Eye disease marker

Abstract

To provide a marker that shows a relationship with changes in the nerve density and shape in the cornea.SOLUTION: In one aspect, the present invention provides a marker of neuropathy in the eye, the expression of which changes in correlation with morphological parameters of the nerves in the eye. In some embodiments, the parameter may include at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity. In a particular embodiment, the parameter may include at least one parameter selected from CNBD and CTBD.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to markers for ocular conditions (eg, ocular neurological disorders, dry eye, etc.) and related technology. [Background technology]

[0002] Dry eye is defined as "a disease in which the stability of the tear film is reduced due to various factors, causing ocular discomfort and visual function abnormalities, and may be accompanied by ocular surface damage" (Non-Patent Document 1, incorporated herein by reference), and its causes and symptoms are diverse. In the past, the main treatment was to replenish moisture with artificial tears, but with progress in elucidating the pathology of dry eye and developing treatments, the cause of the reduced stability of the tear film is now diagnosed according to the stratification of the ocular surface (corneal and conjunctival epithelium, aqueous tear layer, and oily layer), and appropriate treatments are proposed for each (Non-Patent Document 1, incorporated herein by reference). On the other hand, the corneal sensory nerve is an important element for maintaining the homeostasis of the ocular surface. The corneal sensory nerve, which branches off from the first branch of the trigeminal nerve, is distributed throughout the cornea, senses pain and temperature on the ocular surface, transmits them to the brain, and contributes to maintaining the homeostasis of the ocular surface by promoting blinking and tear secretion. Since it became possible to observe corneal sensory nerves in vivo using a confocal laser scanning microscope, it has been reported that changes in nerve density and shape have been observed in dry eye patients (Non-Patent Documents 2 and 3, which are incorporated herein by reference), but it has not yet been clarified whether these changes are the cause of dry eye.

[0003] Furthermore, with the recent advances in proteomics analysis technology, there has been an increase in research into the search for biomarkers in tear components from dry eye patients (Non-Patent Documents 4 and 5), but no markers that show a relationship with changes in nerve density or shape have yet been found. In addition, there are currently no therapeutic drugs for corneal sensory nerves. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Dry Eye Study Group, Committee on Definition and Diagnostic Criteria of Dry Eye. Revised definition and diagnostic criteria of dry eye in Japan (2016 edition) [Non-Patent Document 2] Cruzat A, Pavan-Langston D, Hamrah P. In vivo confocal microscopy of corneal nerves: analysis and clinical correlation. Semin Ophthalmol. 2010;25(5-6):171-7. [Non-Patent Document 3] Alhatem A, Cavalcanti B, Hamrah P. In vivo confocal microscopy in dry eye disease and related conditions. Semin Ophthalmol. 2012;27(5-6):138-48. [Non-Patent Document 4] Srinivasan S, Thangavelu M, Zhang L, et al. iTRAQ quantitative proteomics in the analysis of tears in dry eye patients. Invest Ophthalmol Vis Sci. 2012;53(8):5052-9. [Non-Patent Document 5] Perumal N, Funke S, Pfeiffer N, et al. Proteomics analysis of human tears from aqueous-deficient and evaporative dry eye patients. Sci Rep. 2016;6:29629. Summary of the Invention [Means for solving the problem]

[0005] The inventors observed and measured the shapes of the corneal sensory nerves of dry eye patients and healthy adults using a confocal laser scanning microscope, and also performed measurements of corneal sensitivity and ophthalmological examinations of the ocular surface, which are general dry eye test items, and discovered a new relationship between the shape of the corneal sensory nerves and corneal sensitivity and dry eye symptoms. As a result of extensive research, the inventors discovered a marker that shows an association with changes in nerve density and shape in the cornea, and completed the present invention.

