax of 27.91 μM or more.

[0289] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein flufenamic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0290] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein flufenamic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0291] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein mefenamic acid or a salt thereof is administered in an amount that produces a Cmax of 38.54 μM or more.

[0292] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein mefenamic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0293] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein mefenamic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0294] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein sulindac or a salt thereof is administered in an amount that produces a Cmax of 10.10 μM or more.

[0295] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein sulindac or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0296] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein sulindac or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0297] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein epalrestat or a salt thereof is administered in an amount that produces a Cmax of 12.2 μM or more.

[0298] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein epalrestat or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0299] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein epalrestat or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0300] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein zafirlukast or a salt thereof is administered in an amount that produces a Cmax of 0.8 μM or more.

[0301] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein zafirlukast or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0302] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein zafirlukast or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0303] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein amlexanox or a salt thereof is administered in an amount that produces a Cmax of 16.0 μM or more.

[0304] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein amlexanox or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0305] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization, wherein amlexanox or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0306] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein seratrolast or a salt thereof is administered in an amount that produces a Cmax of 31.03 μM or more.

[0307] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein seratrolast or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0308] The aforementioned agent for preventing and / or treating choroidal neovascularization, wherein seratrolast or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0309] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 25 mg to 360 mg of zaltoprofen or a salt thereof as a daily dose.

[0310] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 100 mg to 700 mg of oxaprozin or a salt thereof as a daily dose.

[0311] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 60 mg to 900 mg of tepefic acid or a salt thereof as a daily dose.

[0312] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 75 mg to 1250 mg of flufenamic acid or a salt thereof as a daily dose.

[0313] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 150 mg to 2250 mg of mefenamic acid or a salt thereof as a daily dose.

[0314] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 30 mg to 500 mg of sulindac or a salt thereof as a daily dose.

[0315] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 10 mg to 400 mg of epalrestat or a salt thereof as a daily dose.

[0316] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 10 mg to 200 mg of zafirlukast or a salt thereof as a daily dose.

[0317] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 10 mg to 150 mg of amlexanox or a salt thereof as a daily dose.

[0318] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization contains 8 mg to 120 mg of seratrodast or a salt thereof as a daily dose.

[0319] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization is administered once to three times a day.

[0320] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization is administered once or twice a day.

[0321] The aforementioned preventive and / or therapeutic agent for choroidal neovascularization is administered once a day.

[0322] A VEGF production inhibitor comprising as an active ingredient at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof.

[0323] A VEGF expression inhibitor comprising as an active ingredient at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, flufenamic acid, mefenamic acid, sulindac, epalrestat, zafirlukast, amlexanox, serotonin and pharmaceutically acceptable salts thereof.

[0324] A drusen inhibitor contains a compound having an epithelial-mesenchymal transition inhibitory activity on retinal pigment epithelial cells as an active ingredient.

[0325] The drusen inhibitor as described above, wherein the compound having epithelial-mesenchymal transition inhibitory activity is an agent that inhibits the expression of a mesenchymal marker or an extracellular matrix in the retinal pigment epithelial cells.

[0326] The drusen inhibitor as described above, wherein the compound having an epithelial-mesenchymal transition inhibitory activity is an agent that inhibits the enhancement of the motility of the retinal pigment epithelial cells.

[0327] The drusen inhibitor as described above, wherein the mesenchymal marker is at least one selected from the group consisting of Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2 and VIM.

[0328] The drusen inhibitor as described above, wherein the extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR, TNC, COL1A1, COL1A2, SRGN, FN1, COL5A2, COL13A1 and LAMB3.

[0329] As described above, the drusen inhibitor, wherein the mesenchymal marker is at least one selected from the group consisting of Snail, Slug, E-cadherin 3, MMP1 and MMP7 and / or the extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR and TNC.

[0330] As for the aforementioned drusen inhibitor, wherein the aforementioned compound having epithelial-mesenchymal transition inhibitory activity is a non-steroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor and / or a thromboxane A2 receptor antagonist.

[0331] As described above, the drusen inhibitor, wherein the non-steroidal anti-inflammatory agent is a propionic acid-based non-steroidal anti-inflammatory agent, an aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent, or an arylacetic acid-based non-steroidal anti-inflammatory agent, and the leukotriene receptor antagonist is a cysteinyl leukotriene type 1 (CysLT1) receptor antagonist or a 5-lipoxygenase inhibitor.

[0332] As described above, the drusen inhibitor, wherein the propionic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of zaltoprofen, oxaprozin, teipervic acid, ibuprofen, flurbiprofen and their pharmaceutically acceptable salts; the aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of flufenamic acid, mefenamic acid and their pharmaceutically acceptable salts; the arylacetic acid-based non-steroidal anti-inflammatory agent is sulindac. and / or its pharmaceutically acceptable salts; the aforementioned aldose reductase inhibitor is epalrestat and / or its pharmaceutically acceptable salts; the aforementioned leukotriene receptor antagonist is at least one selected from the group consisting of zafirlukast, montelukast, pranlukast and their pharmaceutically acceptable salts; the aforementioned chemical mediator release inhibitor is amlexanox and / or its pharmaceutically acceptable salts; and / or the thromboxane A2 receptor antagonist is seratrokast and / or its pharmaceutically acceptable salts.

[0333] As described above, the drusen inhibitor, wherein the compound having epithelial-mesenchymal transition inhibitory activity is selected from alisertib, MK-0457 (Tozasertib), PHA-739358 (danusertib), AMG-900, barasertib, CYC116, MLN8054, baicalin, baicalein, lupeol, and istanbulin. A (9a-hydroxy-3,4a,5-trimethyl-4,5,6,7,8a,9-hexahydrobenzo[f][1]benzofuran-2,8-dione), Phytol, EF24, Crucmin, Phloroglucinol, Plumbagin, Rapamycin, FK506 (Tacrolimus), Thalidomide, LY550410, SB-505124, SD-208, TAPI-0, TAPI-1, JNJ-38877605, PF-04217903, AG1478 (Tyrphostin), Erlotinib, Gefitinib, Lapatinib, PD153035, PD158780, WHI-P154, BMS-536924, A83-01, D4476, LY-364947, SB-431542, SD-208, AZD6244 (Selumetinib), CI-1040, PD0325901, GDC-0941 (Pictilisib (4-[2-(1H-indazole-4- 1-[(4-methylsulfonylpiperazin-1-yl)methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine), PI-103, PIK-90, ZSTK474, API-2, AZD0530 (Saracatinib), PP1, 2-Hydroxycinnamaldehyde, 5-aza-dC, BI 5700, Celecoxib, CX-4945 (Silmitasertib (5-[(3-chlorophenyl)amino]benzo[c][2,6] naphthyridine-8-carboxylic acid), disulfiram, eribulin mesyate, evodiamine, EW-7203, fasudil, nintedanib, fuzheng huayu recipe, grape seed proanthocyanidins, vorinostat, herbimycin A, entinostat, honokiol, NPI-0052, methacycline, dasatinib, Ki26894, NSC 74859, NVP-LDE-225 (Erismodegib), Palbociclib, Pinocembrin, Salvianolic Acid B, Sorafenib, Resveratrol, S-Allylcysteine, Silibinin meglumine, Simvastatin, Centchroman, ML327, GN-25, Trichostatin A, Sarasinoside A1, Panobinostat, Danusertib, Cystatin C, Thymoquinone, Ulinastatin, Dendrofalconerol A (DF-A), ginsenoside (ginseng saponin), Celastrus orbiculatus seed extract, salicin (Willow white willow extract), salicylic acid, scutellaria extract, osthole, muscadine grape skin extract, Tongxinluo, procyanidin C1 (cinnamon bark), Withania somnifera root extract, Qingyihuaji, roselle extract, epigallocatechin gallate, proanthocyanidins (grape seed extract) and salvianolic acid B.

[0334] As the aforementioned drusen inhibitor, it is an oral agent or an injection.

[0335] As described above, the drusen inhibitor, wherein the oral preparation is a solid preparation.

[0336] As mentioned above, the drusen inhibitor, wherein the solid preparation is a tablet.

[0337] As the aforementioned drusen inhibitor, it is a liquid preparation.

[0338] The drusen inhibitor as described above, wherein the liquid preparation is an ophthalmic preparation or a capsule preparation.

[0339] As in the aforementioned drusen inhibitor, zaltoprofen or a salt thereof is administered in an amount that gives a Cmax of 20.4 μM or more.

[0340] As in the aforementioned drusen inhibitor, zaltoprofen or a salt thereof is administered in an amount that gives a Cmax of 16.8 μM or more.

[0341] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0342] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the above Cmax.

[0343] As in the aforementioned drusen inhibitor, oxaprozin or a salt thereof is administered in an amount that produces a Cmax of 340.9 μM or more.

[0344] As described above, the drusen inhibitor is a drug in which oxaprozin or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0345] As described above, the drusen inhibitor is a drug in which oxaprozin or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the Cmax described above.

[0346] As in the aforementioned drusen inhibitor, teipfic acid or a salt thereof is administered in an amount that gives a Cmax of 69.15 μM or more.

[0347] As in the aforementioned drusen inhibitor, teipfic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0348] As in the aforementioned drusen inhibitor, teipfic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0349] As in the aforementioned drusen inhibitor, flufenamic acid or a salt thereof is administered in an amount that produces a Cmax of 27.91 μM or more.

[0350] As described above, the drusen inhibitor is a drug in which flufenamic acid or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0351] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the above Cmax.

[0352] As in the aforementioned drusen inhibitor, mefenamic acid or a salt thereof is administered in an amount that gives a Cmax of 38.54 μM or more.

[0353] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0354] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the above Cmax.

[0355] As in the aforementioned drusen inhibitor, sulindac or a salt thereof is administered in an amount that gives a Cmax of 10.10 μM or more.

[0356] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 2 or more of the Cmax described above.

[0357] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the Cmax described above.

[0358] As in the aforementioned drusen inhibitor, epalrestat or a salt thereof is administered in an amount that gives a Cmax of 12.2 μM or more.

[0359] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0360] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the above Cmax.

[0361] As in the aforementioned drusen inhibitor, zafirlukast or a salt thereof is administered in an amount that gives a Cmax of 0.8 μM or more.

[0362] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0363] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the Cmax described above.

[0364] As in the aforementioned drusen inhibitor, amlexanox or a salt thereof is administered in an amount that produces a Cmax of 16.0 μM or more.

[0365] As in the aforementioned drusen inhibitor, amlexanox or a salt thereof is administered in an amount that produces a blood concentration of 1 / 2 or more of the aforementioned Cmax.

[0366] As in the aforementioned drusen inhibitor, amlexanox or a salt thereof is administered in an amount that produces a blood concentration of 1 / 4 or more of the aforementioned Cmax.

[0367] As in the aforementioned drusen inhibitor, seratrolast or a salt thereof is administered in an amount that gives a Cmax of 31.03 μM or more.

[0368] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 2 or more of the above Cmax.

[0369] As described above, the drusen inhibitor is administered in an amount that produces a blood concentration of 1 / 4 or more of the above Cmax.

[0370] As the aforementioned drusen inhibitor, it contains 25 mg to 360 mg of zaltoprofen or a salt thereof as a daily dose.

[0371] As the aforementioned drusen inhibitor, it contains 100 mg to 700 mg of oxaprozin or a salt thereof as a daily dose.

[0372] As the aforementioned drusen inhibitor, it contains 60 mg to 900 mg of teiperic acid or a salt thereof as a daily dose.

[0373] As the aforementioned drusen inhibitor, it contains 75 mg to 1250 mg of flufenamic acid or a salt thereof as a daily dose.

[0374] As the aforementioned drusen inhibitor, it contains 150 mg to 2250 mg of mefenamic acid or a salt thereof as a daily dose.

[0375] As the aforementioned drusen inhibitor, it contains 30 mg to 500 mg of sulindac or a salt thereof as a daily dose.

[0376] As the aforementioned drusen inhibitor, it contains 10 mg to 400 mg of epalrestat or a salt thereof as a daily dose.

[0377] As the aforementioned drusen inhibitor, it contains 10 mg to 200 mg of zafirlukast or a salt thereof as a daily dose.

[0378] As the aforementioned drusen inhibitor, it contains 10 mg to 150 mg of amlexanox or a salt thereof as a daily dose.

[0379] As the aforementioned drusen inhibitor, it contains 8 mg to 120 mg of seratrodast or a salt thereof as a daily dose.

[0380] As with the aforementioned drusen inhibitor, it is administered once to three times a day.

[0381] As with the aforementioned drusen inhibitors, the drug is administered once or twice a day.

[0382] As the above-mentioned drusen inhibitor, it is administered once a day.

[0383] A preventive and / or therapeutic agent for age-related macular degeneration, comprising the above-mentioned drusen inhibitor as an active ingredient.

[0384] A preventive and / or therapeutic agent for age-related macular degeneration, comprising a propionic acid-based non-steroidal anti-inflammatory agent, an aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent, an arylacetic acid-based non-steroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor, or a thromboxane A2 receptor antagonist as an active ingredient, wherein the aldose reductase inhibitor is epalrestat and / or a pharmaceutically acceptable salt thereof.

[0385] As described above, the agent for preventing and / or treating age-related macular degeneration, wherein the propionic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of zaltoprofen, oxaprozin, ibuprofen, flurbiprofen and pharmaceutically acceptable salts thereof; the aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of flufenamic acid, mefenamic acid and pharmaceutically acceptable salts thereof; the arylacetic acid-based non-steroidal anti-inflammatory agent is sulindac and / or a pharmaceutically acceptable salt thereof; the leukotriene receptor antagonist is at least one selected from the group consisting of zafirlukast, montelukast, pranlukast and pharmaceutically acceptable salts thereof; the chemical mediator release inhibitor is amlexanox and / or a pharmaceutically acceptable salt thereof; the thromboxane A2 receptor antagonist is serotonin and / or a pharmaceutically acceptable salt thereof.

[0386] The aforementioned preventive and / or therapeutic agent for age-related macular degeneration is an oral agent or an injectable agent.

[0387] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the oral preparation is a solid preparation.

[0388] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the solid preparation is a tablet.

[0389] The aforementioned preventive and / or therapeutic agent for age-related macular degeneration is a liquid preparation.

