Eye disease markers

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] This disclosure relates to markers and related technologies for eye conditions (e.g., ocular nerve abnormalities, dry eye, etc.). [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 dysfunction, and sometimes accompanied by damage to the ocular surface" (Non-Patent Literature 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 advances in understanding the pathogenesis of dry eye and the development of therapeutic drugs, it is now possible to diagnose the cause of reduced tear film stability by layering the ocular surface (corneal and conjunctival epithelium, aqueous tear film, and lipid tear film), and to propose appropriate treatments for each layer (Non-Patent Literature 1, incorporated herein by reference). On the other hand, corneal sensory nerves are an important element in maintaining the homeostasis of the ocular surface. Corneal sensory nerves, which branch off from the first branch of the trigeminal nerve, are distributed throughout the cornea, sense pain and temperature on the ocular surface, transmit this information to the brain, and contribute 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 confocal laser scanning microscopes, changes in nerve density and shape have been reported in patients with dry eye (Non-Patent Documents 2 and 3, as incorporated herein by reference), but it remains unclear whether these changes are the cause of dry eye.

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

[0004] [Non-Patent Document 1] Dry Eye Research Society, Committee on Definition and Diagnostic Criteria for Dry Eye. Revision of the Definition and Diagnostic Criteria for 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. [Overview of the project] [Means for solving the problem]

[0005] The inventors observed and measured the shape of corneal sensory nerves in dry eye patients and healthy adults using a confocal laser scanning microscope. They also measured corneal sensation and performed ophthalmic examinations of the ocular surface, which are common dry eye examination items, and newly discovered a relationship between the shape of corneal sensory nerves, corneal sensation, and dry eye symptoms. As a result of diligent research, the inventors discovered markers that show a correlation with changes in nerve density and shape in the cornea, and completed the present invention.