[0006] Thus, the present disclosure provides: (Item 1) A marker of neurological abnormality in the eye, the expression of which changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity rate. (Item 2) The marker of item 1, wherein the parameters include at least one parameter selected from the group consisting of CNBD and CTBD. (Item 3) The markers are cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, α1-acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10, haptoglobin, protein S100-A6, paralemmin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin, cytoplasmic 2, α-enolase, mammaglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid binding protein, cytokeratin 75, 75), histone H1.4, keratin 84 (Keratin, type II cuticular Hb4), galectin-7, cathepsin D, cytokeratin 6B (Keratin, type II cytoskeletal 6B), histone H1.1, cytokeratin 17 (Keratin, type I cytoskeletal 17), neutrophil defensin 3, cytokeratin 79 (Keratin, type II cytoskeletal 79), histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 4) 4. The marker according to any one of items 1 to 3, wherein the marker is selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof. (Item 5) 5. The marker according to any one of items 1 to 4, which is a marker for neurological abnormalities in dry eye. (Item 6) A detection agent for detecting the marker according to any one of items 1 to 5. (Item 7) 1. A method for identifying a marker of a neurological abnormality in the eye, comprising: (a) obtaining parameters selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity in healthy subjects and dry eye patients; (b) measuring the expression level of one or more genes or proteins in samples obtained from the healthy subject and the dry eye patient; (c) correlating said measurements with said one or more gene or protein expression levels; A method comprising: (Item 8) 8. The method according to item 7, wherein the parameters include at least CNBD or CTBD. (Item 9) 9. The method according to item 7 or 8, wherein the sample is tear fluid. (Item 10) 10. The method according to any one of items 7 to 9, wherein the marker is a marker for dry eye associated with neurological abnormality. (Item 11) A dry eye marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5. (Item 12) 14-3-3 protein θ, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, α-actin 1 (Actin, alpha skeletal muscle), α1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid-binding protein, fibrinogen β chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin alpha 5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, trypsin-3, and any combination thereof. (Item 13) 13. The marker according to item 11 or 12, wherein the marker is selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof. (Item 14) A detection agent for detecting the marker according to any one of items 11 to 13. (Item 15) A marker for dry eye associated with neurological abnormality, comprising at least one marker according to any one of items 1 to 5 and at least one marker according to any one of items 11 to 13. (Item 1A) 1. A method for diagnosing whether a subject has a neurological abnormality in the eye, the method comprising: obtaining a sample from said subject; Measuring the amount of a marker of ocular neuropathies in the sample, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity rate; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy individual, and determining that the subject has a neurological abnormality in the eye based on the measured amount of the marker; A method comprising: (Item 2A) The method of claim 1A, wherein if the amount of the measured marker is higher or lower than the amount of the marker in the sample obtained from the healthy individual, the subject may be diagnosed as having or likely to have a neurological disorder. (Item 3A) The method according to any one of items 1A to 2A, wherein the parameters include at least one parameter selected from the group consisting of CNBD and CTBD. (Item 4A) The markers are cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, α1-acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10, haptoglobin, protein S100-A6, paralemmin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin, cytoplasmic 2, α-enolase, mammaglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid binding protein, cytokeratin 75, 75), histone H1.4, keratin 84 (Keratin, type II cuticular Hb4), galectin-7, cathepsin D, cytokeratin 6B (Keratin, type II cytoskeletal 6B), histone H1.1, cytokeratin 17 (Keratin, type I cytoskeletal 17), neutrophil defensin 3, cytokeratin 79 (Keratin, type II cytoskeletal 79), histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 5A) The method of any one of items 1A to 4A, wherein the marker is selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof. (Item 6A) The method according to any one of items 1A to 5A, which is a marker for neurological abnormalities in dry eye. (Item 7A) The method according to any one of items 1A to 6A, wherein the sample is tear fluid. (Item 8A) 1. A method for diagnosing whether a subject has dry eye, the method comprising: obtaining a sample from said subject; measuring the amount of a marker for dry eye in the sample, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject, and determining that the subject has dry eye based on the measured amount of the marker; A method comprising: (Item 9A) The method of claim 8A, wherein if the amount of the measured marker is higher or lower than the amount of the marker in the sample obtained from the healthy individual, the subject may be diagnosed as having or likely to have a neurological disorder. (Item 10A) The markers are 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, alpha actin 1 (Actin, alpha skeletal muscle), alpha 1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid binding protein, fibrinogen beta chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin alpha 5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, trypsin-3, and any combination thereof. (Item 11A) The method according to any one of items 8A to 10A, wherein the marker is selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), Galectin-3-binding protein, and any combination thereof. (Item 12A) The method according to any one of items 8A to 11A, wherein the sample is tear fluid. (Item 13A) 1. A method for diagnosing whether a subject has dry eye associated with a neurological abnormality, the method comprising: obtaining a sample from the subject; Measuring at least one marker defined in any one of items 1A to 7A and at least one marker defined in any one of items 8A to 12A in the sample; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject, and determining that the subject has dry eye associated with a neurological abnormality based on the amount of the marker; A method comprising: (Item 1B) 1. A method of treating an eye in a subject, the method comprising: obtaining a sample from said subject; Measuring the amount of a marker of ocular neuropathies in the sample, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity rate; Comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy individual to diagnose whether or not the individual has a neurological abnormality in the eye; administering a pharmaceutical agent for preventing or treating the neurological abnormality to a subject determined to have the neurological abnormality in the diagnosis step; A method comprising: (Item 2B) The method of claim 1B, wherein if the amount of the measured marker is higher or lower than the amount of the marker in the sample obtained from the healthy individual, the subject can be diagnosed as having or likely to have a neurological disorder. (Item 3B) The method according to any one of items 1B to 2B, wherein the parameters include at least one parameter selected from the group consisting of CNBD and CTBD. (Item 4B) The markers are cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, α1-acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10, haptoglobin, protein S100-A6, paralemmin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin, cytoplasmic 2, α-enolase, mammaglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid binding protein, cytokeratin 75, 75), histone H1.4, keratin 84 (Keratin, type II cuticular Hb4), galectin-7, cathepsin D, cytokeratin 6B (Keratin, type II cytoskeletal 6B), histone H1.1, cytokeratin 17 (Keratin, type I cytoskeletal 17), neutrophil defensin 3, cytokeratin 79 (Keratin, type II cytoskeletal 79), histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 5B) The method according to any one of items 1B to 4B, wherein the marker is selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof. (Item 6B) The method according to any one of items 1B to 5B, which is a marker for neurological abnormalities in dry eye. (Item 7B) The method according to any one of items 1B to 6B, wherein the sample is tear fluid. (Item 8B) 1. A method of treating dry eye in a subject, the method comprising: obtaining a sample from said subject; measuring the amount of a marker for dry eye in the sample, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject, and determining that the subject has dry eye based on the amount of the marker; administering a medicament for preventing or treating dry eye to a subject determined to have dry eye in the diagnostic step; A method comprising: (Item 9B) The method of claim 8B, wherein if the amount of the measured marker is higher or lower than the amount of the marker in the sample obtained from the healthy individual, the subject may be diagnosed as having or likely to have a neurological disorder. (Item 10B) The markers are 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, alpha actin 1 (Actin, alpha skeletal muscle), alpha 1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid binding protein, fibrinogen beta chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin alpha 5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, trypsin-3, and any combination thereof. (Item 11B) The method according to any one of items 8B to 10B, wherein the marker is selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof. (Item 12B) The method according to any one of items 8B to 11B, wherein the sample is tear fluid. (Item 13B) 1. A method for treating dry eye associated with a neurological abnormality in a subject, the method comprising: obtaining a sample from the subject; Measuring at least one marker defined in any one of items 1B to 7B and at least one marker defined in any one of items 8B to 12B in the sample; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject, and determining that the subject has dry eye associated with a neurological abnormality based on the amount of the marker; administering a pharmaceutical agent for preventing or treating dry eye associated with neurological abnormality to a subject determined to have dry eye associated with neurological abnormality in the diagnosis step; A method comprising: (Item 1C) 1. An in vitro method for diagnosing whether a subject has a neurological abnormality in the eye, the method comprising: measuring in vitro a marker of ocular neuropathies in a sample from the subject, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity rate; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy individual, and determining that the subject has a neurological abnormality in the eye based on the amount of the marker; A method comprising: (Item 2C) The method of claim 1C, wherein if the amount of the measured marker is higher or lower than the amount of the marker in the sample obtained from the healthy individual, the subject can be diagnosed as having or likely to have a neurological disorder. (Item 3C) The method according to item 1C or 2C, wherein the parameters include at least one parameter selected from the group consisting of CNBD and CTBD. (Item 4C) The markers are cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, α1-acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10, haptoglobin, protein S100-A6, paralemmin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin, cytoplasmic 2, α-enolase, mammaglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid binding protein, cytokeratin 75, 75), histone H1.4, keratin 84 (Keratin, type II cuticular Hb4), galectin-7, cathepsin D, cytokeratin 6B (Keratin, type II cytoskeletal 6B), histone H1.1, cytokeratin 17 (Keratin, type I cytoskeletal 17), neutrophil defensin 3, cytokeratin 79 (Keratin, type II cytoskeletal 79), histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 5C) The method of any one of items 1C to 4C, wherein the marker is selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof. (Item 6C) The method according to any one of items 1C to 5C, which is a marker for neurological abnormalities in dry eye. (Item 7C) The method according to any one of items 1A to 6A, wherein the sample is tear fluid. (Item 8C) 1. An in vitro method for diagnosing whether a subject has dry eye, the method comprising: measuring in vitro a marker for dry eye in a sample from the subject, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject, and determining that the subject has dry eye in the eye based on the amount of the marker; A method comprising: (Item 9C) The method of claim 8C, wherein if the amount of the measured marker is higher or lower than the amount of the marker in the sample obtained from the healthy individual, the subject may be diagnosed as having or likely to have a neurological disorder. (Item 10C) The markers are 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, alpha actin 1 (Actin, alpha skeletal muscle), alpha 1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid binding protein, fibrinogen beta chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin alpha 5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, trypsin-3, and any combination thereof. (Item 11C) The method according to any one of items 8C to 10C, wherein the marker is selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof. (Item 12C) The method according to any one of items 8C to 11C, wherein the sample is tear fluid. (Item 13C) 1. A method for diagnosing whether a subject has dry eye associated with a neurological abnormality, the method comprising: obtaining a sample from the subject; Measuring at least one marker defined in any one of items 1C to 7C and at least one marker defined in any one of items 8C to 12C in the sample; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject, and determining that the subject has dry eye based on the measured amount of the marker; A method comprising: (Item 1D) 1. A method for detecting a neurological abnormality in an eye of a subject, the method comprising: Measuring the amount of a marker for ocular neurological abnormality in a sample obtained from the subject, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity rate; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy individual; A method comprising: (Item 2D) The method according to item 1D, wherein the parameters include at least one parameter selected from the group consisting of CNBD and CTBD. (Item 3D) The markers are cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, α1-acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10, haptoglobin, protein S100-A6, paralemmin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin, cytoplasmic 2, α-enolase, mammaglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid binding protein, cytokeratin 75, 75), histone H1.4, keratin 84 (Keratin, type II cuticular Hb4), galectin-7, cathepsin D, cytokeratin 6B (Keratin, type II cytoskeletal 6B), histone H1.1, cytokeratin 17 (Keratin, type I cytoskeletal 17), neutrophil defensin 3, cytokeratin 79 (Keratin, type II cytoskeletal 79), histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 4D) The method of any one of items 1D to 3D, wherein the marker is selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof. (Item 5D) The method according to any one of items 1D to 4D, which is a marker for neurological abnormalities in dry eye. (Item 6D) The method according to any one of items 1D to 5D, wherein the sample is tear fluid. (Item 7D) 1. A method for detecting dry eye in a subject, the method comprising: measuring the amount of a marker for dry eye in the sample, the marker being a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy individual; A method comprising: (Item 8D) The markers are 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, alpha actin 1 (Actin, alpha skeletal muscle), alpha 1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid binding protein, fibrinogen beta chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin alpha 5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, trypsin-3, and any combination thereof. (Item 9D) The method according to item 7D or 8D, wherein the marker is selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof. (Item 10D) The method according to any one of items 7D to 9D, wherein the sample is tear fluid. (Item 11D) 1. A method for detecting dry eye associated with a neurological abnormality in a subject, the method comprising: Measuring at least one marker as defined in any one of items 1D to 6D and at least one marker as defined in any one of items 7D to 10D in the sample; comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy individual; A method comprising:

[0007] In the present invention, it is intended that one or more of the above features may be provided in combination in addition to the combinations shown. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. Effect of the Invention

[0008] The markers disclosed herein are indicative of ocular neurological disorders, and may be used to diagnose conditions associated with ocular neurological disorders and to screen for drugs effective in treating ocular neurological disorders. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows an overview of the study design in Example 1. [Diagram 2] FIG. 2 shows the background of the subjects studied in Example 1. [Diagram 3] Figure 3 shows the results of the questionnaire regarding subjective symptoms. [Figure 4]FIG. 4 shows the visual analog scale (VAS) results for dry eye symptoms (blurred vision, photophobia, and eye irritation). [Diagram 5] FIG. 5 shows the results of the ophthalmological examination. [Figure 6] Figure 6 shows the results of each neurological parameter measured by confocal laser scanning microscopy. Statistical analysis was performed using Welch's t-test. [Figure 7] Figure 7 shows a correlation graph between CNBD and CTBD and BUT. Statistical analysis was performed using the Pearson correlation coefficient. [Figure 8] FIG. 8 shows a list of proteins detected in tears that were correlated with neurological parameters. [Figure 9] 9 shows a correlation graph between cystatin-S and CNFD, CNBD, CNFL, CTBD, and meandering rate. Statistical analysis was performed using the Pearson correlation coefficient. [Figure 10] Figure 10 shows the correlation between each parameter and profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, and protein S100-A9. Statistical analysis was performed using the Pearson correlation coefficient. [Figure 11] FIG. 11 shows a list of proteins that correlate with BUT and subjective symptoms (DEQ5 and OSDI). [Figure 12] Figure 12 shows the correlation between the parameters of secretory leukocyte protease inhibitor (Antileukoproteinase) and galectin-3-binding protein BUT, OSDI and DEQ5. Statistical analysis was performed using the Pearson correlation coefficient. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described below. Throughout this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the case of conflict, the present specification (including definitions) will take precedence. In this specification, "about" means ±10% of the following value.