[0390] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the liquid preparation is an ophthalmic preparation or a capsule preparation.

[0391] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the age-related macular degeneration is dry AMD.

[0392] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the age-related macular degeneration is wet AMD.

[0393] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the age-related macular degeneration is non-AMD, early AMD and / or intermediate AMD.

[0394] A preventive and / or therapeutic agent for age-related macular degeneration is a combination of an angiogenesis inhibitor and the aforementioned drusen inhibitor.

[0395] The preventive and / or therapeutic agent for age-related macular degeneration as described above, wherein the angiogenesis inhibitor is an anti-VEGF antibody or a fragment of the VEGF antibody, a fusion protein of an anti-VEGF antibody fragment and the Fc region of an IgG molecule, and / or an anti-angiogenesis aptamer.

[0396] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the angiogenesis inhibitor is at least one selected from the group consisting of bevacizumab, ranibizumab, aflibercept, pegaptanib sodium, ESBA-1008, lambazumab, MC-1101, doxycycline hydrochloride, and amitraz hydrochloride.

[0397] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the drusen inhibitor is an oral agent or an injectable agent.

[0398] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the oral preparation is a solid preparation.

[0399] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the solid preparation is a tablet.

[0400] The aforementioned preventive and / or therapeutic agent for age-related macular degeneration is a liquid preparation.

[0401] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the liquid preparation is an ophthalmic preparation or a capsule preparation.

[0402] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the age-related macular degeneration is dry AMD.

[0403] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the age-related macular degeneration is wet AMD.

[0404] The aforementioned agent for preventing and / or treating age-related macular degeneration, wherein the age-related macular degeneration is non-AMD, early AMD and / or intermediate AMD.

[0405] A method for inducing drusen in vitro comprises contacting cells with at least one selected from the group consisting of cytokines, growth factors, and chemokines.

[0406] The method as described above, wherein the cells are retinal pigment epithelial cells.

[0407] The method as described above, wherein the cells are ARPE-19.

[0408] The method as described above, wherein the cytokine is at least one selected from the group consisting of IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-16, TNF-α and IFN-γ.

[0409] The method as described above, wherein the growth factor is at least one selected from the group consisting of TGF-β, FGFs, HGF, EGF and PDGF.

[0410] The method as described above, wherein the chemokine is at least one selected from the group consisting of IL-8, MIP-1α, MCP-1, RANTES, and eotaxin.

[0411] The method as described above, wherein the contact time is from 10 minutes to 108 hours.

[0412] The method as mentioned above, wherein the contacting temperature is 4°C to 50°C.

[0413] As in the aforementioned method, wherein the induction of drusen comprises evaluating using epithelial markers, mesenchymal markers, ECM secretion, increased motility and / or Focus formation as indicators.

[0414] A method for evaluating or screening drusen inhibitors comprises: measuring the inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells in vitro in the presence of a test agent.

[0415] A method for evaluating or screening drusen inhibitors comprises: measuring the inhibition of retinal pigment epithelial cell aggregation in vitro in the presence of a test agent.

[0416] A method for evaluating or screening a drug for preventing and / or treating age-related macular degeneration comprises: measuring the inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells in vitro in the presence of a test drug.

[0417] A method for evaluating or screening a drug for preventing and / or treating age-related macular degeneration comprises: measuring the inhibition of aggregation of retinal pigment epithelial cells in vitro in the presence of a test drug.

[0418] The aforementioned evaluation or screening method comprises: contacting the retinal pigment epithelial cells with at least one selected from the group consisting of cytokines, growth factors, and chemokines.

[0419] As in the aforementioned evaluation or screening method, wherein the aforementioned retinal pigment epithelial cells are ARPE-19.

[0420] As in the aforementioned evaluation or screening method, wherein the aforementioned cytokine is at least one selected from the group consisting of IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-16, TNF-α and IFN-γ.

[0421] The aforementioned evaluation or screening method, wherein the growth factor is at least one selected from the group consisting of TGF-β, FGFs, HGF, EGF and PDGF.

[0422] The aforementioned evaluation or screening method, wherein the chemokine is at least one selected from the group consisting of IL-8, MIP-1α, MCP-1, RANTES, and eotaxin.

[0423] As in the aforementioned evaluation or screening method, wherein the aforementioned contact time is from 10 minutes to 108 hours.

[0424] As in the aforementioned evaluation or screening method, wherein the contacting temperature is 4°C to 50°C.

[0425] A use of a compound having epithelial-mesenchymal transition inhibitory activity for preparing a preventive and / or therapeutic agent for choroidal neovascularization.

[0426] A use of a compound having epithelial-mesenchymal transition inhibitory activity for preparing a VEGF production inhibitor.

[0427] A use of a compound having epithelial-mesenchymal transition inhibitory activity for preparing a drusen inhibitor.

[0428] A use of a compound having epithelial-mesenchymal transition inhibitory activity for preparing an agent for preventing and / or treating age-related macular degeneration.

[0429] The use of the aforementioned compound, wherein the aforementioned compound having epithelial-mesenchymal transition inhibitory activity is an agent that inhibits the expression of mesenchymal markers or extracellular matrix in the aforementioned retinal pigment epithelial cells.

[0430] The use of the compound as described above, wherein the mesenchymal marker is at least one selected from the group consisting of Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2 and VIM.

[0431] The use of the compound as described above, wherein the extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR, TNC, COL1A1, COL1A2, SRGN, FN1, COL5A2, COL13A1 and LAMB3.

[0432] The use of the aforementioned compound, wherein the aforementioned mesenchymal marker is at least one selected from the group consisting of Snail, Slug, E-cadherin 3, MMP1 and MMP7, and / or the aforementioned extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR and TNC.

[0433] The use of the aforementioned compound, wherein the aforementioned compound having epithelial-mesenchymal transition inhibitory activity is a non-steroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor and / or a thromboxane A2 receptor antagonist.

[0434] The use of the aforementioned compound, wherein the aforementioned non-steroidal anti-inflammatory agent is a propionic acid-based non-steroidal anti-inflammatory agent, an aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent, or an arylacetic acid-based non-steroidal anti-inflammatory agent; and the aforementioned leukotriene receptor antagonist is a cysteinyl leukotriene type 1 (CysLT1) receptor antagonist or a 5-lipoxygenase inhibitor.

[0435] The use of the aforementioned compound, wherein the aforementioned propionic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of zaltoprofen, oxaprozin, teiprenic acid, ibuprofen, flurbiprofen and their pharmaceutically acceptable salts; the aforementioned aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of flufenamic acid, mefenamic acid and their pharmaceutically acceptable salts; the aforementioned arylacetic acid-based non-steroidal anti-inflammatory agent is sulindac and / or its pharmaceutically acceptable salt; the aforementioned aldose reductase inhibitor is epalrestat and / or its pharmaceutically acceptable salt; the aforementioned leukotriene receptor antagonist is at least one selected from the group consisting of zafirlukast, montelukast, pranlukast and their pharmaceutically acceptable salts; the aforementioned chemical mediator release inhibitor is amlexanox and / or its pharmaceutically acceptable salt; and / or the thromboxane A2 receptor antagonist is seratrokast and / or its pharmaceutically acceptable salt.

[0436] The use of the aforementioned compound, wherein the aforementioned compound having epithelial-mesenchymal transition inhibitory activity is selected from alisertib, MK-0457 (Tozasertib), PHA-739358 (danusertib), AMG-900, Barasertib, CYC116, MLN8054, Baicalin, Baicalein, Lupeol, Istanbulin A (9a-hydroxy-3,4a,5-trimethyl-4,5,6,7,8a,9-hexahydrobenzo[f][1]benzofuran-2,8-dione), Phytol, EF24, Crucmin, Phloroglucinol, Plumbagin, Rapamycin, FK506 (Tacrolimus), Thalidomide, LY550410, SB-505124, SD-208, TAPI-0, TAPI-1, JNJ-38877605, PF-04217903, AG1478 (Tyrphostin), Erlotinib, Gefitinib, Lapatinib, PD153035, PD158780, WHI-P154, BMS-536924, A83-01, D4476, LY-364947, SB-431542, SD-208, AZD6244 (Selumetinib), CI-1040, PD0325901, GDC-0941 (Pictilisib (4-[2-(1H-indazole-4- 1-[(4-methylsulfonylpiperazin-1-yl)methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine), PI-103, PIK-90, ZSTK474, API-2, AZD0530 (Saracatinib), PP1, 2-Hydroxycinnamaldehyde, 5-aza-dC, BI 5700, Celecoxib, CX-4945 (Silmitasertib (5-[(3-chlorophenyl)amino]benzo[c][2,6] naphthyridine-8-carboxylic acid), disulfiram, eribulin mesyate, evodiamine, EW-7203, fasudil, nintedanib, fuzheng huayu recipe, grape seed proanthocyanidins, vorinostat, herbimycin A, entinostat, honokiol, NPI-0052, methacycline, dasatinib, Ki26894, NSC 74859, NVP-LDE-225 (Erismodegib), Palbociclib, Pinocembrin, Salvianolic Acid B, Sorafenib, Resveratrol, S-Allylcysteine, Silibinin meglumine, Simvastatin, Centchroman, ML327, GN-25, Trichostatin A, Sarasinoside A1, Panobinostat, Danusertib, Cystatin C, Thymoquinone, Ulinastatin, Dendrofalconerol A (DF-A), ginsenoside (ginseng saponin), Celastrus orbiculatus seed extract, salicin (Willow white willow extract), salicylic acid, scutellaria extract, osthole, muscadine grape skin extract, Tongxinluo, procyanidin C1 (cinnamon bark), Withania somnifera root extract, Qingyihuaji, roselle extract, epigallocatechin gallate, proanthocyanidins (grape seed extract) and salvianolic acid B.

[0437] The use of the aforementioned compound is oral or injection.

[0438] The use of the aforementioned compound, wherein the aforementioned oral preparation is a solid preparation.

[0439] The use of the compound as mentioned above, wherein the solid preparation is a tablet.

[0440] As for the use of the aforementioned compound, it is a liquid preparation.

[0441] The use of the aforementioned compound, wherein the aforementioned liquid preparation is an ophthalmic preparation or a capsule preparation.

[0442] A compound having epithelial-mesenchymal transition inhibitory activity, used for the prevention and / or treatment of choroidal neovascularization.

[0443] A compound having epithelial-mesenchymal transition inhibitory activity, used to inhibit the production of VEGF.

[0444] A compound with epithelial-mesenchymal transition inhibitory activity for inhibiting drusen.

[0445] A compound having epithelial-mesenchymal transition inhibitory activity, for preventing and / or treating age-related macular degeneration.

[0446] The compound as described above, wherein the compound having epithelial-mesenchymal transition inhibitory activity is an agent that inhibits the expression of a mesenchymal marker or an extracellular matrix in the retinal pigment epithelial cells.

[0447] The compound as described above, wherein the mesenchymal marker is at least one selected from the group consisting of Snail, Slug, CDH3, MMP1, MMP7, MMP3, ZEB2, CDH2 and VIM.

[0448] The compound as described above, wherein the extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR, TNC, COL1A1, COL1A2, SRGN, FN1, COL5A2, COL13A1 and LAMB3.

[0449] As in the aforementioned compound, wherein the aforementioned mesenchymal marker is at least one selected from the group consisting of Snail, Slug, E-cadherin 3, MMP1 and MMP7; and / or the aforementioned extracellular matrix is ​​at least one selected from the group consisting of COL5A3, COL6A3, LAMC2, HMMR and TNC.

[0450] As mentioned above, the compound having epithelial-mesenchymal transition inhibitory activity is a non-steroidal anti-inflammatory agent, an aldose reductase inhibitor, a leukotriene receptor antagonist, a chemical mediator release inhibitor and / or a thromboxane A2 receptor antagonist.

[0451] As described above, the non-steroidal anti-inflammatory agent is a propionic acid-based non-steroidal anti-inflammatory agent, an aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent, or an arylacetic acid-based non-steroidal anti-inflammatory agent; and the leukotriene receptor antagonist is a cysteinyl leukotriene type 1 (CysLT1) receptor antagonist or a 5-lipoxygenase inhibitor.

[0452] As described above, the propionic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of zaltoprofen, oxaprozin, teipervic acid, ibuprofen, flurbiprofen and their pharmaceutically acceptable salts; the aminoarylcarboxylic acid-based non-steroidal anti-inflammatory agent is at least one selected from the group consisting of flufenamic acid, mefenamic acid and their pharmaceutically acceptable salts; the arylacetic acid-based non-steroidal anti-inflammatory agent is sulindac and / or or a pharmaceutically acceptable salt thereof; the aforementioned aldose reductase inhibitor is epalrestat and / or a pharmaceutically acceptable salt thereof; the aforementioned leukotriene receptor antagonist is at least one selected from the group consisting of zafirlukast, montelukast, pranlukast and pharmaceutically acceptable salts thereof; the aforementioned chemical mediator release inhibitor is amlexanox and / or a pharmaceutically acceptable salt thereof; and / or the thromboxane A2 receptor antagonist is seratrokast and / or a pharmaceutically acceptable salt thereof.