[0006] Therefore, this disclosure provides the following: (Item 1) A marker of ocular neurological abnormalities whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity. (Item 2) The marker according to item 1, wherein the parameter includes at least one parameter selected from the group consisting of CNBD and CTBD. (Item 3) The aforementioned markers include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin (Actin, cytoplasmic 2), α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, and cytokeratin 75 (Keratin, type II cytoskeletal Markers as described in item 1 or 2, selected from the group consisting of 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 4) The marker described in 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-related lipocalin, phospholipid transport protein, protein S100-A9, and any combination thereof. (Item 5) A marker for nerve abnormalities in dry eye, as described in one of items 1-4. (Item 6) A detection agent for detecting any one of the markers listed in items 1 to 5. (Item 7) A method for identifying markers of nerve abnormalities in the eye, (a) A step of obtaining a parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL and tortuosity in healthy individuals and patients with dry eye, (b) A step of measuring the expression level of one or more genes or proteins in samples obtained from the healthy person and the dry eye patient, (c) A step of correlating the measured value with the expression level of one or more genes or proteins. Methods that include... (Item 8) The method according to item 7, wherein the parameter includes at least CNBD or CTBD. (Item 9) The method according to item 7 or 8, wherein the sample is tear fluid. (Item 10) The method according to any one of items 7 to 9, wherein the marker is a marker for dry eye associated with neurological abnormalities. (Item 11) 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. (Item 12) A marker according to item 11, selected from the group consisting of 14-3-3 protein θ, secretory leukoprotease 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 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, trypsin-3, and any combination thereof. (Item 13) The marker according to item 11 or 12, wherein the marker is selected from the group consisting of 14-3-3 protein θ, secretory leukoprotease 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 related to dry eye associated with nerve 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) A method for diagnosing whether a subject has a nerve abnormality in the eye, the method comprising: obtaining a sample of the subject; A step of measuring the amount of a marker of nerve abnormality in the eye in the sample, wherein the marker is 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, the step; 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 nerve abnormality in the eye based on the measured amount of the marker A method comprising. (Item 2A) If the measured amount of the marker is higher or lower than the amount of the marker in the sample obtained from the healthy subject, it can be diagnosed that the subject has or is likely to have nerve abnormality. The method according to claim 1A. (Item 3A) The method according to Item 1A or 2A, wherein the parameter includes at least one parameter selected from the group consisting of CNBD and CTBD. (Item 4A) The aforementioned markers include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin (Actin, cytoplasmic 2), α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, and cytokeratin 75 (Keratin, type II cytoskeletal The method described in any one of items 1A to 3A, selected from the group consisting of 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 5A) The method according to 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-related lipocalin, phospholipid transport protein, protein S100-A9, and any combination thereof. (Item 6A) A marker for nerve abnormalities in dry eye, as described in any one of items 1A to 5A. (Item 7A) The method according to any one of items 1A to 6A, wherein the sample is tear fluid. (Item 8A) A method for diagnosing whether a subject has dry eye, the method being: The steps include obtaining a sample of the subject, A step of measuring the amount of a dry eye marker in the sample, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI, and DEQ5. The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has dry eye based on the measured amount of the marker. Methods that include... (Item 9A) The method according to claim 8A, wherein if the measured amount of the marker is higher or lower than the amount of the marker in the sample obtained from the healthy person, it can be diagnosed that the person has or is likely to have a neurological abnormality. (Item 10A) The aforementioned markers include 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), and cytokeratin 79 (Keratin, type II cytoskeletal). 79) The method described in item 8A or 9A, selected from the group consisting of laminin α5, non-histone chromosome protein HMG-17, nucleobingin-2, peroxiredoxin-1, multimeric 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 θ, secretory leukocyte protease inhibitors (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) A method for diagnosing whether a subject has dry eye associated with a neurological disorder, the method being: The steps include obtaining a sample from the subject, The steps include measuring at least one marker specified in any one of items 1A to 7A and at least one marker specified in any one of items 8A to 12A in the sample, The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining, based on the amount of the marker, that the subject has dry eye associated with a neurological disorder. Methods that include... (Item 1B) A method for treating the eyes of a subject, wherein the method is The steps include obtaining a sample of the subject, A step of measuring the amount of a marker for ocular neurological abnormalities in the sample, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity. A step of diagnosing whether there is a nerve abnormality in the eye by comparing the amount of the marker measured with the amount of the marker in a sample obtained from a healthy person, The diagnostic step involves administering a drug to a subject who is determined to have a neurological abnormality, for the purpose of preventing or treating the neurological abnormality. Methods that include... (Item 2B) The method according to claim 1B, wherein if the measured amount of the marker is higher or lower than the amount of the marker in the sample obtained from the healthy person, it can be diagnosed that the person has or is likely to have a neurological abnormality. (Item 3B) The method according to item 1B or 2B, wherein the parameter includes at least one parameter selected from the group consisting of CNBD and CTBD. (Item 4B) The aforementioned markers include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin (Actin, cytoplasmic 2), α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, and cytokeratin 75 (Keratin, type II cytoskeletal The method described in any one of items 1B to 3B, selected from the group consisting of 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin 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-related lipocalin, phospholipid transport protein, protein S100-A9, and any combination thereof. (Item 6B) A marker for nerve abnormalities in dry eye, as described in any one of items 1B to 5B. (Item 7B) The method according to any one of items 1B to 6B, wherein the sample is tear fluid. (Item 8B) A method for treating dry eye in a subject, wherein the method is The steps include obtaining a sample of the subject, A step of measuring the amount of a dry eye marker in the sample, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI, and DEQ5. The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has dry eye based on the amount of the marker. The diagnostic step involves administering a drug for the prevention or treatment of dry eye to a subject who is determined to have dry eye in the diagnostic step. Methods that include... (Item 9B) The method according to claim 8B, wherein if the measured amount of the marker is higher or lower than the amount of the marker in the sample obtained from the healthy person, it can be diagnosed that the person has or is likely to have a neurological abnormality. (Item 10B) The aforementioned markers include 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), and cytokeratin 79 (Keratin, type II cytoskeletal). 