[0011] (definition) In this specification, "nerve abnormality" in the eye refers to abnormality of the corneal sensory nerve. The corneal sensory nerve is the trigeminal nerve, and nerve abnormality in the eye is accompanied by symptoms caused by the decreased function of the trigeminal nerve, such as photophobia, decreased heat and cold sensation, eye burning sensation, eye pain due to wind, light and temperature changes, and decreased pain tolerance.

[0012] As used herein, "corneal nerve fibre density" (CNFD) refers to the density of nerve fibres per mm 2 This refers to the number of nerve fibers per unit area.

[0013] As used herein, the term "corneal nerve branch density" (CNBD) refers to the density of the nerve branch of the cornea per mm 2 This refers to the number of branch points on the main fibers per axial axis.

[0014] As used herein, the term "corneal nerve fibre length" (CNFL) refers to the length of the nerve fibres per mm 2 Refers to the total length of the nerves per unit area.

[0015] As used herein, the term "corneal nerve fibre total branch density (CTBD)" refers to the density of the nerve fibres in the cornea per mm 2 This refers to the total number of branching points per

[0016] As used herein, the term "corneal nerve fibre area (CNFA)" refers to the area of ​​the cornea that is 1 mm 2 Total area of ​​nerve fibers per mm 2 )

[0017] In this specification, the term "corneal nerve fibre width" (CNFW) refers to the width of the cornea per mm 2 This refers to the average nerve fiber width (mm) per

[0018] As used herein, "nerve tortuosity" refers to the average curvature of the nerve trunk in the cornea. Tortuosity has measurements based on SLD and TCI, where SLD is 1 minus the length of the straight line connecting the start and end of the trunk divided by the actual length of the trunk, and TCI refers to the change in the slope of the trunk.

[0019] As used herein, "marker" refers to an indicator, e.g., a predictive, diagnostic and / or prognostic indicator, that can be detected in a sample, and includes polynucleotides (e.g., DNA and / or RNA), proteins, and polypeptides. A marker can serve as an indicator of a particular subtype of a disease or disorder, characterized by certain molecular, pathological, histological and / or clinical characteristics.

[0020] As used herein, "dry eye" refers to a condition accompanied by ocular discomfort and visual abnormalities caused by a decrease in tears or a change in the composition thereof.

[0021] As used herein, the term "subject" refers to a subject to which the disclosed therapeutic and preventive compositions or combinations or methods are administered, and examples of subjects include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, goats, pigs, etc.).

[0022] A description of preferred embodiments will be given below, but it should be understood that this embodiment is an example of the present invention, and the scope of the present invention is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications and changes within the scope of the present invention by referring to the following preferred examples. Regarding these embodiments, those skilled in the art can combine any embodiments as appropriate.

[0023] (neurological abnormality marker) In one aspect, the present disclosure provides a marker of ocular nerve abnormality, whose expression changes (increases or decreases) in correlation with morphological parameters of ocular nerve. The present inventors have found that there is a correlation between dry eye patients and morphological parameters of ocular nerve (e.g., CNBD, CTBD, CNFD, CNFL and meandering rate). In particular, CNBD and CTBD are significantly higher in dry eye patients compared to healthy subjects. Thus, in some embodiments, the parameter may include at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and meandering rate. In certain embodiments, the parameter may include at least one parameter selected from CNBD and CTBD.

[0024] In some embodiments, the neurological abnormality may be corneal neurological abnormality. In certain embodiments, the neurological abnormality may be dry eye neurological abnormality. As shown in Figure 7, there are healthy subjects with BUT of 5 seconds and high CNBD and CTBD, and these subjects are not dry eye patients but may be treated as those with neurological abnormality.

[0025] In some embodiments, the markers are cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, alpha 1 acid glycoprotein 1, alpha 1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, keratin, type I cytoskeletal 10, haptoglobin, protein S100-A6, paralemmin-1, laminin alpha 5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, gamma actin, cytoplasmic 2, alpha enolase, mammaglobin-B, 14-3-3 protein zeta / delta, epithelial fatty acid binding protein, cytokeratin, type II cytoskeletal 75), histone H1.4, keratin 84 (Keratin, type II cuticular Hb4), galectin-7, cathepsin D, cytokeratin 6B (Keratin, type II cytoskeletal 6B), histone H1.1, cytokeratin 17 (Keratin, type I cytoskeletal 17), neutrophil defensin 3, cytokeratin 79 (Keratin, type II cytoskeletal 79), histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (peptidyl-prolyl cis-trans isomerase A), and any combination thereof.

[0026] In a preferred embodiment, the marker may be selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof. These markers are correlated with both CNBD and CTBD.

[0027] (Method of identifying markers) In another aspect, the disclosure provides a method for identifying markers of neurological abnormalities in the eye, comprising: (a) obtaining a parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity rate in a healthy subject and a dry eye patient; (b) measuring one or more gene expression levels or protein expression levels in samples obtained from the healthy subject and the dry eye patient; and (c) correlating the measurements with the one or more gene expression levels or protein expression levels.

[0028] In some embodiments, the parameter may include at least one of CNBD and CTBD. In some embodiments, the sample is tear fluid. In some embodiments, the ocular neurological abnormality may be corneal neurological abnormality and / or dry eye neurological abnormality.

[0029] (Dry eye marker) In another embodiment, the disclosure provides a marker for dry eye whose expression is altered (increased or decreased) in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5.

[0030] In some embodiments, the markers are 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, alpha actin 1 (Actin, alpha skeletal muscle), alpha 1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid binding protein, fibrinogen beta chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin alpha 5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, trypsin-3, and any combination thereof.

[0031] In a preferred embodiment, the marker may be selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof.

[0032] In some embodiments, at least one of the markers for ocular neurological abnormalities and at least one of the markers for dry eye may be used in combination.

[0033] (Detection Agent) In another aspect, the present disclosure provides a detection agent for detecting the protein or nucleic acid whose expression changes (increases or decreases) in correlation with the morphological parameters of the nerve in eye.In some embodiments, the morphological parameters of the nerve are selected from the group consisting of CNBD, CTBD, CNFD, CNFL and meandering rate, and preferably include at least one parameter of at least CNBD and CTBD.