[0453] As described above, the compound having epithelial-mesenchymal transition inhibitory activity is selected from alisertib, MK-0457 (Tozasertib), PHA-739358 (danusertib), AMG-900, Barasertib, CYC116, MLN8054, Baicalin, Baicalein, Lupeol, Istanbulin A (9a-hydroxy-3,4a,5-trimethyl-4,5,6,7,8a,9-hexahydrobenzo[f][1]benzofuran-2,8-dione), Phytol, EF24, Crucmin, Phloroglucinol, Plumbagin, Rapamycin, FK506 (Tacrolimus), Thalidomide, LY550410, SB-505124, SD-208, TAPI-0, TAPI-1, JNJ-38877605, PF-04217903, AG1478 (Tyrphostin), Erlotinib, Gefitinib, Lapatinib, PD153035, PD158780, WHI-P154, BMS-536924, A83-01, D4476, LY-364947, SB-431542, SD-208, AZD6244 (Selumetinib), CI-1040, PD0325901, GDC-0941 (Pictilisib (4-[2-(1H-indazole-4- 1-[(4-methylsulfonylpiperazin-1-yl)methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine), PI-103, PIK-90, ZSTK474, API-2, AZD0530 (Saracatinib), PP1, 2-Hydroxycinnamaldehyde, 5-aza-dC, BI 5700, Celecoxib, CX-4945 (Silmitasertib (5-[(3-chlorophenyl)amino]benzo[c][2,6] naphthyridine-8-carboxylic acid), disulfiram, eribulin mesyate, evodiamine, EW-7203, fasudil, nintedanib, fuzheng huayu recipe, grape seed proanthocyanidins, vorinostat, herbimycin A, entinostat, honokiol, NPI-0052, methacycline, dasatinib, Ki26894, NSC 74859, NVP-LDE-225 (Erismodegib), Palbociclib, Pinocembrin, Salvianolic Acid B, Sorafenib, Resveratrol, S-Allylcysteine, Silibinin meglumine, Simvastatin, Centchroman, ML327, GN-25, Trichostatin A, Sarasinoside A1, Panobinostat, Danusertib, Cystatin C, Thymoquinone, Ulinastatin, Dendrofalconerol A (DF-A), ginsenoside (ginseng saponin), Celastrus orbiculatus seed extract, salicin (Willow white willow extract), salicylic acid, scutellaria extract, osthole, muscadine grape skin extract, Tongxinluo, procyanidin C1 (cinnamon bark), Withania somnifera root extract, Qingyihuaji, roselle extract, epigallocatechin gallate, proanthocyanidins (grape seed extract) and salvianolic acid B.

[0454] As the aforementioned compound, it is an oral agent or an injection.

[0455] The compound as described above, wherein the oral preparation is provided in the form of a solid preparation.

[0456] The compound as mentioned above, wherein the solid preparation is a tablet.

[0457] The aforementioned compound is provided in the form of a liquid.

[0458] The compound as described above, wherein the liquid preparation is an ophthalmic preparation or a capsule preparation.

[0459] [Example]

[0460] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0461] Test Examples 1 to 3 and Reference Test Examples 2 to 4 represent tests on the prevention and / or treatment of CNV.

[0462] Test Examples 4 to 8 represent tests on the prevention and / or treatment of drusen.

[0463] [Test Example 1: Construction of EMT cell model]

[0464] Experimental Example 1-1: Study on the induction conditions of EMT in RPE cells

[0465] The inventors conducted an experiment to stimulate RPE cells (retinal pigment epithelial cell line: ARPE-19, the same below) under 10 conditions using cytokines or growth factors (TNF-α / IL-1β / TGF-β), and found that significant EMT induction was induced. It is believed that the EMT-induced cell model using cytokines or growth factors reflects the inferred EMT induction mechanism of patients with CNV.

[0466] The 10 conditions are as follows.

[0467] (1) TNF-α 100 ng / mL

[0468] (2) IL-1β 100 ng / mL

[0469] (3) IL-1β 20 ng / mL

[0470] (4)TNF-α500ng / mL+TGF-β5ng / mL

[0471] (5)TNF-α200ng / mL+TGF-β5ng / mL

[0472] (6)TNF-α100ng / mL+TGF-β5ng / mL

[0473] (7)TNF-α100ng / mL+TGF-β10ng / mL

[0474] (8)TNF-α100ng / mL+TGF-β25ng / mL

[0475] (9)IL-1β100ng / mL+TGF-β5ng / mL

[0476] (10)IL-1β20ng / mL+TGF-β5ng / mL

[0477] Experimental Example 1-2: Verification of EMT by Expression Changes of EMT Marker Molecules

[0478] RNA was extracted from stimulated RPE cells and expression changes were verified.

[0479] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 1-1, and after 48 hours of EMT induction, cells were recovered. The recovered cells were washed with PBS, 350 μL of RLT buffer (QIAGEN company) was added, and RNA collection was lysate was made. Using RNeasy micro kit (QIAGEN company #74004), total RNA was extracted from the lysate, and SuperScript III First-Strand Synthesis SuperMix (Invitrogen company #11752-250) for qRT-PCR was used to make cDNA from the RNA 2 μg obtained. Using the obtained cDNA 50ng as a template, real-time PCR (ABIPrism 7500 sequence detection system) was performed according to the manufacturer's draft, and the expression of the mRNA of each gene was quantitatively analyzed. Specific primers for each gene were purchased from Invitrogen company. The Ct (Threshold Cycle) value of each gene was normalized by the intrinsic control and the ratio relative to the untreated sample was calculated. The results are shown in Figure 1 .

[0480] like Figure 1 A and Figure 1 As shown in Figure B, the expression of E-cadherin, a marker of epithelial lineage, was decreased in stimulated RPE cells ( Figure 1 A), the expression of N-cadherin, a mesenchymal marker, was upregulated ( Figure 1 B in the figure). This cadherin conversion is a representative phenomenon of EMT. In addition, the mesenchymal marker represented by fibronectin ( Figure 1C) and the hyperregulation of EMT-related transcription factors (Snail, Slug, ZEB1) ( Figure 1 E to Figure 1 G in the figure) and the expression of ECM represented by type I collagen (COL1A1) is increased ( Figure 1 D) Induction of EMT in RPE cells was confirmed by stimulation.

[0481] Test Example 1-3: Verification of temporal changes in EMT marker expression using microarray The EMT induction state at this point in the EMT induction time was analyzed.

[0482] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 1-1, and cells were recovered after 1 hour, 4 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours of EMT induction. The recovered cells were washed with PBS, 350 μL of RLT buffer was added, and RNA was collected and lysate was made. Using RNeasy micro kit (QIAGEN company #74004), total RNA was extracted from the lysate, and fluorescent labeling was performed using Low InputQuickAmp calibration kit (Agilent Technologies company #5190-2305). Labeled cRNA was hybridized using a human whole genome oligo DNA microarray kit (Agilent Technologies company #G4112F) and in accordance with the protocol specified by the manufacturer. The hybridized slides were cleaned using the Gene Expression Wash Pack (Agilent Technologies #5188-5327) and scanned using the Agilent microarray scanner (Agilent Technologies #G2505B). The scanned images were digitized using Feature Extraction software version 9.5.1 (Agilent Technologies). The numerical data were normalized using the Grobal normalization method. The results are shown in Figure 2 .

[0483] like Figure 2 As shown, the peak of the decrease or increase of EMT markers was 12 hours or 72 hours after EMT induction depending on each gene. Although there were variations, the decreased epithelial markers and the increased mesenchymal markers and ECM were as follows.

[0484] As epithelial markers decreased by EMT induction, ID1, ID2, MUC1, cytokeratin 18 (KRT18), THBS1, VIL2, and E-cadherin (CDH1) were confirmed. In addition, a decrease in TGFB2, a growth factor, was confirmed.

[0485] As mesenchymal markers and ECMs that are hyperregulated due to EMT induction, TWIST1, VIM, CD44, ZEB1, RDX, MMP9, FN1, RHOA, ZEB2, MMP2, TJP1, CTNNB1, MMP3, ETS1, SNAI1, SNAI2, HAS2, SERPINE1, CDH2, MSN, TCF3, SDC1, ITGAV, COL1A1, and SPARC were confirmed. In addition, the hyperregulation of growth factors FGF1, FGF2, and TGFB1 was confirmed.

[0486] [Test Example 2: CNV inhibition test]

[0487] Experimental Example 2-1: Expression changes of EMT markers 1

[0488] The EMT inhibitory effects of five compounds (oxaprozin, epalrestat, zafirlukast, amlexanox, and zaltoprofen) were evaluated using the aforementioned cell model.

[0489] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 1-1, and each compound (oxaprozin, epalrestat, zafirlukast, amlexanox, zaltoprofen) was added at the same time. After 48 hours of EMT induction, the cells were recovered. As a control, a sample without the addition of the evaluation compound (tested agent) was used. The recovered cells were washed with PBS, 350 μL of RLT buffer was added, and RNA was collected and dissolved. Each compound was used at the following concentrations. Oxaprozin: 300 μM, epalrestat: 30 μM, zafirlukast: 3 μM, amlexanox: 30 μM, zaltoprofen: 30 μM. Total RNA was extracted from the lysate using the RNeasy micro kit (QIAGEN #74004), and cDNA was prepared from 2 μg of RNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen #11752-250) for qRT-PCR. 50 ng of the obtained cDNA was used as a template, and real-time PCR (ABI Prism 7500 sequence detection system) was performed according to the manufacturer's protocol to quantitatively analyze the expression of mRNA of each gene. Specific primers for each gene were purchased from Invitrogen. The Ct (Threshold Cycle) value of each gene was normalized by an intrinsic control, and the ratio relative to the non-treated sample was calculated. The results are shown in Figures 3 to 7 .

[0490] exist Figures 3 to 7 In each graph, the bar graph on the left represents the expression level of each gene when EMT is not induced. The bar graph in the middle represents the expression level of each gene when EMT is induced. The bar graph on the right represents the expression level of each gene when an EMT inhibitory compound is given. In addition, Figures 3 to 7 The results of using oxaprozin, epalrestat, zafirlukast, amlexanox, and zaltoprofen as EMT inhibitory compounds are shown.

[0491] In this specification, each EMT marker is also described by the following abbreviations.

[0492] Cadherin: CDH (CDH2, CDH3, etc.)

[0493] Matrix metalloproteinases: MMP (MMP1, MMP3, MMP7, etc.)

[0494] Fibronectin: FN (FN1, etc.)

[0495] Vimentin: VIM

[0496] Collagen type 1α1: COL1A1

[0497] Collagen type 1α2: COL1A2

[0498] Collagen type 5α2: COL5A2

[0499] Collagen type 5α3: COL5A3

[0500] Collagen type 6α3: COL6A3

[0501] Collagen type 13α: COL13A1

[0502] Laminin beta 3: LAMB3

[0503] Laminin gamma 2: LAMC2

[0504] Hyaluronan-mediated motility factor receptor: HMMR

[0505] Tenascin C: TNC

[0506] Zinc Finger E-Box Binding Homeobox 2: ZEB2

[0507] Serglycin: SRGN

[0508] Chondroitin sulfate proteoglycan 4: CSPG4

[0509] exist Figures 3 to 7 In each of the graphs, the first row from the left is Snail, Slug, CDH3, COL5A3, COL6A3, and the second row from the left is MMP1, MMP7, LAMC2, HMMR, TNC.

[0510] Figure 3 (Oxaprozin) The third row from the left is ZEB2, MMP3, COL1A1, COL1A2, CSPG4, and the fourth row is SRGN.

[0511] Figure 4 The third row of (epalrestat) from the left are ZEB2, CDH2, FN1, VIM, COL1A1, and the fourth row from the left are COL1A2, COL5A2, COL13A1, CSPG4, SRGN.

[0512] Figure 5 The third row of (Zafirlukast) from the left are MMP3, COL13A1, LAMB3, and CSPG4.

[0513] Figure 6The third row of (Amlexanox) from the left are ZEB2, CDH2, MMP3, COL1A1, COL13A1.

[0514] Figure 7 (Zaltoprofen) The third row from the left is ZEB2, FN1, COL13A1, CSPG4.

[0515] like Figures 3 to 7 As shown in the figure, among the EMT markers, the expressions of Snail, Slug, CDH3, MMP1, MMP7, which are mesenchymal markers, and COL5A3, COL6A3, LAMC2, HMMR, and TNC, which are ECM markers, were all suppressed when any EMT inhibitory compound was applied. Thus, it was confirmed that these 10 EMT markers were commonly suppressed by EMT inhibitory compounds. Even EMT markers other than these 10 were also suppressed. Figure 3 As shown in Figure 2, the expression of ZEB2, MMP3, COL1A1, COL1A2, CSPG4, and SRGN was confirmed to be decreased in response to oxaprozin. Figure 4 As shown in Figure 2, it was confirmed that the expression of ZEB2, CDH2, FN1, VIM, COL1A1, COL1A2, COL5A2, COL13A1, CSPG4, and SRGN was decreased in epalrestat. Figure 5 As shown in Figure 2, zafirlukast confirmed that the expression of MMP3, COL13A1, LAMB3, and CSPG4 decreased. Figure 6 As shown in Figure 2, it was confirmed that the expression of ZEB2, CDH2, MMP3, COL1A1, and COL13A1 was decreased in Amlexanox. Figure 7 As shown, in the case of zaltoprofen, the expression of ZEB2, FN1, COL13A1, and CSPG4 was confirmed to be decreased. It was confirmed that these EMT markers can also be indicators of inhibition by EMT inhibitory compounds.

[0516] according to Figures 3 to 7 , 5 compounds (oxaprozin, epalrestat, zafirlukast, amlexanox, zaltoprofen) showed significant inhibition of EMT. These compounds inhibit the generation of VEGF and other related to the generation or expansion of CNV by significantly inhibiting EMT, and are believed to be able to fundamentally prevent and / or treat CNV.

[0517] Experimental Example 2-2: Expression changes of EMT markers 2

[0518] For 5 compounds (flufenamic acid aluminum (added concentration: 28μM (Cmax)), mefenamic acid (added concentration: 39μM (Cmax)), sulindac (added concentration: 100μM), teprofenic acid (added concentration: 1000μM), seroquel (added concentration: 31μM (Cmax))), the expression changes of EMT markers were evaluated by the same method as in Experimental Example 2-1. The results are shown in Figures 8 to 12 .

[0519] Figure 8 (Aluminum flufenamate) The first row from the left is Snail, Slug, COL5A3, COL6A3, MMP1, the second row from the left is MMP7, LAMC2, HMMR, TNC, FN1, the third row from the left is COL1A1, COL1A2, COL13A1, CDH2, MMP3, and the fourth row from the left is VIM, CSPG4.

[0520] Fig. 9 (Mefenamic acid) The first row from the left is Snail, CDH3, COL5A3, COL6A3, MMP1, the second row from the left is MMP7, LAMC2, HMMR, TNC, FN1, the third row from the left is COL1A1, COL1A2, COL13A1, MMP3, VIM, and the fourth row is CSPG4.

[0521] Fig.10 (Sulindac) The first row from the left is Snail, Slug, CDH3, COL5A3, COL6A3, the second row from the left is MMP1, MMP7, LAMC2, HMMR, TNC, the third row from the left is FN1, CDH2, MMP3, VIM, COL1A1, and the fourth row from the left is COL1A2, COL13A1, CSPG4, ZEB2.