79) The method described in item 8B or 9B, selected from the group consisting of laminin α5, non-histone chromosome protein HMG-17, nucleobingin-2, peroxiredoxin-1, multimeric 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 θ, secretory leukocyte protease inhibitors (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) A method for treating dry eye associated with neurological abnormalities in a subject, the method is: The steps include obtaining a sample from the subject, The steps include measuring at least one marker specified in any one of items 1B to 7B and at least one marker specified in any one of items 8B to 12B in the sample, The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has dry eye associated with a neurological disorder based on the amount of the marker, In the diagnostic step, a subject is determined to have dry eye associated with a neurological abnormality, and the step is to administer a medicine for the prevention or treatment of dry eye associated with a neurological abnormality. Methods that include... (Item 1C) An in vitro method for diagnosing whether a subject has a nerve abnormality in the eye, wherein the method is A step of measuring in vitro a marker of neuronal abnormality in the eye of a subject, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity; The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has a nerve abnormality in the eye based on the amount of the marker. Methods that include... (Item 2C) The method according to claim 1C, wherein if the measured amount of the marker is higher or lower than the amount of the marker in the sample obtained from the healthy person, it can be diagnosed that the person has or is likely to have a neurological abnormality. (Item 3C) The method according to item 1C or 2C, wherein the parameter includes at least one parameter selected from the group consisting of CNBD and CTBD. (Item 4C) The aforementioned markers include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin (Actin, cytoplasmic 2), α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, and cytokeratin 75 (Keratin, type II cytoskeletal The method described in any one of items 1C to 3C, selected from the group consisting of 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 5C) The method according to 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-related lipocalin, phospholipid transport protein, protein S100-A9, and any combination thereof. (Item 6C) The method described in any one of items 1C to 5C, which are markers for nerve 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) An in vitro method for diagnosing whether a subject has dry eye, wherein the method is: A step of measuring a dry eye marker in vitro in a sample of the subject, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI, and DEQ5. The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has dry eye in the eye based on the amount of the marker. Methods that include... (Item 9C) The method according to claim 8C, wherein if the measured amount of the marker is higher or lower than the amount of the marker in the sample obtained from the healthy person, it can be diagnosed that the person has or is likely to have a neurological abnormality. (Item 10C) The aforementioned markers include 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), and cytokeratin 79 (Keratin, type II cytoskeletal). 79) The method described in item 8C or 9C, selected from the group consisting of laminin α5, non-histone chromosome protein HMG-17, nucleobingin-2, peroxiredoxin-1, multimeric 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 θ, secretory leukocyte protease inhibitors (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) A method for diagnosing whether a subject has dry eye associated with a neurological disorder, the method being: The steps include obtaining a sample from the subject, The steps include measuring at least one marker specified in any one of items 1C to 7C and at least one marker specified in any one of items 8C to 12C in the sample, The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining that the subject has dry eye based on the measured amount of the marker. Methods that include... (Item 1D) A method for detecting nerve abnormalities in the eye of a subject, the method is: A step of measuring the amount of a marker for ocular neurological abnormalities in a sample obtained from the subject, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity. The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person. Methods that include... (Item 2D) The method according to item 1D, wherein the parameter includes at least one parameter selected from the group consisting of CNBD and CTBD. (Item 3D) The aforementioned markers include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin (Actin, cytoplasmic 2), α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, and cytokeratin 75 (Keratin, type II cytoskeletal The method described in item 1D or 2D, selected from the group consisting of 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof. (Item 4D) The method according to 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-related lipocalin, phospholipid transport protein, protein S100-A9, and any combination thereof. (Item 5D) The method described in any one of items 1D to 4D, which is a marker for nerve 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) A method for detecting dry eye in a subject, wherein the method is A step of measuring the amount of a dry eye marker in the sample, wherein the marker is a marker whose expression changes in correlation with at least one parameter selected from the group consisting of BUT, OSDI, and DEQ5. The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person. Methods that include... (Item 8D) The aforementioned markers include 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), and cytokeratin 79 (Keratin, type II cytoskeletal). 79) The method according to item 7D, selected from the group consisting of laminin α5, non-histone chromosome protein HMG-17, nucleobingin-2, peroxiredoxin-1, multimeric 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 θ, secretory leukocyte protease inhibitors (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) A method for detecting dry eye associated with neurological abnormalities in a subject, wherein the method is: The steps include measuring at least one marker specified in any one of items 1D to 6D and at least one marker specified in any one of items 7D to 10D in the sample, The steps include comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person. Methods that include...