[0034] In some embodiments, proteins or nucleic acids whose expression is altered (increased or decreased) in correlation with morphological parameters of nerves in the eye include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, lipocalin-1, alpha 1 acid glycoprotein 1, alpha 1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, keratin, type I cytoskeletal 10, haptoglobin, protein S100-A6, paralemmin-1, laminin alpha 5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, gamma actin, cytoplasmic 2), α-enolase, mammaglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid binding protein, cytokeratin 75, histone H1.4, keratin 84, galectin-7, cathepsin D, cytokeratin 6B, histone H1.1, cytokeratin 17, neutrophil defensin 3, cytokeratin 79, histone H2B 1-N type, peroxiredoxin-1, cytokeratin 12 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophilin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof.

[0035] In a preferred embodiment, the protein or nucleic acid whose expression changes (increases or decreases) in correlation with morphological parameters of the nerves in the eye may be selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof.

[0036] Examples of detection agents include, but are not limited to, antibodies that specifically bind to the above proteins, nucleic acids that specifically hybridize to the above nucleic acids, etc. Detection is also possible using a mass spectrometer.

[0037] In some embodiments, the detection agent of the present disclosure can be used as a diagnostic agent for ocular neurological abnormality.The detection agent of the present disclosure is used to measure the amount of the protein or nucleic acid in a sample obtained from a subject, and the measured value is compared with the average value of healthy subjects.If the measured value is significantly different from the average value of healthy subjects, the subject can be diagnosed as having or possibly having ocular neurological abnormality.Samples include, but are not limited to, tears, blood, and conjunctival scrapings.In a preferred embodiment, the sample is tears.

[0038] In a further aspect, the present disclosure provides a detection agent for detecting a protein or nucleic acid whose expression is altered in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5.

[0039] Examples of proteins or nucleic acids whose expression changes (increases or decreases) in correlation with at least one parameter selected from the group consisting of BUT, OSDI, and DEQ5 include 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, alpha actin 1 (Actin, alpha skeletal muscle), alpha 1-antichymotrypsin, calmodulin-3, cathepsin D, epithelial fatty acid binding protein, fibrinogen β chain, glucose-6-phosphate isomerase, histone H1.4, histone H2A 1-C type, and histone H2B 1-J type, keratin 84 (Keratin, type II cuticular Hb4), cytokeratin 79 (Keratin, type II cytoskeletal 79), laminin α5, non-histone chromosomal protein HMG-17, nucleobindin-2, peroxiredoxin-1, polymeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, and trypsin-3.

[0040] In certain embodiments, the protein or nucleic acid whose expression changes (increases or decreases) in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5 may be selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof.

[0041] (Diagnostic Method) In a further aspect, the present disclosure provides a method for diagnosing an ocular condition (e.g., neurological abnormality, dry eye, or dry eye associated with neurological abnormality) in a subject, the method comprising the steps of obtaining a sample from the subject, measuring the amount of a marker (protein or nucleic acid) described herein in the sample, comparing the amount of the measured marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has an ocular neurological abnormality based on the amount of the measured marker.If the amount of the measured marker is higher or lower than the amount of the marker in a sample obtained from a healthy person, the subject can be diagnosed as having or likely to have an ocular condition.

[0042] In another aspect, the disclosure provides a method of using a marker described herein as an indicator for diagnosing an ocular condition in a subject, the method comprising measuring an amount of a marker described herein in a sample obtained from the subject and comparing the measured amount of the marker to the amount of the marker in a sample obtained from a healthy individual.

[0043] In some embodiments, the ocular condition can be dry eye, an ocular neurological disorder, a corneal neurological disorder, or a dry eye neurological disorder, hi some embodiments, the sample can be a tear sample.

[0044] (Therapeutic drug screening method) In one aspect, a method is provided for screening for a drug to treat an ocular condition (e.g., dry eye, neurological disorders, etc.) in a subject, the method comprising administering a candidate drug to the eye of a model animal and measuring the amount of a marker described herein in a sample obtained from the model animal before and after administration. If the marker in the sample obtained from the model animal after administration is elevated or decreased compared to before administration, the candidate drug can be identified as effective in treating the ocular condition.

[0045] The model animal used in this screening method is a non-human animal, and examples of the model animal include mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.). In a specific embodiment, the model animal may be a lacrimal gland removal model, a forced eye opening model, a tear secretion inhibition model, a corneal flap model, or a Sjogren's syndrome model.

[0046] (Detection method) Provided is a method for detecting an ocular condition (e.g., neurological disorder, dry eye, or dry eye associated with neurological disorder), comprising measuring the amount of a marker (protein or nucleic acid) described herein in the sample, and comparing the amount of the measured marker with the amount of the marker in a sample obtained from a healthy individual. In some embodiments, the subject may be determined to have dry eye based on the amount of the measured marker. In a specific embodiment, the ocular condition may be detected in the sample if the amount of the measured marker is higher or lower than the amount of the marker in a sample obtained from a healthy individual.

[0047] (Companion Diagnostics) Provided is a method for treating an ocular condition in a subject (e.g., a neurological disorder, dry eye, or dry eye associated with a neurological disorder), the method comprising the steps of measuring the amount of a marker (protein or nucleic acid) described herein in the sample, comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy subject to diagnose whether or not the subject has the ocular condition, and administering a pharmaceutical agent for preventing or treating the ocular condition to a subject determined to have the ocular condition in the diagnosing step.

[0048] Examples of medications for preventing or treating ocular conditions (e.g., neurological disorders, dry eye, or dry eye associated with neurological disorders) include, but are not limited to, Diquas ophthalmic solution (dicafossol sodium), Mucosta ophthalmic solution (rebamipide), and Hyalein ophthalmic solution (purified sodium hyaluronate).

[0049] The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0050] The present invention will be described in more detail below with reference to the following examples. The research in these examples will be conducted in accordance with the ethical principles based on the Declaration of Helsinki and in compliance with the "Ethical Guidelines for Medical Research Involving Human Subjects."