[0522] Fig.11 (Tepfiic acid) The first row from the left is Snail, Slug, CDH3, COL5A3, COL6A3, the second row from the left is MMP1, MMP7, LAMC2, HMMR, TNC, the third row from the left is FN1, CDH2, MMP3, VIM, COL1A1, and the fourth row from the left is COL1A2, COL13A1, CSPG4, ZEB2.

[0523] Fig.12The first row of (Serrolast) from the left is Snail, CDH3, COL5A3, COL6A3, MMP1, the second row from the left is MMP7, LAMC2, HMMR, TNC, FN1, the third row from the left is CDH2, MMP3, VIM, COL1A1, COL1A2, and the fourth row from the left is COL13A1, CSPG4, ZEB2.

[0524] like Figures 8 to 12 As shown, 5 compounds (flufenamic acid aluminum, mefenamic acid, sulindac, teipfic acid, serotonin) also showed a significant inhibition of the expression of EMT markers during EMT induction. These compounds inhibit the generation of VEGF and the like associated with the generation or expansion of CNV by significantly inhibiting EMT, and it is believed that CNV can be fundamentally prevented and / or treated.

[0525] Experimental Example 2-3: Changes in VEGF expression 1

[0526] The VEGF inhibitory effects of the three compounds (oxaprozin, epalrestat, and zaltoprofen) were evaluated using the aforementioned cell model.

[0527] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 1-1, and oxaprozin (Cmax: 340.9 μM), epalrestat (Cmax: 12.2 μM), or zaltoprofen (Cmax: 20.4 μM) were added at the same time. After 48 hours of EMT induction, the cells were recovered. Each compound was added at a concentration of 1 / 2 of Cmax and 1 / 4 of Cmax. As a control, a sample to which the evaluation compound (tested agent) was not added was used. The recovered cells were washed with PBS, 350 μL of RLT buffer was added, and RNA was collected and dissolved. Total RNA was extracted from the lysate using the RNeasy micro kit (QIAGEN #74004), and cDNA was prepared from 2 μg of the obtained RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen #11752-250). Using 50 ng of the obtained cDNA as a template, real-time PCR (ABIPrism 7500 sequence detection system) was performed according to the manufacturer's protocol to quantitatively analyze the expression of mRNA of each gene. Specific primers for each gene were purchased from Invitrogen. The Ct (Threshold Cycle) value of each gene was normalized by an intrinsic control, and the ratio relative to the non-treated sample was calculated. In addition, a significant difference test (Student t test) was performed for each result, and "*" was marked when p < 0.05, and "**" was marked when p < 0.01. The results are shown in Figures 13 to 15 .

[0528] like Fig.13 As shown in Figure 2, in RPE cells, VEGF mRNA expression is increased due to the induction of EMT. Oxaprozin inhibits the increased expression of VEGF in a concentration-dependent manner. A significant VEGF inhibitory effect was also observed at a concentration of 1 / 4 of Cmax ( Fig.13 A in the figure). The expression of fibronectin, Snail, and vimentin, which are mesenchymal markers, is increased due to the induction of EMT. Oxaprozin inhibited the increased expression of these mesenchymal markers in a concentration-dependent manner (respectively Fig.13 B to Fig.13 D).

[0529] like Fig.14 As shown in Figure 2, in RPE cells, VEGF mRNA expression is increased due to the induction of EMT. Epalrestat inhibits the increased expression of VEGF in a concentration-dependent manner ( Fig.14A in the figure). Fibronectin, Snail, and vimentin, which are mesenchymal markers, are expressed at elevated levels due to the induction of EMT. Epalrestat inhibits the elevated expression of these mesenchymal markers in a concentration-dependent manner (respectively Fig.14 B to Fig.14 D).

[0530] like Fig.15 As shown in Figure 2, in RPE cells, VEGF mRNA expression is increased due to the induction of EMT. Zaltoprofen inhibits the increased expression of VEGF in a concentration-dependent manner. A significant VEGF inhibitory effect was also observed at a concentration of 1 / 4 of Cmax ( Fig.15 A in the figure). Fibronectin, Snail, and vimentin, which are mesenchymal markers, are expressed at elevated levels due to the induction of EMT. Zaltoprofen concentration-dependently inhibited the elevated expression of these mesenchymal markers (respectively Fig.15 B to Fig.15 D).

[0531] according to Figures 13 to 15 , 3 compounds (oxaprozin, epalrestat, zaltoprofen) by significantly inhibiting EMT, and show the situation of suppressing the hyperexpression of mRNA expression of VEGF. These compounds can fundamentally prevent and / or treat CNV because they can suppress the VEGF that is hyperexpressed due to EMT. In addition, according to these results, it is believed that zafirlukast or amlexanox, which can significantly inhibit EMT, can also inhibit VEGF, and fundamentally prevent and / or treat CNV.

[0532] Reference Test Example 2-4: Changes in VEGF expression 2

[0533] Regarding NSAIDs different from oxaprozin and zaltoprofen, etodolac (Cmax: 42.5 μM) and indomethacin (Cmax: 2.79 μM) were evaluated for their VEGF inhibitory effects by the same method as in Test Example 2-3 using the aforementioned cell model. The results are shown in Fig.16 or Fig.17 .

[0534] like Fig.16 As shown in Figure 2, in RPE cells, VEGF mRNA expression was increased due to the induction of EMT. However, etodolac did not inhibit the increased expression of VEGF ( Fig.16 A in the figure). Fibronectin, Snail, and vimentin, which are mesenchymal markers, were expressed at elevated levels due to the induction of EMT. However, etodolac did not inhibit the elevated expression of these mesenchymal markers. No significant differences were observed under all conditions (respectively Fig.16 B to Fig.16 D).

[0535] like Fig.17 As shown in Figure 2, in RPE cells, VEGF mRNA expression was increased due to the induction of EMT. However, indomethacin did not inhibit the increased expression of VEGF ( Fig.17 A in the figure). The expression of fibronectin, Snail, and vimentin, which are mesenchymal markers, was increased due to the induction of EMT. However, indomethacin did not inhibit the increased expression of these mesenchymal markers. No significant differences were observed under all conditions (respectively Fig.17 B to Fig.17 D).

[0536] Based on the results of etodolac and indomethacin, it is speculated that compounds that cannot inhibit the EMT of RPE cells cannot inhibit the hyperexpression of VEGF and are therefore ineffective for the prevention and / or treatment of CNV.

[0537] Experimental Example 2-5: Changes in VEGF expression 3

[0538] The VEGF inhibitory effect of the three compounds (aluminum flufenamate (Cmax: 27.91 μM), mefenamic acid (Cmax: 38.54 μM), and serostat (Cmax: 31.03 μM)) was evaluated using the aforementioned cell model. The VEGF inhibitory effect was evaluated using the aforementioned cell model in the same manner as in Experimental Example 2-3. The results are shown in Figures 18 to 20 .

[0539] like Fig.18 As shown in Figure 2, in RPE cells, VEGF mRNA expression is increased due to the induction of EMT. Flufenamic acid aluminum inhibits the increased expression of VEGF in a concentration-dependent manner. A significant VEGF inhibitory effect was also observed at a concentration of 1 / 4 of Cmax ( Fig.18 A in the figure). Fibronectin, Snail, and vimentin, which are mesenchymal markers, are overexpressed due to the induction of EMT. Flufenamic acid aluminum inhibits the overexpression of these mesenchymal markers (respectively Fig.18 B to Fig.18 D).

[0540] like Fig.19 As shown in Figure 2, in RPE cells, VEGF mRNA expression is increased due to the induction of EMT. Mefenamic acid inhibits the increased expression of VEGF in a concentration-dependent manner. A significant VEGF inhibitory effect was also observed at a concentration of 1 / 4 of Cmax ( Fig.19 A in the figure). Fibronectin, Snail, and vimentin, which are mesenchymal markers, are overexpressed due to the induction of EMT. Mefenamic acid inhibits the overexpression of these mesenchymal markers (respectively Fig.19 B to Fig.19 D).

[0541] like Fig. 20 As shown in Figure 2, in RPE cells, VEGF mRNA expression is increased due to the induction of EMT. Setrolast inhibits the increased expression of VEGF in a concentration-dependent manner. A significant VEGF inhibitory effect was also observed at a concentration of 1 / 4 of Cmax ( Fig. 20 A in the figure). Fibronectin, Snail, and vimentin, which are mesenchymal markers, are overexpressed due to the induction of EMT. Setrolast inhibits the overexpression of these mesenchymal markers (respectively Fig. 20 B to Fig. 20 D).

[0542] like Figures 18 to 20 As shown, 3 compounds (flufenamic acid aluminum, mefenamic acid, serolast) by significantly inhibiting EMT, and show the situation of suppressing the hyperexpression of mRNA expression of VEGF. These compounds are owing to being able to suppress the VEGF that is hyperexpressed due to EMT, it is inferred that CNV can be fundamentally prevented and / or treated. These compounds are owing to being able to suppress the VEGF that is hyperexpressed due to EMT, and therefore can fundamentally prevent and / or treat CNV. In addition, according to these results, it is believed that zafirlukast or amlexanox that can significantly suppress EMT can also suppress VEGF, and fundamentally prevent and / or treat CNV.

[0543] [Test Example 3: In vivo model test for evaluating CNV inhibition]

[0544] It is known that laser irradiation of the mouse fundus induces choroidal angiogenesis and EMT (reference: Ishikawa, et al., Exp Eye Res. 2016 Jan; 142: 19-25). Therefore, the inhibitory effects of zaltoprofen on CNV and EMT were evaluated using a mouse laser irradiation model.

[0545] A zaltoprofen suspension was prepared by stirring 0.15 g of zaltoprofen (manufactured by Tokyo Chemical Industry Co., Ltd.) and 0.75 g of gum arabic (manufactured by Wako Pure Chemical Industries, Ltd.) in 60 mL of water. A vehicle (Vehicle) was prepared by dissolving 0.75 g of gum arabic in 60 mL of water and stirring.

[0546] Next, the fundus of C57BL / 6J mice (8 weeks old, N=13) was irradiated with laser light at four locations in each eye under a slit lamp using a multicolor laser photocoagulation device. Immediately after irradiation, the medium or zaltoprofen solution (25 mg / kg) was orally administered once a day for 3 weeks, and the following evaluations were performed. It should be noted that (1) and (3) were performed on the same individual.

[0547] (1) CNV evaluation (N=3)

[0548] (2) EMT evaluation: type 1 collagen (N = 10)

[0549] (3) EMT evaluation: Vimentin (N=3)

[0550] Test Example 3-1: Inhibitory effect of zaltoprofen on choroidal neovascularization (CNV) induced by laser irradiation

[0551] Two weeks after laser irradiation, 500 mg of fluorescein sodium (Fluorescite) (1 mL / kg) was administered via the tail vein, and fluorescent fundus photographs were taken about 1 minute after administration. Based on the fluorescent fundus photographs taken, scores were given for the four sites of each eye based on the following evaluation criteria. The results are shown in Fig.21 , the representative image is shown in Fig. 22 .

[0552] [Evaluation criteria]

[0553] ·No fluorescence······0

[0554] ·Slightly fluorescent······1

[0555] Moderate fluorescence observed······2

[0556] ·See strong tendency······3

[0557] like Fig.21 As shown, regarding the CNV score caused by laser irradiation, the result of the zaltoprofen-administered group was significantly lower than that of the vehicle-administered group.

[0558] exist Fig. 22 In the figure, the arrow indicates the part that leaks fluorescence due to CNV. Fig. 22 As shown, CNV caused by laser irradiation was significantly suppressed in the zaltoprofen-administered group (right photograph) compared with the vehicle-administered group (left photograph).

[0559] Test Example 3-2: Zaltoprofen's inhibitory effect on EMT (EMT evaluation: type 1 collagen) induced by laser irradiation

[0560] Three weeks after laser irradiation, the animals were euthanized by cervical dislocation and the eyeballs were removed and fixed by immersion in 4% polyoxymethylene-phosphate buffer (Wako Pure Chemical Industries, Ltd.). The cornea, lens, and retina were removed from the eyeball to form an eye cup, and then washed three times with PBST. Next, dehydration was performed using 50% methanol and then 100% methanol solution, and incubation was performed with a blocking solution (1% BSA, 0.5% TritonX-100 / PBS) for 60 minutes. Furthermore, after standing overnight in the primary antibody (anti-collagen antibody, Rockland), it was reacted in the secondary antibody (Alexa488 antibody, Life technologies) for 1 hour and placed on a slide to prepare a flat stained specimen. The specimen was quantitatively evaluated after taking a photograph of the staining image obtained using an anti-type 1 collagen antibody using a microscope. The results are shown in Fig.23 .

[0561] like Fig.23 As shown in the figure, the amount of type 1 collagen produced by laser irradiation was significantly reduced in the zaltoprofen-administered group compared with the vehicle-administered group (p<0.05). That is, it was found that zaltoprofen exhibited a significant inhibitory effect on EMT.

[0562] Test Example 3-3: Inhibitory effect of zaltoprofen on EMT induced by laser irradiation (EMT evaluation: vimentin)

[0563] Three weeks after laser irradiation, the animals were euthanized by cervical dislocation and the eyeballs were removed and fixed by immersion in 4% paraformaldehyde-phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.). The fixed eyeballs were immersed in 10% sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) and 25% sucrose for 3 hours each, and eye cups were made. Frozen sections were made and attached to slides. The sections were observed under a microscope, and the sections in which the laser irradiation site could be confirmed were selected. Next, the slide with the sections attached was placed in HistoVT One (manufactured by Nacalai Tesque Co., Ltd.) diluted 10 times with water, and immersed at 70°C for 15 minutes. TrueBlack (manufactured by Biotium Co., Ltd.) diluted 20 times with 70% ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the sections, and after standing at room temperature for several minutes, 5% goat serum (manufactured by Dako Japan Co., Ltd.) was added to the sections, and they were left standing at room temperature for 30 minutes for blocking. Next, Can Get Immunostain Solution B (manufactured by Toyobo Co., Ltd.) was added to the sections and left to stand overnight. Can Get was added to the secondary antibody (Alexa Fluor 546, manufactured by Thermo Fisher Scientific) and Hoechst 33342 (manufactured by Dojin Chemical Laboratories, Ltd.). Immunohistochemical staining solution B was added to the sections and allowed to stand for 30 minutes. The sections were observed and photographed using a fluorescence microscope under the following conditions.