[0007] In the present invention, the one or more of the above features are intended to be provided in combination with or without expressly stated combinations. Further embodiments and advantages of the present invention will be recognized by those skilled in the art by reading and understanding the detailed description below as needed. [Effects of the Invention]

[0008] The markers described herein serve as indicators of ocular nerve abnormalities. According to this disclosure, it is possible to diagnose conditions related to ocular nerve abnormalities and to screen for drugs that are effective in treating ocular nerve abnormalities. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows a schematic of the research design in Example 1. [Figure 2] Figure 2 shows the background of the subjects who were included in the study in Example 1. [Figure 3] Figure 3 shows the results of the questionnaire regarding subjective symptoms. [Figure 4]Figure 4 shows the results of a visual analog scale (VAS) for dry eye symptoms (blurred vision, photophobia, and eye irritation). [Figure 5] Figure 5 shows the results of the ophthalmic examination. [Figure 6] Figure 6 shows the results of each nerve parameter measured by confocal laser scanning microscopy. Statistical analysis was performed using Welch's t-test. [Figure 7] Figure 7 shows the correlation graphs between CNBD and CTBD and BUT. Statistical analysis was performed using the Pearson correlation coefficient. [Figure 8] Figure 8 shows a list of proteins detected in tear fluid that correlated with neuronal parameters. [Figure 9] Figure 9 shows the correlation graphs 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 correlations between profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, and protein S100-A9 and each parameter. Statistical analysis was performed using Pearson correlation coefficients. [Figure 11] Figure 11 shows a list of proteins that correlate with BUT and subjective symptoms (DEQ5 and OSDI). [Figure 12] Figure 12 shows the correlation between secretory leukocyte protease inhibitors (Antileukoproteinase) and galectin-3-binding protein parameters BUT, OSDI, and DEQ5, respectively. Statistical analysis was performed using Pearson correlation coefficients. [Modes for carrying out the invention]

[0010] The present invention is described below. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of any conflict, this specification (including definitions) shall prevail. In this specification, "about" means ±10% of the following value.

[0011] (definition) In this specification, "neurological abnormalities" in the eye refer to abnormalities of the corneal sensory nerve. The corneal sensory nerve is the trigeminal nerve, and neurological abnormalities in the eye are accompanied by symptoms resulting from a decrease in the function of the trigeminal nerve, such as decreased sensitivity to photophobia, heat and cold, burning sensation in the eye, eye pain due to wind, light and temperature changes, and decreased pain tolerance.

[0012] In this specification, "corneal nerve fiber density (CNFD)" refers to the corneal area per millimeter of the cornea. 2 This refers to the number of nerve fibers per unit area.

[0013] In this specification, "corneal nerve branch density" (CNBD) refers to the corneal area within 1 mm². 2 This refers to the number of branching points on the main fiber per unit area.

[0014] In this specification, "corneal nerve fiber length" (CNFL) refers to 1 mm in the cornea. 2 This refers to the total length of nerves in a given area.

[0015] In this specification, "corneal nerve fiber total branch density (CTBD)" refers to the total branch density of corneal nerve fibers in a 1 mm² area. 2 This refers to the total number of branching points in a given area.

[0016] In this specification, the "corneal nerve fiber area (CNFA)" refers to the area of ​​the cornea within 1 mm². 2 Total area of ​​nerve fibers per unit (mm²) 2 ) refers to.

[0017] In this specification, "corneal nerve fiber width" (CNFW) refers to 1 mm in the cornea. 2 This refers to the average nerve fiber width (mm) per unit area.

[0018] In this specification, "nerve tortuosity" refers to the mean curvature of the nerve trunk in the cornea. Nerve tortuosity can be measured based on SLD and TCI, where SLD is calculated by subtracting the value obtained by dividing the length of the straight line connecting the start and end points of the trunk by the actual length of the trunk from 1, and TCI refers to the change in the slope of the trunk.

[0019] In this specification, “marker” refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and / or prognostic indicator, and includes polynucleotides (e.g., DNA and / or RNA), proteins, and polypeptides. Markers can serve as indicators of specific subtypes of disease or disorder characterized by certain molecular, pathological, histological, and / or clinical characteristics.

[0020] In this specification, "dry eye" refers to a condition characterized by eye discomfort or visual disturbances caused by a decrease in or alteration of the composition of tears.

[0021] In this specification, “Subject” means a subject to which a composition or combination or method for the treatment and prevention of the present disclosure is administered, and subjects include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, horses, sheep, monkeys, goats, pigs, etc.).

[0022] The following describes preferred embodiments, but it should be understood that these embodiments are illustrative of the present invention and the scope of the invention is not limited to such preferred embodiments. Those skilled in the art should also understand that modifications, changes, etc., within the scope of the invention can be easily made by referring to the following preferred embodiments. Those skilled in the art may combine any of these embodiments as appropriate.