[0051] (Example 1: Relationship between homeostasis of the ocular surface and corneal sensory nerves) The purpose of this example is to clarify the relationship between the shape of the corneal sensory nerve and corneal sensation / dry eye symptoms. The shapes of the corneal sensory nerves of dry eye patients and healthy adults were observed and measured using a confocal laser scanning microscope, and corneal sensitivity was measured, as well as subjective symptoms and ophthalmological examinations of the ocular surface, which are general dry eye examination items, to analyze the effect of the shape of the corneal sensory nerve on sensation and its relationship with dry eye symptoms.

[0052] (method) (subject) Dry eye patients and healthy adults (Selection Criteria) Subjects who met all of the following criteria were included in this study. (1) Individuals who have given voluntary written consent to participate in this study (2) Those aged 20 years or older and under 65 years old on the day of consent (the day the patient agrees to participate in this clinical trial) <Criteria for dry eye patients> (3) Subjects who have had subjective symptoms related to dry eye for one year as of the date of consent (4) Subjects with a total score of 6 or more on the Dry Eye Questionnaire (DEQ-5 Japanese version) in the screening test (5) Subjects with a tear film breakup time (BUT) of 5 seconds or less in the screening test <Standards for healthy adults> (6) Subjects with no history of dry eye within one year prior to the date of consent (7) Subjects who are not receiving continuous eye drop treatment for other ophthalmic diseases (8) Subjects with a total score of 6 or less on the Dry Eye Questionnaire (DEQ-5 Japanese version) in the screening test (9) Those who meet the following criteria in the screening test: 1) Tear breakup time (BUT) ≥ 8 seconds in at least one eye and > 5 seconds in the other eye 2) Fluorescein staining score for corneal and conjunctival epithelial damage <3 points (exclusion criteria) (1) Those with autoimmune diseases, including atopic dermatitis (2) Those with severe eye disorders due to Stevens-Johnson syndrome or ocular pemphigoid (3) Those who have had ophthalmic surgery (including corneal refractive surgery (LASIK) and punctal plug insertion) (4) Glaucoma patients (currently undergoing treatment) (5) Subjects with anterior segment diseases other than dry eye (6) Those with an infectious disease (7) Subjects who are unable to discontinue or are expected to discontinue use of eye drops (all prescription and over-the-counter medications except artificial tears) from the screening test until the end of the study period. (8) Subjects who cannot stop wearing contact lenses one week prior to each visit (9) Any other person who is judged by the principal investigator or other relevant person to be inappropriate for this study (Stopping criteria) The principal investigator or other person in charge of the study will discontinue the study on the subject if any of the following conditions apply: (1) If the subject requests to discontinue participation in the study (2) Any other case in which the principal investigator or other person in charge of the study deems it inappropriate. (Research design) partial shielding * , Intergroup comparative study (prospective observational study) * The photographers of the confocal laser scanning microscopy examination and the image analyzers were kept confidential from the subjects' pathological conditions.

[0053] (Research flow) Dry eye patients and healthy adult volunteers were enrolled in this study and various observations and tests were performed according to the following procedure (Figure 1).

[0054] (1) Visit 1 The principal investigator and co-investigator of the medical institution (hereinafter referred to as the principal investigators, etc.) thoroughly explained the contents of the study and other matters related to the study to the dry eye patients or healthy adults using an explanatory document, and obtained their voluntary, written consent to participate in the study.

[0055] (2) Visit 2 The study investigators conducted screening tests for all subjects who provided written consent, including a medical interview, subjective symptoms (using the Japanese version of the Dry Eye Questionnaire 5 (DEQ-5)), and ophthalmological tests (BUT test, corneal and conjunctival epithelial disorders) to confirm the eligibility of the subjects. The background of the subjects is shown in Figure 2.

[0056] Visit 2 may also be conducted on the same day as Visit 1.

[0057] (3) Visit 3 After Visit 2, the study investigators had the subjects return to the medical institution to collect tears (twice from each eye) and conduct a medical interview, subjective symptoms (using a dry eye questionnaire (DEQ-5 Japanese version, Ocular Surface Disease Index; OSDI Japanese version), a neuropathic ocular pain (NOP) questionnaire, and a visual analog scale (VAS) for dry eye symptoms), and an ophthalmological examination.

[0058] The two tear samples were to be taken at least one hour apart, and the ophthalmological examination was to be carried out at least one hour after the second tear sample. The order of the interview and the subjective symptoms / questionnaire survey was not important.

[0059] (4) Visit 4 After Visit 3, the study investigators had the subjects return to the medical institution where the study was conducted and a confocal laser scanning microscope examination was performed.

[0060] (Evaluation items) (1) Primary endpoint Corneal sensory nerve parameters based on confocal laser scanning microscopy images [Corneal nerve fiber length (CNFL), Corneal nerve fiber density (CNFD), Corneal nerve branch density (CNBD), Corneal nerve fiber total branch density (CTBD), Corneal nerve fiber area (CNFA), Corneal nerve fiber width (CNFW)] (2) Secondary endpoints 1) Parameter for corneal sensory nerve degeneration based on confocal laser scanning microscope images [Nerve Fiber Tortuosity (NFT)] 2) Comprehensive comparative quantification of tear proteins using iTRAQ (Example 2) 3) Tear breakup time (BUT) 4) Schirmer Test I 5) Fluorescein staining score for corneal and conjunctival epithelial damage 6) Corneal Sensitivity 7) Subjective symptoms [Dry eye questionnaire (DEQ-5 Japanese version, Ocular Surface Disease Index (OSDI Japanese version), Neuropathic Ocular Pain (NOP) questionnaire, visual analog scale (VAS) for dry eye symptoms] (Subjective symptoms) The following questionnaire will be used to investigate subjective symptoms related to dry eye and neuropathic eye pain. (1) Dry Eye Questionnaire (DEQ-5 Japanese version) (implemented at Visits 2 and 3) (2) Dry eye questionnaire (OSDI Japanese version) (implemented at Visit 3) (3) Questionnaire on neuropathic ocular pain (NOP) (implemented at Visit 3) (4) VAS for dry eye symptoms (blurred vision, photophobia, eye irritation) (Performed at Visit 3) (Tear collection) The study investigators followed the procedure below at Visit 3 to collect tear fluid for iTRAQ and oxidative stress marker measurement, twice from each eye for a total of four times. The second tear fluid collection (oxidative stress marker) was conducted at least one hour after the first tear fluid collection (iTRAQ). The results of iTRAQ are described in detail in Example 2. (1) 50 μL of saline was instilled into the conjunctival sac of the lower eyelid using a sterile micropipette. (2) Without blinking, the eyeballs were moved left and right and up and down four times to allow the instilled saline to mix with tears. (3) The saline solution was collected from the conjunctival sac of the lower eyelid into a low-adsorption container using a micropipette used for instilling saline into the eye. (4) The collected samples were promptly frozen and stored at -80°C. (5) The frozen samples were then sent frozen on dry ice to each measurement institution.