[0564] Vimentin: excitation wavelength 520-550nm, detection wavelength 580nm

[0565] ·Nuclear: excitation wavelength 330-385nm, detection wavelength 420nm

[0566] The shooting results are shown in Fig.24 and Fig.25 . Fig.24 indicates the vehicle-administered group. Fig.24 The left photo shows a bright field image, and the right photo shows an overlapping image of vimentin (red) and nuclei (blue). Fig.25 In the figure, the left photo shows vimentin in the vehicle-administered group, and the right photo shows vimentin in the zaltoprofen-administered group. Fig.24 and Fig.25 In the figure, the circle indicates the vimentin-positive area caused by laser irradiation.

[0567] like Fig.24 As shown in the figure, a less pigmented part appeared near the RPE at the laser irradiation site (left photo), and staining of vimentin, a mesenchymal marker, was confirmed (right photo). It is believed that laser irradiation causes RPE to express vimentin and induce epithelial-mesenchymal transition (EMT). In addition, Fig.25 As shown, the RPE of the vehicle-administered group was vimentin-positive (left photo). On the other hand, in the zaltoprofen-administered group, the vimentin-positive area was reduced compared to the vehicle-administered group (right photo).

[0568] It is thus clear that zaltoprofen inhibits EMT in the laser irradiation model. That is, it is believed that zaltoprofen inhibits drusen formation by inhibiting EMT of RPE. In addition, even other compounds different from zaltoprofen, as long as they can significantly inhibit EMT, are speculated to be able to prevent and / or treat CNV.

[0569] [4. Demonstration of EMT in human specimens]

[0570] Test Example 4-1: Observation of the morphology of drusen in patients with AMD

[0571] Healthy control samples and retinal pigment epithelial cell (RPE cell) samples of AMD patients were obtained from the National Disease Research Interchange. These tissue samples were dewaxed by treating with xylene for 5 minutes × 3 times, 100% ethanol for 1 minute × 2 times, 90% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, and water for 5 minutes. Stained with Mei's hematoxylin solution for 4 minutes and eosin solution for 1 minute. Treated in running water for 5 minutes, then treated with 70% ethanol for 1 minute, 80% ethanol for 1 minute, 90% ethanol for 1 minute, 100% ethanol for 1 minute × 2 times, and xylene for 5 minutes × 3 times, and sealed with dronabinol. Dry it at room temperature for more than 15 minutes, and then observe the RPE cells of healthy people and the RPE cells of AMD patients. The staining results obtained by hematoxylin-eosin staining are shown in Fig.26 .

[0572] Through careful research Fig.26 Observation results, the inventors of the present invention etc. found: in the macula of healthy people's control, the morphology of RPE cells is cubic, neatly arranged as a single layer by intercellular adhesion; in the macula of AMD patients where drusen exists, the morphological changes of RPE cells are spindle-shaped, and RPE cells obtain motor ability, and cells are stratified. According to these observation results, the inventors of the present invention etc. speculate that in the macula of AMD patients, the intercellular adhesion of RPE cells weakens and produces morphological changes, thus establishing the following hypothesis: RPE cells have produced the phenomenon of losing epitheliality, i.e. EMT (Epithelial-mesencyamal transition, epithelial-mesenchymal transition). In the following Test Example 4-2 to Test Example 4-4, based on the hypothesis, a test for producing EMT in RPE cells was carried out.

[0573] Test Example 4-2: Disappearance of epithelial markers in drusen of AMD patients

[0574] The disappearance of epithelial markers in healthy control samples and retinal pigment epithelial cell (RPE cell) samples of AMD patients was verified by immunohistochemistry (IHC). After the tissue samples were incubated at 56°C for 30 minutes in a gas phase incubator, they were treated with xylene for 5 minutes × 3 times, 100% ethanol for 1 minute × 2 times, 90% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, and water for 5 minutes to dewax. After removing melanin from the tissue according to the manufacturer's protocol using the Delicate Melanin Bleach Kit for Special Stains and IHC (Polysciences #24909-1), the antigen was activated by microwave treatment for 10 minutes using a citrate buffer pH 6.0. After standing at room temperature for 30 minutes, the intrinsic peroxidase was inactivated by treatment with PBS for 5 minutes, 3% hydrogen peroxide / PBS solution for 5 minutes, and PBS for 5 minutes. The tissue was blocked by standing in a 3% BSA / PBS solution at room temperature for 30 minutes. As the primary antibody, 200 μl of anti-E-cadherin antibody (BD Biosience #610181) diluted 500 times with a 1.5% BSA / PBS solution and anti-cytokeratin 18 antibody (Cell signaling Technology #4548) diluted 1000 times were added to each tissue and reacted with the tissue at 4°C for 17 hours. The tissue reacted with the primary antibody was washed with PBS for 5 minutes × 3 times, and then reacted with the secondary antibody at room temperature for 30 minutes using the Mouse on Mouse (MOM) Elite Peroxidase Kit (Vector Laboratories #PK-2200), washed with PBS for 5 minutes × 3 times, and then ABC reaction was performed at room temperature for 30 minutes according to the manufacturer's protocol. After the tissue was washed with PBS for 5 minutes × 3 times, it was stained at room temperature for 3 minutes using ImmPACT DAB peroxidase (HRP) substrate (Vector Laboratories #SK-4105). After the stained tissue was stained with M-type neohematoxylin (Muto Chemical Co., Ltd. #30141), it was allowed to stand in running water for 3 minutes, treated with 70% ethanol for 1 minute, 80% ethanol for 1 minute, 90% ethanol for 1 minute, 100% ethanol for 1 minute × 2 times, and xylene for 5 minutes × 3 times, and sealed with dronabinol. It was dried at room temperature for more than 15 minutes, and then the RPE cells of healthy people and AMD patients were observed. The results are shown in Fig. 27 .

[0575] like Fig. 27 As shown, for healthy controls (Control), staining of E-cadherin (E-Cadherin) as an epithelial marker was confirmed at the intercellular adhesion site, but in the peripheral part of AMD patient's drusen, staining of E-cadherin was not confirmed between cells (left side staining photo). In addition, for healthy controls, strong staining of cytokeratin 18 (Cytokeratin18) as an epithelial marker was confirmed in the whole RPE cells, but in the peripheral part of AMD patient's drusen, signal reduction (right side staining photo) was compared with it.

[0576] This confirmed that RPE cells in AMD patients had lost and decreased epithelial properties.

[0577] Experimental Example 4-3: Hyperexpression of Mesenchymal Markers in Drusen of AMD Patients

[0578] The hyperactivity of mesenchymal markers in healthy human control samples and retinal pigment epithelial cell (RPE cell) samples of AMD patients was verified by immunohistochemistry (IHC). After the tissue samples were incubated at 56°C in a gas phase incubator for 30 minutes, they were dewaxed by treating with xylene for 5 minutes × 3 times, 100% ethanol for 1 minute × 2 times, 90% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, and water for 5 minutes. Antigens were activated by microwave treatment for 10 minutes using a citric acid buffer pH 6.0. After standing at room temperature for 30 minutes, the intrinsic peroxidase was inactivated by treating with PBS for 5 minutes, 3% hydrogen peroxide / PBS solution for 5 minutes, and PBS for 5 minutes. The tissue was blocked by standing at room temperature for 30 minutes in a 3% BSA / PBS solution. As the primary antibody, 200 μl of anti-fibronectin antibody (abcam #45688) diluted to 100 times with 1.5% BSA / PBS solution and anti-vimentin antibody (Sigma-Aldrich #6630) diluted to 10,000 times were added to each tissue and reacted with the tissue at 4°C for 17 hours. The tissue reacted with the primary antibody was washed with PBS for 5 minutes × 3 times, and then reacted with the secondary antibody at room temperature for 30 minutes using VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories #PK-6101) for anti-fibronectin antibody and Mouse on Mouse (MOM) Elite Peroxidase Kit (Vector Laboratories #PK-2200) for anti-vimentin antibody. After washing with PBS for 5 minutes × 3 times, ABC reaction was performed at room temperature for 30 minutes according to the manufacturer's protocol. After the tissue was washed with PBS for 5 minutes × 3 times, ImmPACT AMEC red peroxidase (HRP) substrate (Vector Laboratories company #SK-4285) was used to develop the color at room temperature for 3 minutes and washed with distilled water for 5 minutes. After the developed tissue was stained with M-type new hematoxylin (Muto Chemical Co., Ltd. #30141), it was left to stand in running water for 3 minutes and sealed with VectaMount AQ water-soluble mounting medium (Vector Laboratories company #H-5501). It was dried at room temperature for more than 15 minutes, and then the RPE cells of healthy people and AMD patients were observed. The results are shown in Fig.28 .

[0579] like Fig.28Shown in, in the normal RPE cell of healthy person control (Control), do not see the dyeing of fibronectin (Fibronectin) as interstitial system mark.On the other hand, in drusen and RPE cell of AMD patient, fibronectin demonstrates positive.In addition, in the RPE cell of healthy person control, do not see the dyeing of vimentin (vimentin) as interstitial system mark.The RPE cell of AMD patient drusen demonstrates vimentin positive.

[0580] The above analysis results clearly show that the RPE cells of AMD patients lose or weaken the epithelial characteristics of health, and acquire mesenchymal characteristics and hypertrophy in AMD. In other words, it is clear that in AMD, RPE cells undergo a transformation from epithelial characteristics to mesenchymal characteristics, epithelial-mesenchymal transition (EMT).

[0581] Test Example 4-4: Verification using cynomolgus monkey model

[0582] Sections were prepared from healthy cynomolgus macaque control samples and retinal pigment epithelial cell (RPE cell) samples of AMD cynomolgus macaque models, and the disappearance of epithelial markers and the hyperplasia of mesenchymal markers were verified by immunohistochemistry (IHC). Tissue samples were dewaxed by treating with xylene for 5 minutes × 3 times, 100% ethanol for 1 minute × 2 times, 90% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, and distilled water for 10 minutes. Antigens were activated by microwave treatment for 10 minutes using a citric acid buffer pH 6.0. After standing at room temperature for 30 minutes, the endogenous peroxidase was inactivated by treating with PBS for 5 minutes, 3% hydrogen peroxide / PBS solution for 5 minutes, and PBS for 5 minutes. The tissue was blocked by standing at room temperature for 20 minutes in a 3% BSA / PBS solution. As the primary antibody, 200 μl of anti-E-cadherin antibody (BD Biosience #610181) diluted 500-fold with 1.5% BSA / PBS solution, anti-cytokeratin 18 antibody (abcam #ab668) diluted 2000-fold, and anti-fibronectin antibody (Epitomics #1574-1) diluted 600-fold were added to each tissue and allowed to react with the tissue at 4°C for 17 hours. The tissue reacted with the primary antibody was washed with PBS for 5 minutes × 3 times, and then reacted with the secondary antibody at room temperature for 30 minutes using VECTASTAIN Elite ABC Mouse IgG Kit (Vector Laboratories #PK-6102) for anti-E-cadherin antibody and anti-cytokeratin 18 antibody, and VECTASTAIN EliteABC Rabbit IgG Kit (Vector Laboratories #PK-6101) for anti-fibronectin antibody, and then washed with PBS for 5 minutes × 3 times, and then ABC reaction was performed at room temperature for 30 minutes according to the attached protocol. The tissue was washed with PBS for 5 minutes × 3 times, and then color was developed at room temperature for 3 minutes using ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories #SK-4105) for anti-E-cadherin antibody, and TMB Peroxidase Substrate Kit (Vector Laboratories #SK-4400) for anti-cytokeratin 18 antibody and anti-fibronectin antibody, and then washed with distilled water for 5 minutes.About the tissue through color development, use M type new hematoxylin (Muto Chemical Co., Ltd. #30141) for anti-E-cadherin antibody, use VECTOR nuclear fast red (Vector Laboratories company #H-3403) for anti-cytokeratin 18 antibody and anti-fibronectin antibody to dye.Stand in running water for 10 minutes, utilize 70% ethanol treatment for 1 minute, utilize 80% ethanol treatment for 1 minute, utilize 90% ethanol treatment for 1 minute, utilize 100% ethanol treatment for 1 minute × 2 times, utilize xylene treatment for 5 minutes × 3 times, use dronabinol to be sealed.Make it dry at room temperature for more than 15 minutes, then observe the RPE cell of the RPE cell of the RPE cell of the RPE cell model of AMD of cynomolgus macaque control.Result is shown in. Fig.29 .

[0583] The macula is present only in higher primates, and macular degeneration is also present only in certain primates. To verify the pathology of macular degeneration, the pathology of cynomolgus monkeys, which are known as a model animal for macular degeneration, was analyzed.

[0584] like Fig.29 Shown, about the morphology of RPE cell, in healthy cynomolgus monkey control (Control), confirm that RPE cell is cubic, is neatly arranged as single layer by cell-cell adhesion.On the other hand, in the RPE cell of cynomolgus monkey of AMD, morphological change is spindle-shaped, confirms the stratification of cell at the edge of drusen.

[0585] In addition, about epithelial markers, in the macula of healthy cynomolgus monkey control (Control), the staining of E-cadherin was confirmed at the intercellular adhesion site of RPE cells, but in the RPE cells of cynomolgus monkeys with AMD where drusen existed, the staining of E-cadherin was not confirmed between cells. In addition, in healthy cynomolgus monkey control, the staining of stronger cytokeratin 18 was confirmed in the whole RPE cells, but in the drusen of cynomolgus monkeys with AMD, compared with the control, the signal decreased.

[0586] In addition, regarding the mesenchymal marker, fibronectin was stained in drusen of cynomolgus monkeys with AMD, and accumulation in RPE cells and drusen was confirmed.

[0587] According to the result of the aforementioned test example, EMT was produced in the RPE cells of drusen. Therefore, the inventors of the present invention etc. speculate that if the EMT of RPE cells can be suppressed, the formation of drusen can be suppressed. In the following test example 5-1, in order to retrieve the compound that suppresses the EMT of RPE cells, or in order to evaluate the influence of candidate agents on EMT in RPE cells, the cell model that can induce EMT to RPE cells in vitro was studied.