[0023] (Neurological abnormality markers) In one embodiment, the present disclosure provides markers of ocular nerve abnormalities whose expression changes (increases or decreases) in correlation with morphological parameters of nerves in the eye. The inventors have found a correlation between dry eye patients and morphological parameters of nerves in the eye (e.g., CNBD, CTBD, CNFD, CNFL, and tortuosity). In particular, CNBD and CTBD were significantly higher in dry eye patients compared to healthy individuals. Therefore, in some embodiments, the parameters may include at least one parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity. In a particular embodiment, the parameters may include at least one selected parameter consisting of CNBD and CTBD.

[0024] In some embodiments, the neurological abnormality may be a neurological abnormality in the cornea. In certain embodiments, the neurological abnormality may be a neurological abnormality in dry eye. As shown in Figure 7, there are healthy individuals with a BUT of 5 seconds and high CNBD and CTBD, and such subjects are not dry eye patients but may be candidates for treatment as they have a neurological abnormality.

[0025] In some embodiments, the markers include cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-associated lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, γ-actin (Actin, cytoplasmic 2), α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, and cytokeratin 75 (Keratin, type II cytoskeletal 75) may be selected from the group consisting of 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12), transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin 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 transport protein, protein S100-A9, and any combination thereof. These markers correlate with both CNBD and CTBD.

[0027] (Method for identifying markers) In another embodiment, the present disclosure provides a method for identifying markers of neurological abnormalities in the eye, comprising the steps of (a) obtaining a parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity in healthy individuals and patients with dry eye; (b) measuring the expression levels of one or more genes or proteins in samples obtained from the healthy individuals and patients with dry eye; and (c) correlating the measured values ​​with the expression levels of one or more genes or proteins.

[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 neuropathy in the eye may be corneal neuropathy and / or neuropathy in dry eye.

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

[0030] In some embodiments, the markers include 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), and cytokeratin 79 (Keratin, type II cytoskeletal). 79) The group may be selected from laminin α5, non-histone chromosome protein HMG-17, nucleobingin-2, peroxiredoxin-1, multimeric 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 θ, secretory leukocyte protease inhibitors (Antileukoproteinase), galectin-3-binding proteins, and any combination thereof.

[0032] In some embodiments, at least one marker from the above-mentioned markers for ocular nerve abnormalities and at least one marker from the above-mentioned markers for dry eye may be used in combination.

[0033] (Detector) In another embodiment, the disclosure provides a detection agent for detecting proteins or nucleic acids whose expression changes (increases or decreases) in correlation with morphological parameters of nerves in the eye. In some embodiments, the morphological parameters of nerves are selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity, and preferably include at least one of the parameters of CNBD and CTBD.

[0034] In some embodiments, proteins or nucleic acids whose expression changes (increases or decreases) in correlation with morphological parameters of nerves in the eye include: cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, protein S100-A9, lipocalin-1, α1 acid glycoprotein 1, α1-antichymotrypsin, glyceraldehyde-3-phosphate dehydrogenase, neutrophil elastase, cytokeratin 10 (Keratin, type I cytoskeletal 10), haptoglobin, protein S100-A6, pararemin-1, laminin α5, SEC14-like protein 1, trypsin-2, secretoglobin family 1D member 1, and γ-actin (Actin, cytoplasmic 2) α-enolase, mammoglobin-B, 14-3-3 protein ζ / δ, epithelial fatty acid-binding protein, cytokeratin 75 (Keratin, 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 type 1-N, peroxiredoxin-1, cytokeratin 12 (Keratin, type I cytoskeletal 12) Examples include, but are not limited to, transcobalamin-1, trypsin-1, lysozyme C, phosphoglycerate kinase 1, cyclophyllin A (Peptidyl-prolyl cis-trans isomerase A), and any combination thereof.

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

[0036] Detection agents include, but are not limited to, antibodies that specifically bind to the above-mentioned proteins and nucleic acids that specifically hybridize with the above-mentioned nucleic acids. Detection is also possible using a mass spectrometer.

[0037] In some embodiments, the detection agents of the present disclosure may be used as diagnostic agents for neurological abnormalities in the eye. Using the detection agents of the present disclosure, the amount of the above-mentioned protein or nucleic acid in a sample obtained from a subject is measured, and the measurement is compared to the mean value of a healthy person. If the measurement is significantly different from the mean value of a healthy person, the subject may be diagnosed with or potentially having a neurological abnormality in the eye. Examples of samples include, but are not limited to, tears, blood, and conjunctival swabs. In a preferred embodiment, the sample is tears.