[0061] (Ophthalmological examination) The study investigators performed the following ophthalmological examinations:

[0062] (1) Tear breakup time (BUT) test (Performed at Visits 2 and 3) The time from opening the eyelids to the first occurrence of tear film break anywhere on the cornea was measured three times with the eyelids naturally open, and the average was calculated to one decimal place. In addition, the tear film break patterns were classified into five types (Area break; AB, Spot break; SB, Line break; LB, Dimple break; DB, Random break; RB) and recorded.

[0063] (2) Corneal and conjunctival epithelial disorders (visits 2 and 3) According to the 2006 dry eye diagnostic criteria (Shimazaki Jun, Dry Eye Research Society. 2006 dry eye diagnostic criteria), the cornea and conjunctiva were divided into three sections (temporal bulbar conjunctiva, cornea, and nasal bulbar conjunctiva), and a staining score (fluorescein) of 0 to 3 was determined for each, and the scores were then summed.

[0064] (3) Corneal Sensitivity Test (Performance Time: Visit 3) The cornea was divided into 5 sections (central, superior, inferior, temporal, and nasal), and the corneal sensitivity of each section was recorded using a Cochet-Bonnet aesthesiometer. Pain tolerance was also measured in the central cornea using a Cochet-Bonnet aesthesiometer.

[0065] (4) Schirmer test (Period: Visit 3) Measurements were performed using method I, which does not require topical anesthesia.

[0066] (Confocal laser scanning microscopy) At Visit 4, a Rostock corneal module was attached to a Heidelberg Retina tomograph to image the corneal trigeminal nerve beneath the corneal epithelium. The photographer of the confocal laser scanning microscopy examination was kept anonymous regarding the subjects' pathology.

[0067] (cancel) If any of the following conditions were met, the principal investigator or other person in charge of the study will discontinue the study for that subject. (1) If the subject requests to discontinue participation in the study (2) Any other case in which the principal investigator or other person in charge of the study deems it inappropriate. (Adverse Events) (Definition of adverse events) Adverse events were defined as any undesirable or unintended injury, illness, or symptom thereof that occurred during tear collection, testing, etc., regardless of whether or not there was a causal relationship to the study conducted.

[0068] (Collection and recording of adverse events) If an adverse event occurred, the medical institution conducting the study took appropriate action as necessary, and recorded in the case record the details of the symptoms / findings (diagnosis), whether or not they were serious, the date of onset and resolution, whether or not treatment was administered (details of treatment), and the outcome, etc.

[0069] (Evaluated eye) In principle, for the analysis of each evaluation item except for subjective symptoms, the eye with the shorter BUT at Visit 3 was used as the primary analysis target for dry eye patients, and the eye with the longer BUT was used for healthy adults. If the BUT in both eyes was the same, the right eye was used as the primary evaluation target.

[0070] In addition, eyes that were not evaluated in the main analysis were sometimes evaluated to confirm the reproducibility of the analysis results, for exploratory analysis, etc.

[0071] (Data Handling Standards) In this study, missing values ​​were not imputed. In addition, when abnormal values ​​were found in the measurements of oxidative stress markers, the cause was investigated, and in principle, all measured data was used for analysis, except for abnormal values ​​with clear evidence.

[0072] (Analysis method) (Analysis of primary endpoint) Summary statistics were calculated for corneal sensory nerve parameters based on confocal laser scanning microscope images [corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW)] for dry eye patients and healthy adults, and comparisons were made between the two groups. The significance level for hypothesis testing was set at 0.05 on both sides.

[0073] (Analysis of secondary endpoints) (1) Summary statistics were calculated for the parameter related to corneal sensory nerve degeneration [nerve fiber tortuosity (NFT)] based on confocal laser scanning microscope images in dry eye patients and healthy adults, and a comparison was made between the two groups.

[0074] (2) Differences in tear protein levels quantified by iTRAQ between dry eye patients and healthy adults were examined (Example 2).

[0075] (3) Summary statistics for tear film breakup time (BUT) were calculated for dry eye patients and healthy adults, and a comparison was made between the two groups.

[0076] (4) For the Schirmer I test, summary statistics were calculated for dry eye patients and healthy adults, and a comparison was made between the two groups.

[0077] (5) Summary statistics were calculated for the fluorescein staining scores of corneal and conjunctival epithelial damage in dry eye patients and healthy adults, and a comparison was made between the two groups.

[0078] (6) Regarding corneal sensitivity, summary statistics were calculated for dry eye patients and healthy adults, and a comparison was made between the two groups.

[0079] (7) Summary statistics were calculated for the results of the DEQ-5 (Japanese version), OSDI (Japanese version), NOP questionnaire, and VAS for dry eye patients and healthy adults, and a comparison was made between the two groups.

[0080] (Additional considerations) In addition to the analysis of the primary and secondary endpoints, exploratory analyses were conducted to examine the relationship between the primary and secondary endpoints and the subject's condition (dry eye vs. healthy adults) using appropriate methods such as logistic regression. The relationship between the primary and secondary endpoints was also examined.

[0081] (result) Figure 3 shows a summary of the results of the questionnaire on subjective symptoms. None of the healthy subjects (16 people) met any of the questions about neuropathic eye pain (NOP). On the other hand, not all dry eye patients met any of the questions, and some patients did not meet any of the questions. This suggests that there may be two types of dry eye: those related to nerve abnormalities and those not related to nerve abnormalities.