[0588] [5. Construction of EMT cell model]

[0589] Experimental Example 5-1: Study on the induction conditions of EMT in RPE cells

[0590] The inventors conducted an experiment to stimulate RPE cells (retinal pigment epithelial cell line: ARPE-19, the same below) under 10 conditions using cytokines or growth factors (TNF-α / IL-1β / TGF-β), and found that significant EMT induction was induced. It is believed that the cell model induced by EMT using cytokines or growth factors reflects the EMT induction mechanism inferred in AMD patients.

[0591] The 10 conditions are as follows.

[0592] (1) TNF-α 100 ng / mL

[0593] (2) IL-1β 100 ng / mL

[0594] (3) IL-1β 20 ng / mL

[0595] (4)TNF-α500ng / mL+TGF-β5ng / mL

[0596] (5)TNF-α200ng / mL+TGF-β5ng / mL

[0597] (6)TNF-α100ng / mL+TGF-β5ng / mL

[0598] (7)TNF-α100ng / mL +TGF-β10ng / mL

[0599] (8)TNF-α100ng / mL +TGF-β25ng / mL

[0600] (9)IL-1β100ng / mL+TGF-β5ng / mL

[0601] (10)IL-1β20ng / mL+TGF-β5ng / mL

[0602] Test Example 5-2: Verification of EMT based on expression changes of EMT marker molecules RNA was extracted from stimulated RPE cells, and expression changes were verified.

[0603] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 5-1, and after 48 hours of EMT induction, cells were recovered. The recovered cells were washed with PBS, 350 μL of RLT buffer (QIAGEN company) was added, and RNA collection was lysate was made. Using RNeasy micro kit (QIAGEN company #74004), total RNA was extracted from the lysate, and SuperScript III First-Strand Synthesis SuperMix (Invitrogen company #11752-250) for qRT-PCR was used to make cDNA from the RNA 2 μg obtained. Using the obtained cDNA 50ng as a template, real-time PCR (ABIPrism 7500 sequence detection system) was performed according to the manufacturer's draft, and the expression of mRNA of each gene was quantitatively analyzed. Specific primers for each gene were purchased from Invitrogen company. The Ct (Threshold Cycle) value of each gene was normalized by the internal control, and the ratio relative to the untreated sample was calculated.

[0604] The results are shown in Figure 1 .

[0605] like Figure 1 As shown in A and B in Figure 3, the expression of E-cadherin, a marker of epithelial lineage, was decreased in stimulated RPE cells ( Figure 1 A), the expression of N-cadherin, a mesenchymal marker, was upregulated ( Figure 1 B in the figure). This cadherin conversion is a representative phenomenon of EMT. In addition, the mesenchymal marker represented by fibronectin ( Figure 1 C) and the hyperregulation of EMT-related transcription factors (Snail, Slug, ZEB1) ( Figure 1 E to G in Figure 1) and the expression of ECM represented by type I collagen (COL1A1) ( Figure 1 D) in the figure, it was confirmed that RPE cells induced EMT due to stimulation.

[0606] Test Example 5-3: Verification of changes in EMT marker expression over time using microarray

[0607] The formation of drusen-like structures at the time of EMT induction and the EMT induction status at that time were analyzed.

[0608] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 5-1, and cells were recovered after 1 hour, 4 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours of EMT induction. The recovered cells were washed with PBS, 350 μL of RLT buffer was added, and RNA was collected and lysate was made. Using RNeasy micro kit (QIAGEN company #74004), total RNA was extracted from the lysate, and fluorescent labeling was performed using Low InputQuickAmp calibration kit (Agilent Technologies company #5190-2305). Labeled cRNA is hybridized in the human whole genome oligo DNA microarray kit (Agilent Technologies company #G4112F) according to the protocol specified by the manufacturer. The hybridized slides were cleaned using the Gene Expression Cleaning Kit (Agilent Technologies #5188-5327) and scanned using the Agilent Microarray Scanner (Agilent Technologies #G2505B). The scanned images were digitized using Feature Extraction software version 9.5.1 (Agilent Technologies). The numerical data were normalized using the Grobal normalization method. The results are shown in Figure 2 .

[0609] like Figure 2 As shown, the peak of decrease or increase of EMT markers varies depending on the gene, and occurs 12 hours or 72 hours after EMT induction. The decreased epithelial markers and increased mesenchymal markers and ECM are as follows.

[0610] As epithelial markers decreased by EMT induction, ID1, ID2, MUC1, cytokeratin 18 (KRT18), THBS1, VIL2, E-cadherin (CDH1), and TGFB2 were confirmed.

[0611] As mesenchymal markers and ECMs that are hyperactive due to EMT induction, FGF2, TWIST1, VIM, CD44, ZEB1, RDX, MMP9, FN1, TGFB1, RHOA, ZEB2, MMP2, TJP1, CTNNB1, MMP3, ETS1, SNAI1, SNAI2, HAS2, FGF1, SERPINE1, CDH2, MSN, TCF3, SDC1, ITGAV, COL1A1, and SPARC were confirmed.

[0612] 24 hours after EMT induction, drusen-like structures were formed on the culture plate seeded with RPE cells. The expression of epithelial markers represented by E-cadherin and cytokeratin 18 decreased, while the expression of mesenchymal markers and ECM groups represented by fibronectin / vimentin increased, and a clear correlation was observed between the formation of drusen-like structures and the EMT induction state.

[0613] Therefore, it was confirmed that the formation of drusen-like structures occurred through EMT of RPE cells.

[0614] Test Example 5-4: Verification of EMT induction based on hyperkinetic activity

[0615] The enhanced motility of RPE cells caused by EMT was evaluated using an invasion assay (cell invasion analysis), which is a method for evaluating the motility and invasiveness of cells.

[0616] RPE cells (ARPE-19) were seeded on a culture plate and incubated at 37°C for 5 days. For RPE cells, EMT was induced under the conditions described in Experimental Example 5-1, and the cells were recovered 24 hours after EMT induction. The recovered cells were inoculated into BD Biocoat Matrigel invasion chambers (BD Biosciece #354480) and further incubated at 37°C for 24 hours. After incubation, non-infiltrating cells were removed according to the manufacturer's protocol, and 100% methanol was added to fix the cells at room temperature for 15 minutes. Giemsa staining solution (Nacalai Tesque #37114-35) was added, and after staining at room temperature for 30 minutes, the cells were washed with PBS for 5 minutes × 2 times. After washing, the membrane remaining from the infiltrated cells was recovered and sealed on a glass slide. Allow it to dry for more than 15 minutes, then observe and take images using a microscope, and count the number of cells in the image. The results are shown in Fig.30 .

[0617] like Fig.30 A and Fig.30 Shown in B, the infiltrating cell number of RPE cell increases due to stimulation, confirms that the motor ability of RPE cell is hyperactive. RPE cells as epithelial cells do not usually have motor ability. In this way, the situation that the epithelial cells that originally did not have motor ability obtain motor ability is also a representative phenomenon of EMT.

[0618] Experimental Example 5-5: Morphological observation of RPE cells undergoing EMT

[0619] RPE cells (ARPE-19) were seeded on a culture plate and incubated at 37°C for 5 days. For RPE cells, EMT was induced under the conditions described in Experimental Example 5-1, and the cells were observed 48 hours after EMT induction. In addition, in order to observe the overall image of the culture plate under a low-power field of view, the observed cells were treated with a 4% polyformaldehyde solution at room temperature for 30 minutes and fixed, and then washed 3 times with PBS. Treated with 100% methanol at room temperature for 10 minutes × 2 times, Giemsa staining solution (NacalaiTesque #37114-35) was added, and stained at room temperature for 15 minutes. After washing with methanol 3 times, it was dried at room temperature for 3 hours. The dried cells were observed under a microscope. The results are shown in Fig.31 .

[0620] When EMT was induced in RPE cells under the conditions described in Experimental Example 5-1, Fig. 22 As shown by the arrow in B, aggregates were formed and drusen-like structures were confirmed. On the other hand, when EMT was not induced, Fig.31 As shown in A, PRE cells do not form aggregates but maintain a fusion state. Fig.31 C and Fig.31 D in the figure is to observe whether EMT is induced under low power field. Fig.31 As shown in D in FIG. 1 , it was confirmed that the drusen-like structures (Focus) were morphologically similar to the drusen ( Fig.31 E in). Thus prove, in RPE cells, due to the mesenchymal hypertrophy caused by EMT induction, drusen-like structures are formed. In addition, the drusen-like structures in the RPE cells can be effectively used as a model for reproducing AMD patient's drusen in vitro.

[0621] [6. Evaluation and / or screening of EMT inhibitory compounds]

[0622] Experimental Example 6-1: Expression changes of EMT markers 1

[0623] As previously mentioned, confirm that by stimulating RPE cells, the change of EMT markers or the hyperactivity of motor ability can be caused, and drusen-like structures are formed, thereby constructing a cell model that can induce EMT. Using this cell model, select the compound that inhibits EMT. In addition, by using this cell model, EMT inhibitory compounds can be evaluated.

[0624] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 5-1, and the evaluation compound (also referred to as the tested agent) was added. As an EMT induction control, a sample without the addition of the evaluation compound (tested agent) was used. After EMT induction for 48 hours, the cells were fixed at room temperature for 30 minutes using a 4% polyformaldehyde solution and then washed 3 times with PBS. Treated with 0.2% Triton-X / PBS solution for 5 minutes and washed 3 times with PBS. After staining the cells with a fluorescent staining solution (0.2% Phalloidin-Alexa 568 (Molecular Probes Company #A12380) / 0.05% Hochest33342 (Invitrogen Company #H3570) / 3% BSA-PBS) at room temperature for 1 hour, the cells were washed 5 minutes × 3 times with PBS. Cells that have been fluorescently stained were imaged using ImageExpress Micro (Molecular Devices) and digitized using MetaXpress2.0 (Molecular Devices). The degree of cluster formation (cluster level) was analyzed by numerical data, and the numerical value when no compound was added was used as a benchmark, and the cluster formation inhibition rate was calculated according to the following formula 1 to verify the EMT inhibitory effect of each compound. The degree of cluster formation (cluster level) is obtained by taking the number and volume of clusters as indicators.

[0625] (Formula 1)

[0626]

[0627] The IC50 in the table was calculated using the following formula 2.

[0628] (Formula 2)

[0629] IC50=10^(LOG(A / B)*(0.5-C) / (DC)+LOG(B))

[0630] A: With a high concentration of 50%

[0631] B: sandwiched with 50% low concentration

[0632] C: Inhibition rate at a low concentration of 50%

[0633] D: Inhibition rate at high concentrations of 50%

[0634] As a result, the following compounds were found to have an EMT inhibitory effect.

[0635] Table 1

[0636] Among the EMT-inhibiting compounds listed in Table 1 above, the EMT-inhibiting effects of five representative compounds (oxaprozin, epalrestat, zafirlukast, amlexanox, and zaltoprofen) were evaluated using the above-mentioned cell model.

[0637] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 5-1, and 5 representative compounds (oxaprozin, epalrestat, zafirlukast, amlexanox, zaltoprofen) were added at the same time. After 48 hours of EMT induction, the cells were recovered. As a control, a sample without the addition of the evaluation compound (tested agent) was used. The recovered cells were washed with PBS, 350 μL of RLT buffer was added, and RNA was collected and lysate was made. Using RNeasy micro kit (QIAGEN company #74004), total RNA was extracted from the lysate, and cDNA was made from the obtained RNA 2 μg using SuperScript III First-Strand Synthesis SuperMix (Invitrogen company #11752-250) for qRT-PCR. The obtained cDNA 50ng was used as a template, and real-time PCR (ABIPrism 7500 sequence detection system) was performed according to the manufacturer's protocol to quantitatively analyze the expression of mRNA of each gene. Specific primers for each gene were purchased from Invitrogen. The Ct (Threshold Cycle) value of each gene was normalized by the intrinsic control and the ratio relative to the non-treated sample was calculated. The results are shown in Figures 3 to 7 .

[0638] exist Figures 3 to 7 In each graph, the bar graph on the left represents the expression level of each gene when EMT is not induced. The bar graph in the middle represents the expression level of each gene when EMT is induced. The bar graph on the right represents the expression level of each gene when an EMT inhibitory compound is given. In addition, Figures 3 to 7 The results of using oxaprozin, epalrestat, zafirlukast, amlexanox, and zaltoprofen as EMT inhibitory compounds are shown.

[0639] In this specification, each EMT marker is also described by the following abbreviations.

[0640] Cadherin: CDH (CDH2, CDH3, etc.)

[0641] Matrix metalloproteinases: MMP (MMP1, MMP3, MMP7, etc.)

[0642] Fibronectin: FN (FN1, etc.)

[0643] Vimentin: VIM

[0644] Collagen type 1α1: COL1A1

[0645] Collagen type 1α2: COL1A2

[0646] Collagen type 5α2: COL5A2

[0647] Collagen type 5α3: COL5A3

[0648] Collagen type 6α3: COL6A3

[0649] Collagen type 13α: COL13A1

[0650] Laminin beta 3: LAMB3

[0651] Laminin gamma 2: LAMC2

[0652] Hyaluronan-mediated motility factor receptor: HMMR

[0653] Tenascin C: TNC

[0654] Zinc finger E-box binding protein 2: ZEB2

[0655] Serglycin: SRGN

[0656] Chondroitin sulfate proteoglycan 4: CSPG4

[0657] like Figures 3 to 7 As shown in the figure, among the EMT markers, the expressions of Snail, Slug, CDH3, MMP1, MMP7, which are mesenchymal markers, and COL5A3, COL6A3, LAMC2, HMMR, and TNC, which are ECM markers, were all suppressed when any EMT inhibitory compound was applied. Thus, it was confirmed that these 10 EMT markers were commonly suppressed by EMT inhibitory compounds. Even EMT markers other than these 10 were also suppressed. Figure 3 As shown in Figure 2, the expression of ZEB2, MMP3, COL1A1, COL1A2, CSPG4, and SRGN was confirmed to be decreased in response to oxaprozin. Figure 4 As shown in Figure 2, it was confirmed that the expression of ZEB2, CDH2, FN1, VIM, COL1A1, COL1A2, COL5A2, COL13A1, CSPG4, and SRGN was decreased in epalrestat. Figure 5 As shown in Figure 2, zafirlukast confirmed that the expression of MMP3, COL13A1, LAMB3, and CSPG4 decreased. Figure 6 As shown in Figure 2, it was confirmed that the expression of ZEB2, CDH2, MMP3, COL1A1, and COL13A1 was decreased in Amlexanox. Figure 7 As shown, in the case of zaltoprofen, the expression of ZEB2, FN1, COL13A1, and CSPG4 was confirmed to be decreased. It was confirmed that these EMT markers can also be indicators of inhibition by EMT inhibitory compounds.

[0658] Experimental Example 6-2: Expression changes of EMT markers 2

[0659] IC50 was determined for 5 compounds (aluminum flufenamate, mefenamic acid, sulindac, teipfic acid, and seratrolast) by the same method as in Test Example 6-1. The results are shown in Table 2.

[0660] [Table 2]

[0661]

[0662] As shown in Table 2, for the five compounds (flufenamic acid aluminum (added concentration: 28 μM (Cmax)), mefenamic acid (added concentration: 39 μM (Cmax)), sulindac (added concentration: 100 μM), teprofenic acid (added concentration: 1000 μM), seroquel (added concentration: 31 μM (Cmax))) that confirmed the EMT inhibitory effect, the expression changes of EMT markers were evaluated by the same method as in Test Example 6-1. The results are shown in Figures 8 to 12 .

[0663] like Figures 8 to 12 As shown, it was confirmed that the five EMT inhibitory compounds (flufenamic acid aluminum, mefenamic acid, sulindac, teipfic acid, and seratrolast) also reduced the expression of EMT markers during EMT induction.

[0664] Test Example 6-3: Hyperkinesia Inhibition Test

[0665] Using the invasion assay described in Test Example 5-4 above, the effect of an EMT-inhibiting compound on RPE cells whose motility is enhanced due to EMT was confirmed.

[0666] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 5-1, and 5 compounds (oxaprozin, epalrestat, zafirlukast, amlexanox, zaltoprofen) were added. After 24 hours of EMT induction, the cells were recovered. The recovered cells were inoculated into the BD Biocoat Matrigel invasion chamber (BD Biosciece company #354480) and further incubated at 37 ° C for 24 hours. After incubation, after removing non-infiltrating cells according to the manufacturer's protocol, 100% methanol was added and the cells were fixed at room temperature for 15 minutes. Giemsa staining solution (Nacalai Tesque company #37114-35) was added, and after staining at room temperature for 30 minutes, PBS was used to wash for 5 minutes × 2 times. After washing, the membrane remaining in the infiltrated cells was recovered and sealed on a slide. After drying for more than 15 minutes, the cells were observed and photographed using a microscope, and the number of infiltrating cells was counted. Figure 32 to Figure 34 .

[0667] Figure 32 to Figure 34 are the results of using epalrestat, amlexanox, and zafirlukast as EMT inhibitory compounds. Figure 32 to Figure 34 In the bar graphs, the white bar graph on the left represents the number of infiltrating RPE cells when EMT is induced, and the black bar graphs on the right represent the number of infiltrating RPE cells when EMT inhibitory compounds are added from low concentration to high concentration, from the left.

[0668] like Figure 32 to Figure 34 As shown in the figures, it was confirmed that the motility induced by EMT in RPE cells was significantly inhibited in a concentration-dependent manner by the EMT inhibitory compound.

[0669] [7. Evaluation of drug efficacy by drusen-like structure inhibition test]

[0670] RPE cells (ARPE-19) were inoculated on a culture plate and incubated at 37 ° C for 5 days. For RPE cells, EMT was induced under the conditions described in Test Example 5-1, and 5 compounds (oxaprozin, epalrestat, zafirlukast, amlexanox, zaltoprofen) were added at the same time, and the cells were observed 48 hours after EMT induction. As an EMT induction control, a sample without the addition of the evaluation compound (tested drug) was used. The observed cells were treated with 4% polyformaldehyde solution at room temperature for 30 minutes and fixed, and then washed 3 times with PBS. Treated with 0.2% Triton-X / PBS solution for 5 minutes and washed 3 times with PBS. The cells were stained with a fluorescent staining solution (0.2% Phalloidin-Alexa568 (Molecular Probes #A12380) / 0.05% Hochest 33342 (Invitrogen #H3570) / 3% BSA-PBS) at room temperature for 1 hour and then washed with PBS for 5 minutes × 3 times. The fluorescently stained cells were imaged using Image Express Micro (Molecular Devices) and quantified using MetaXpress2.0 (Molecular Devices). The degree of cluster formation was analyzed by numerical data, and the inhibition rate of cluster formation was calculated based on the absence of compound addition and the absence of EMT induction as the benchmark to verify the EMT inhibitory effect of each compound. For the cells after obtaining the fluorescent image, in order to observe the entire image at a low magnification field of view, they were treated with 100% methanol at room temperature for 10 minutes × 2 times, and Giemsa staining solution (Nacalai Tesque #37114-35) was added, and stained at room temperature for 15 minutes. After washing with methanol three times, the cells were dried at room temperature for 3 hours. The dried cells were observed under a microscope. The results are shown in Figure 35 to Figure 39 The agglomerate formation inhibition rate was calculated according to the formula 1 shown in the above-mentioned Test Example 6-1.

[0671] Figure 35 to Figure 39 The results are as follows: oxaprozin, epalrestat, amlexanox, zafirlukast, and zaltoprofen were used as EMT inhibitory compounds. Figure 35 to Figure 39 As shown, it was confirmed that the EMT inhibitory compounds all inhibited the drusen-like structures formed in the RPE cell model in a concentration-dependent manner. That is, the IC50 values ​​in the drusen-like structure inhibition test were 61.0 μM for oxaprozin, 16.9 μM for epalrestat, 32.5 μM for amlexanox, 3.8 μM for zafirlukast, and 16.5 μM for zaltoprofen.

[0672] Based on the new knowledge that drusen is a structure caused by EMT of RPE cells, it was shown that drusen can be inhibited by applying EMT-inhibiting compounds to drusen. From this result, it is speculated that even known EMT-inhibiting compounds have the same effect on drusen.

[0673] For the five EMT-inhibiting compounds, the peak plasma concentration (Cmax) of the drug was determined from the interview form and accompanying documents of each drug by the following method.

[0674] The results are shown in Table 3.

[0675] [Table 3]

[0676]

[0677] As shown in Table 3, the blood concentrations of the five EMT-inhibiting compounds were sufficient to exert the expected effects on AMD.

[0678] [Test Example 8: In vivo model test for evaluating drusen inhibition]

[0679] Test Example 8-1: Inducing the formation of drusen-like structures in the posterior part of the eye (retinal area)

[0680] (1) Method for inducing drusen-like structures by inducing inflammation in the posterior ocular region (retinal region)

[0681] 400 ng of recombinant mouse TNF-a (R&D Systems #410-MT) was administered to the retinal periphery of the posterior eye of the mouse.

[0682] The method of administration is as follows.

[0683] 1) Prepare 2.5 μL of TNF-a (160 ng / μL) dissolved in PBS.

[0684] 2) Open the eyelids in such a way that the mouse's eyeball can be clearly observed.

[0685] 3) Insert the needle until you feel slight resistance and inject the drug solution (400 ng / eye).

[0686] (2) Method for preparing eyeball tissue samples

[0687] Two weeks after administration, the eyeballs were removed and formalin-fixed paraffin-embedded blocks were prepared.

[0688] (3) Morphological observation of RPE cells

[0689] The tissue samples were dewaxed by treating with xylene for 5 minutes × 3 times, 100% ethanol for 1 minute × 2 times, 90% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, and water for 5 minutes. They were stained with Meier's hematoxylin solution for 4 minutes and eosin solution for 1 minute. They were treated in running water for 5 minutes, then treated with 70% ethanol for 1 minute, 80% ethanol for 1 minute, 90% ethanol for 1 minute, 100% ethanol for 1 minute × 2 times, and xylene for 5 minutes × 3 times, and sealed with dronabinol. They were dried at room temperature for more than 15 minutes, and then the untreated RPE cells and inflammation-induced RPE cells were observed. The results are shown in Fig.40 .

[0690] like Fig.40 Shown in, by inflammation induction and form drusen sample structure (part surrounded by dotted line) in retinal area.Confirm that the retinal pigment epithelial cell of no treatment group presents cubic form, contacts closely with adjacent cell, and demonstrates 1 layer of arrangement (photo above).On the other hand, confirm that the retinal pigment epithelial cell that is arranged as 1 layer after inflammation induction is not observed, and is present in the inside, surrounding of drusen sample structure body, disappears with the close connection of adjacent cell, and its form becomes spindle-shaped (photo below) from cubic shape.

[0691] (4) Evaluation method of each marker by immunohistochemistry

[0692] The eyeball tissue sections were made from the blocks. After the tissue samples were incubated at 56°C in a gas phase incubator for 30 minutes, they were treated with xylene for 5 minutes × 3 times, 100% ethanol for 1 minute × 2 times, 90% ethanol for 1 minute, 80% ethanol for 1 minute, 70% ethanol for 1 minute, and water for 5 minutes to dewax. After removing the melanin in the tissue according to the manufacturer's protocol using a refined melanin kit for special staining and IHC (Polysciences #24909-1), the antigen was activated by microwave treatment for 10 minutes using a citrate buffer pH 6.0. After standing at room temperature for 30 minutes, the intrinsic peroxidase was inactivated by treatment with PBS for 5 minutes, 3% hydrogen peroxide / PBS solution for 5 minutes, and PBS for 5 minutes. The tissue was blocked by standing at room temperature for 30 minutes in a 3% BSA / PBS solution. As the primary antibody, 200 μL of anti-E-cadherin antibody (BD Biosience #610182) diluted 500-fold with 1.5% BSA / PBS solution, anti-cytokeratin antibody (Sigma #C2931) diluted 1000-fold, anti-fibronectin antibody (abcam #45688) diluted 1000-fold, and anti-vimentin antibody (Cell signaling Technology #5741) diluted 100-fold were added to each tissue and reacted with the tissue at 4°C for 17 hours. The tissue reacted with the primary antibody was washed with PBS for 5 minutes × 3 times, and then reacted with the secondary antibody (Mouse on Mouse (MOM) Elite Peroxidase Kit (Vector Laboratories #PK-2200) for anti-E-cadherin antibody and anti-cytokeratin antibody, and VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories #PK-6101) for anti-fibronectin antibody and anti-vimentin antibody) at room temperature for 30 minutes, washed with PBS for 5 minutes × 3 times, and then ABC reaction was performed at room temperature for 30 minutes according to the manufacturer's protocol. The tissue was washed with PBS for 5 minutes × 3 times, and then color was developed at room temperature for 3 minutes using ImmPACT AMEC Red Peroxidase (HRP) Substrate (Vector Laboratories #SK-4285). The stained tissue was stained with M-type neo-hematoxylin (Muto Chemical Co., Ltd. #30141), then allowed to stand in running water for 3 minutes, treated with 70% ethanol for 1 minute, 80% ethanol for 1 minute, 90% ethanol for 1 minute, and 100% ethanol for 1 minute × 2 times, treated with xylene for 5 minutes × 3 times, and sealed with dronabinol.The samples were dried at room temperature for 15 minutes or longer and then observed. The results for E-cadherin are shown in FIG. Fig.41 The results for cytokeratin are shown in Fig.42 The results for fibronectin are shown in Fig.43 The results for vimentin are shown in Fig.44 .

[0693] like Fig.41 As shown, in the untreated retinal epithelial cells, strong positive E-cadherin staining was observed between cells, and strong cell-cell adhesion was maintained (upper photo). On the other hand, in the retinal pigment epithelial cells inside and around the drusen-like structures formed by inflammation induction, staining disappeared (lower photo).

[0694] like Fig.42 As shown, in the untreated retinal epithelial cells, strong positive staining of cytokeratin was observed between cells (upper photograph). On the other hand, in the retinal pigment epithelial cells, especially around the drusen-like structures formed by inflammation induction, the staining disappeared (lower photograph).

[0695] like Fig.43 As shown, in the untreated retinal epithelial cells (the part surrounded by the dotted line), the signal was negative, and no expression of fibronectin was observed (upper photo). On the other hand, the staining was positive inside and around the drusen-like structures formed by inflammation induction, and fibronectin expression was observed (lower photo).

[0696] like Fig.44 As shown, in the untreated retinal epithelial cells (the part surrounded by the dotted line), the signal was negative and no vimentin expression was observed (upper photo). On the other hand, in the inside and around the drusen-like structures formed by inflammation induction, the staining was positive and vimentin expression was observed (lower photo).

[0697] Test Example 8-2: Pharmacological evaluation: Inhibitory effect on the formation of drusen-like structures

[0698] (1) Method for inducing drusen-like structures by inducing inflammation in the posterior ocular region (retinal region)

[0699] By the method described in Test Example 8-1, 400 ng of recombinant mouse TNF-a (R&D Systems, #410-MT) was administered to the retinal periphery of the posterior eye of the mouse.

[0700] (2) Administration of evaluation compounds

[0701] The compounds to be evaluated were suspended in a 0.5% carboxymethylcellulose aqueous solution and orally administered using a sonde for 14 days at a dose of 600 mg / kg / day for oxaprozin, 600 mg / kg / day for epalrestat, and 40 mg / kg / day for zaltoprofen.

[0702] (3) Morphological observation of RPE cells

[0703] Ocular tissue samples were prepared by the method described in Test Example 8-1, and untreated RPE cells and inflammation-induced RPE cells were observed by HE staining, immunostaining of E-cadherin, or immunostaining of fibronectin.

[0704] The pharmacological evaluation of oxaprozin is shown in Figure 45 to Figure 47 The pharmacological evaluation of epalrestat is shown in Figures 48 to 50 The pharmacological evaluation of zaltoprofen is shown in Figure 51 to Figure 53 .

[0705] like Fig.45 As shown, drusen-like structures (part surrounded by dotted lines) were formed in the retinal area by inflammation induction (upper photograph). In the oxaprozin-administered group, the formation of drusen-like structures was significantly suppressed (lower photograph).

[0706] After inflammation induction, retinal pigment epithelial cells were not observed arranged in a single layer, but were present inside and around drusen-like structures, with the close connection with adjacent cells disappearing and their morphology changing from cubic to spindle-like. On the other hand, in the oxaprozin-administered group, a large number of cells maintaining a cubic morphology were observed.