[0082] Figure 4 shows a summary of the VAS results for dry eye symptoms (blurred vision, photophobia, eye irritation). As shown, the VAS was significantly higher in dry eye patients.

[0083] A summary of the ophthalmological examination results is shown in Figure 5. The BUT and pain tolerance were significantly higher in dry eye patients than in healthy subjects, but there were no significant differences between dry eye patients and healthy subjects in corneal and conjunctival epithelial damage, corneal sensitivity test, and Schirmer test.

[0084] Figure 6 shows the results of each neurological parameter measured by confocal laser scanning microscopy. In this study, the following parameters were measured: Corneal nerve fiber density (CNFD: 1mm 2 (number of fibers per Corneal nerve branching density (CNBD: 1mm 2 (number of branch points on the main fiber per Corneal nerve fiber length (CNFL: 1mm 2 (total nerve length per Corneal nerve fiber total branching density (CTBD: 1 mm 2 (total number of branch points per Corneal nerve fiber area (CNFA: 1mm 2 Total area of ​​nerve fibers per mm 2 )) Corneal nerve fiber width (CNFW: 1mm 2 Average nerve fiber width per Nerve tortuosity (mean curvature of the nerve trunk in the cornea) CNBD and CTBD were found to be significantly higher in dry eye patients than in healthy subjects, and these parameters were shown to be highly correlated with BUT (Figure 7).

[0085] (Example 2: Comprehensive measurement of proteins in human tears) This example aims to identify proteins that correlate with the neurological parameters measured in Example 1.

[0086] (material and method) The tear fluid samples collected in Example 1 from 30 healthy subjects (15 right eyes, 15 left eyes) and 67 dry eye patients (34 right eyes, 33 left eyes) were used for measurement ( FIG. 1 ). The tear fluid samples were frozen and stored (−80° C.) until measurement.

[0087] [Table 1]

[0088] [Table 2]

[0089] [Table 3]

[0090] [Table 4]

[0091] [Table 5]

[0092] [Table 6]

[0093] [Table 7]

[0094] [Table 8]

[0095] Measurement procedure (total protein concentration measurement) Calibration standards and QC samples are run in duplicate, and test samples are run once.

[0096] [Table 9]

[0097] Preparation of control samples A portion of each tear sample from healthy subjects was pooled to prepare the pool. The tear samples from healthy subjects to be used for pooling were determined based on the results of total protein concentration measurements.

[0098] [Table 10-1]

[0099] [Table 10-2]

[0100] [Table 11]

[0101] [Table 12]

[0102] [Table 13]

[0103] [Table 14]

[0104] (result) Figure 8 shows the protein ratios detected that correlated with neurological parameters (P<0.1). Among the proteins shown in Figure 8, cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, and protein S100-A9 were specifically correlated with both CNBD and CTBD, indicating that these proteins are strongly correlated with neurological abnormalities.

[0105] Cysteine-S correlated with five parameters, CNFD, CNBD, CNFL, CTBD, and tortuosity, and is considered to be one of the markers reflecting the state of ocular nerve abnormalities (Figure 9). Profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, and protein S100-A9 also correlated with nerve fiber branching density (in CNBD and CTBD) and nerve fiber length (CNFL), and are considered to be markers reflecting the state of ocular nerve abnormalities (Figure 10).

[0106] Figure 11 shows the proteins correlated with BUT and subjective symptoms (DEQ5 and OSDI). Among these, 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), and galectin-3-binding protein were correlated with BUT, OSDI, and DEQ5. Figure 12 shows the correlation of secretory leukocyte protease inhibitor (Antileukoproteinase) and galectin-3-binding protein with each parameter of BUT, OSDI, and DEQ5. These proteins showed significant correlation with each parameter of BUT, OSDI, and DEQ5.

[0107] Example 3: Diagnosis of neurological disorders in the eye The tear fluid of the subject is collected, and the concentration of cystatin-S in the tear fluid is measured by ELISA or PCR. If the measured concentration of cystatin-S in the tear fluid is significantly higher or lower than the reference value of a healthy person, the subject can be diagnosed as having or being likely to have a neurological abnormality. In addition to cystatin-S, the markers identified in Example 2 may also be measured.

[0108] Example 4: Screening of drugs to treat neurological disorders in the eye Candidate drugs are instilled into dry eye model animals, such as lacrimal gland removal model, forced eye opening model, tear secretion inhibition model, corneal flap model, and Sjogren's syndrome model. Tear samples are collected before and after instillation, and the concentrations of cystatin-S in tears before and after instillation are compared. If the concentration of cystatin-S in tears after instillation is significantly different from the concentration of cystatin-S in tears before instillation, the candidate drug is identified as effective in treating neurological abnormalities in the eye.

[0109] Although the present invention has been illustrated using the preferred embodiments thereof, it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications and literature cited in this specification are incorporated by reference in their entirety as if the contents themselves were specifically set forth in this specification.

[0110] This application claims the benefit of priority from Japanese Patent Application No. 2019-085809, filed on April 26, 2019, the contents of which are incorporated herein by reference in their entirety. [Industrial Applicability]

[0111] A marker correlating with ocular abnormalities (particularly neurological abnormalities and dry eye) is provided. This marker is useful as an indicator for diagnosis and for determining the effectiveness of drugs, and can be used in the fields of pharmaceuticals, etc.

Claims

1. 1. A method for detecting corneal sensory abnormalities in an eye, comprising measuring a marker of corneal sensory nerves in the eye, the expression of which correlates with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity, A method for detecting corneal neuropathy in the eye, wherein the marker is selected from the group consisting of cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transfer protein, protein S100-A9, and any combination thereof.

2. A method for detecting dry eye in an eye, comprising measuring a marker for dry eye whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI and DEQ5, A method for detecting dry eye in an eye, wherein the marker is selected from the group consisting of 14-3-3 protein theta, secretory leukocyte protease inhibitor (Antileukoproteinase), Galectin-3-binding protein, and any combination thereof.

3. A method for detecting dry eye associated with corneal neuropathy in an eye, comprising measuring at least one marker according to claim 1 and at least one marker according to claim 2.

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