[0707] like Fig.46 As shown in the figure, E-cadherin staining disappeared in the retinal pigment epithelial cells inside and around the drusen-like structures induced by inflammation (upper photo). On the other hand, in the retinal epithelial cells of the oxaprozin-administered group, strong positive staining was seen between cells, and strong cell-cell adhesion was maintained (lower photo).

[0708] like Fig.47 As shown in the figure, the staining was positive inside and around the drusen-like structures formed by inflammation induction, and the expression of fibronectin was observed (upper photo). On the other hand, in the oxaprozin-administered group, the formation of drusen-like structures was confirmed to be inhibited, and the staining signal intensity was also weak (lower photo).

[0709] like Fig.48As shown, drusen-like structures (part surrounded by dotted lines) were formed in the retinal area by inflammation induction (upper photograph). In the epalrestat-administered group, the formation of drusen-like structures was significantly suppressed (lower photograph).

[0710] After inflammation induction, retinal pigment epithelial cells were not observed arranged in a single layer, but were present inside and around drusen-like structures, with the close connection with adjacent cells disappearing, and their morphology changed from cubic to spindle-like. On the other hand, in the epalrestat-administered group, a large number of cells maintaining a cubic morphology were observed.

[0711] like Fig.49 As shown in the figure, E-cadherin staining disappeared in the retinal pigment epithelial cells inside and around the drusen-like structures formed by inflammation induction (upper photo). On the other hand, in the retinal epithelial cells of the epalrestat-administered group, strong positive staining was seen between cells, and strong cell-cell adhesion was maintained (lower photo).

[0712] like Fig.50 As shown in the figure, the staining was positive inside and around the drusen-like structures formed by inflammation induction, and the expression of fibronectin was observed (upper photo). On the other hand, in the epalrestat-administered group, the formation of drusen-like structures was confirmed to be inhibited, and the staining signal intensity was also weak (lower photo).

[0713] like Fig.51 As shown, drusen-like structures (part surrounded by dotted lines) were formed in the retinal area by inflammation induction (upper photograph). In the zaltoprofen-administered group, the formation of drusen-like structures was significantly suppressed (lower photograph).

[0714] After inflammation induction, retinal pigment epithelial cells were not observed arranged in a single layer, but were present inside and around drusen-like structures, with the close connection with adjacent cells disappearing, and their morphology changed from cubic to spindle-like. On the other hand, in the zaltoprofen-administered group, a large number of cells maintaining a cubic morphology were observed.

[0715] like Fig.52 As shown in the figure, E-cadherin staining disappeared in the retinal pigment epithelial cells inside and around the drusen-like structures induced by inflammation (upper photo). On the other hand, in the retinal epithelial cells of the zaltoprofen-administered group, strong positive staining was seen between cells, and strong cell-cell adhesion was maintained (lower photo).

[0716] like Fig.53As shown in the figure, the staining was positive inside and around the drusen-like structures formed by inflammation induction, and the expression of fibronectin was observed (upper photo). On the other hand, in the zaltoprofen-administered group, the formation of drusen-like structures was confirmed to be inhibited, and the staining signal intensity was also weak (lower photo).

[0717] [Preparation Example]

[0718] (Prescription Example 1 Tablet)

[0719] Tablets are manufactured by tableting based on a common tablet manufacturing method. Carboxymethylcellulose calcium, hypromellose, crystalline cellulose, magnesium aluminosilicate, magnesium stearate, etc. are used as additives to manufacture plain tablets containing 100 mg or 200 mg of oxaprozin. Generally, 400 mg per day can be divided into 1 or 2 times for oral administration for adults. It should be noted that the dosage can be appropriately increased or decreased according to age and symptoms, and the maximum dosage per day is 600 mg.

[0720] (Prescription Example 2-1 tablet)

[0721] Tablets are manufactured by tableting based on a common tablet manufacturing method. Carboxymethylcellulose calcium, crystalline cellulose, titanium oxide, magnesium stearate, corn starch, lactose hydrate, hydroxypropyl cellulose, hypromellose, and polyethylene glycol 6000 are used as additives, and film-coated tablets containing 80 mg of zaltoprofen are manufactured. Generally, 80 mg can be orally administered three times a day to adults. In the case of single use, 80 mg to 160 mg are orally administered at one time.

[0722] (Prescription Example 2-2 tablets)

[0723] Tablets are manufactured by tableting based on a common tablet manufacturing method. Carnauba wax, carboxymethylcellulose calcium, crystalline cellulose, titanium oxide, magnesium stearate, corn starch, lactose hydrate, hydroxypropyl cellulose, and hypromellose are used as additives to manufacture film-coated tablets containing 80 mg of zaltoprofen. Generally, 80 mg can be orally administered three times a day to adults. In the case of single use, 80 mg to 160 mg are orally administered at one time.

[0724] (Prescription Example 3 Tablets)

[0725] Tablets are made based on the manufacturing method of common tablets. Lactose hydrate, crystalline cellulose, povidone, cross-linked sodium carboxymethyl cellulose, magnesium stearate, hypromellose, titanium oxide, etc. are used as additives in manufacturing, and film-coated tablets including 20 mg of zafirlukast, etc., are usually divided into 2 times of oral administration after breakfast and before going to bed for adults. It should be noted that the daily dosage of the elderly is 40 mg, and the maximum amount of 1 day for adults (except the elderly) is 80 mg.

[0726] (Prescription Example 4 Tablets)

[0727] Tablets are manufactured by tableting based on a common tablet manufacturing method. Corn starch, hydroxypropylcellulose, carboxymethylcellulose calcium, magnesium stearate, lactose hydrate, etc. are used as additives to manufacture plain tablets containing 25 mg or 50 mg of Amlexanox. Generally, for adults, 25 mg to 50 mg can be taken orally three times a day in the morning, evening, and before going to bed, or in the morning, daytime, and evening, depending on the symptoms.

[0728] (Prescription Example 5 tablets)

[0729] Tablets are manufactured by tableting based on a conventional tablet manufacturing method. D-mannitol, hydroxypropylcellulose, carboxymethylcellulose calcium, magnesium stearate, hypromellose, titanium oxide, polyoxyethylene (105) polyoxypropylene (5) glycol, etc. are used as additives, and film-coated tablets containing 50 mg of epalrestat are manufactured. Generally, for adults, 50 mg can be orally administered three times a day before each meal. It should be noted that the dosage can be appropriately increased or decreased according to age and symptoms.

[0730] (Prescription Example 6 tablets)

[0731] Tablets are manufactured by tableting based on a common tablet manufacturing method. Corn starch, magnesium stearate, talc, hydroxypropyl methylcellulose, titanium oxide, hydroxypropyl cellulose, propylene glycol, polyoxyethylene

[160] polyoxypropylene

[30] glycol, etc. are used as additives to manufacture film-coated tablets containing 100 mg or 200 mg of tepoxilic acid. Generally, 200 mg can be orally administered three times a day to adults. It should be noted that the dosage can be appropriately increased or decreased according to age and symptoms.

[0732] (Prescription Example 7 tablets)

[0733] Tablets are manufactured by tableting based on a common tablet manufacturing method. Carboxymethylcellulose calcium, hypromellose, crystalline cellulose, magnesium aluminosilicate, magnesium stearate, etc. are used as additives to manufacture plain tablets containing 125 mg or 250 mg of aluminum flufenamate. Generally, for adults, 125 mg to 250 mg can be divided into 3 times a day for oral administration. It should be noted that the dosage can be appropriately increased or decreased according to age and symptoms, and the maximum dosage per day is 750 mg.

[0734] (Prescription Example 8 tablets)

[0735] Tablets are manufactured by tableting based on a common tablet manufacturing method. Corn starch, hypromellose, magnesium aluminosilicate, talc, titanium oxide, etc. are used as additives to manufacture plain tablets containing 250 mg of mefenamic acid. Generally, for adults, 500 mg can be orally administered once, and then 250 mg can be orally administered once every 6 hours. It should be noted that the dosage can be appropriately increased or decreased according to age and symptoms, and the maximum daily dosage is 1500 mg.

[0736] (Prescription Example 9 tablets)

[0737] Tablets are manufactured by tableting based on a conventional tablet manufacturing method. Cellulose, alpha-starch, magnesium stearate, etc. are used as additives to manufacture plain tablets containing 50 mg or 100 mg of sulindac. Generally, for adults, 300 mg per day can be divided into 2 doses per day for oral administration. It should be noted that the dosage may be appropriately increased or decreased according to age and symptoms.

[0738] (Prescription Example 10 tablets)

[0739] Tablets are manufactured by tableting based on a common tablet manufacturing method. Hydroxypropylcellulose, magnesium stearate, corn starch, lactose hydrate, etc. are used as additives to manufacture plain tablets containing 40 mg or 80 mg of serolast. Generally, 80 mg can be orally administered once a day to adults.

Claims

1. A preventive and / or therapeutic agent for age-related macular degeneration, comprising only zaltoprofen and / or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 1, characterized in that Inhibits EMT in RPE cells.

3. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 1, characterized in that The age-related macular degeneration is accompanied by one or more symptoms selected from the group consisting of decreased visual acuity, choroidal neovascularization, bleeding, hemorrhage and drusen.

4. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 1, characterized in that The age-related macular degeneration is one or more selected from dry AMD, wet AMD, no AMD, early AMD and intermediate AMD.

5. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 1, characterized in that: It is available as an oral agent, injection, eye drop or eye ointment.

6. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 1, characterized in that: Zaltoprofen and / or its salt is contained in an amount of 0.03 to 1% by weight based on the total weight of the preparation so as to obtain a dosage that gives a Cmax of 20.4 μM or more.

7. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 1, characterized in that: Laser irradiation model animals showed an inhibitory effect on epithelial-mesenchymal transition (EMT).

8. A preventive and / or therapeutic agent for age-related macular degeneration, comprising only oxaprozin and / or a pharmaceutically acceptable salt thereof as an active ingredient.

9. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 8, which has one or more of the following characteristics a to f: aInhibit EMT in RPE cells; b) The age-related macular degeneration is accompanied by one or more symptoms selected from decreased vision, choroidal neovascularization, bleeding, hemorrhage and drusen; c. The age-related macular degeneration is one or more selected from dry AMD, wet AMD, no AMD, early AMD and intermediate AMD; d is oral preparation, injection, eye drops or eye ointment; e. containing oxaprozin and / or its salt in an amount of 0.03 to 1% by weight relative to the total weight of the preparation so as to achieve a dosage that produces a Cmax of 340.9 μM or more; f showed an inhibitory effect on epithelial-mesenchymal transition (EMT) in laser irradiated model animals.

10. A preventive and / or therapeutic agent for age-related macular degeneration, comprising only epalrestat and / or a pharmaceutically acceptable salt thereof as an active ingredient.

11. The preventive and / or therapeutic agent for age-related macular degeneration according to claim 10, which has one or more of the following characteristics a to f: aInhibit EMT in RPE cells; b) The age-related macular degeneration is accompanied by one or more symptoms selected from decreased vision, choroidal neovascularization, bleeding, hemorrhage and drusen; c. The age-related macular degeneration is one or more selected from dry AMD, wet AMD, no AMD, early AMD and intermediate AMD; d is oral preparation, injection, eye drops or eye ointment; e. containing epalrestat and / or its salt in an amount of 0.03 to 1% by weight relative to the total weight of the preparation so as to achieve a dosage that produces a Cmax of 12.2 μM or more; f showed an inhibitory effect on epithelial-mesenchymal transition (EMT) in laser irradiated model animals.

12. A use, which is the use of zaltoprofen and / or its pharmaceutically acceptable salt in the preparation of a medicament for preventing and / or treating age-related macular degeneration, The active ingredient of the pharmaceutical product is only the zaltoprofen and / or its pharmaceutically acceptable salt, The medicine is an oral preparation, an injection, an eye drop or an eye ointment.

13. The use according to claim 12, which has one or more of the following features a to e: aInhibit EMT in RPE cells; b) The age-related macular degeneration is accompanied by one or more symptoms selected from decreased vision, choroidal neovascularization, bleeding, hemorrhage and drusen; c. The age-related macular degeneration is one or more selected from dry AMD, wet AMD, no AMD, early AMD and intermediate AMD; d. containing zaltoprofen and / or its salt in an amount of 0.03 to 1% by weight relative to the total weight of the preparation so as to obtain a dosage that produces a Cmax of 20.4 μM or more; e showed an inhibitory effect on epithelial-mesenchymal transition (EMT) in laser irradiated model animals.

14. A use, namely, use of oxaprozin and / or its pharmaceutically acceptable salt in the preparation of a pharmaceutical for preventing and / or treating age-related macular degeneration, The active ingredient of the pharmaceutical product is only the oxaprozin and / or its pharmaceutically acceptable salt, The medicine is an oral preparation, an injection, an eye drop or an eye ointment.

15. The use according to claim 14, which has one or more of the following features a to e: aInhibit EMT in RPE cells; b) The age-related macular degeneration is accompanied by one or more symptoms selected from decreased vision, choroidal neovascularization, bleeding, hemorrhage and drusen; c. The age-related macular degeneration is one or more selected from dry AMD, wet AMD, no AMD, early AMD and intermediate AMD; d. containing zaltoprofen and / or its salt in an amount of 0.03 to 1% by weight relative to the total weight of the preparation so as to obtain a dosage that produces a Cmax of 340.9 μM or more; e showed an inhibitory effect on epithelial-mesenchymal transition (EMT) in laser irradiated model animals.

16. A use, which is the use of epalrestat and / or its pharmaceutically acceptable salt in the preparation of a pharmaceutical for preventing and / or treating age-related macular degeneration, The active ingredient of the pharmaceutical product is only the epalrestat and / or its pharmaceutically acceptable salt, The medicine is an oral preparation, an injection, an eye drop or an eye ointment.

17. The use according to claim 16, which has one or more of the following features a to e: aInhibit EMT in RPE cells; b) The age-related macular degeneration is accompanied by one or more symptoms selected from decreased vision, choroidal neovascularization, bleeding, hemorrhage and drusen; c. The age-related macular degeneration is one or more selected from dry AMD, wet AMD, no AMD, early AMD and intermediate AMD; d. containing zaltoprofen and / or its salt in an amount of 0.03 to 1% by weight relative to the total weight of the preparation so as to achieve a dosage that produces a Cmax of 12.2 μM or more; e showed an inhibitory effect on epithelial-mesenchymal transition (EMT) in laser irradiated model animals.

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