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

[0039] 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 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 types 1-C, and histone H2B types 1-J, keratin 84 (Keratin, type II cuticular Hb4), and cytokeratin 79 (Keratin, type II cytoskeletal). 79) Examples include laminin α5, non-histone chromosome protein HMG-17, nucleobingin-2, peroxiredoxin-1, multimeric immunoglobulin receptor, protein S100-A7, SEC14-like protein 1, serotransferrin, thioredoxin, trypsin-2, and trypsin-3.

[0040] In certain embodiments, 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 may be selected from the group consisting of 14-3-3 protein θ, secretory leukocyte protease inhibitors (Antileukoproteinase), galectin-3-binding proteins, and any combination thereof.

[0041] (Diagnostic method) In a further embodiment, the present disclosure provides a method for diagnosing an eye 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: obtaining a sample from the subject; measuring the amount of a marker (protein or nucleic acid) described herein in the sample; and comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person, and determining, based on the measured amount of the marker, that the subject has a neurological disorder in the eye. If the measured amount of the marker is higher or lower than the amount of the marker in a sample obtained from a healthy person, the subject may be diagnosed with or likely to have an eye condition.

[0042] In another embodiment, the present disclosure provides a method for using the markers described herein as indicators for diagnosing the condition of a subject's eye, comprising the steps of: measuring the amount of the markers described herein in a sample obtained from the subject; and comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person.

[0043] In some embodiments, the eye condition may be dry eye, ocular nerve abnormality, corneal nerve abnormality, or ocular nerve abnormality in dry eye. In some embodiments, the sample may be a tear fluid sample.

[0044] (Methods for screening for therapeutic drugs) In one embodiment, a method is provided for screening for therapeutic drugs for eye conditions (such as dry eye or neurological disorders) in a subject, the method comprising the steps of 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 instillation is elevated or decreased compared to before instillation, the candidate drug can be identified as effective in treating the eye condition.

[0045] The model animals used in this screening method are non-human animals, and examples of model animals include mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, horses, sheep, monkeys, etc.). In certain embodiments, the model animal may be a lacrimal gland excision model, a forced eye opening model, a tear secretion suppression model, a corneal flap model, or a Sjögren's syndrome model.

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

[0047] (Companion Diagnosis) A method for treating an eye condition (e.g., a neurological disorder, dry eye, or dry eye associated with a neurological disorder) in a subject, the method comprising the steps of: measuring the amount of a marker (protein or nucleic acid) described herein in a sample; diagnosing whether the subject has the eye condition by comparing the measured amount of the marker with the amount of the marker in a sample obtained from a healthy person; and administering a medicine for the prevention or treatment of the eye condition to a subject who is determined to have the eye condition in the diagnostic step.

[0048] Examples of medicines for preventing or treating eye conditions (e.g., nerve abnormalities, dry eye, or dry eye associated with nerve abnormalities) include, but are not limited to, Diquas eye drops (diquafosol sodium), Mucosta eye drops (rebamipide), and Hyalein eye drops (purified sodium hyaluronate).

[0049] The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and not to limit the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Examples]

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

[0051] (Example 1: Relationship between ocular surface homeostasis and corneal sensory nerves) This embodiment aims to clarify the relationship between the shape of corneal sensory nerves, corneal sensation, and dry eye symptoms. The shape of corneal sensory nerves in dry eye patients and healthy adults was observed and measured using a confocal laser scanning microscope. Corneal sensation was measured, and general dry eye examination items such as subjective symptoms and ophthalmic examination of the ocular surface were performed to analyze the influence of corneal sensory nerve shape on sensation and its relationship with dry eye symptoms.

[0052] (method) (subject) Dry eye patients and healthy adults (Selection Criteria) Participants in this study were those who met all of the following criteria. (1) Those who have given their free and written consent to participate in this study. (2) Persons who are 20 years of age or older and under 65 years of age on the date of obtaining consent (the date on which the patient consents to participate in this clinical trial) <Criteria for patients with dry eye> (3) Persons who, as of the date of obtaining consent, have experienced subjective symptoms related to dry eye for one year. (4) Those whose total score on the dry eye questionnaire (DEQ-5 Japanese version) in the screening test exceeds 6. (5) In the screening test, persons with a tear film breakup time (BUT) of 5 seconds or less <Standards for healthy adults> (6) Persons who have not had a history of dry eye within one year prior to the date of obtaining consent. (7) Persons who are not receiving continuous eye drop treatment for other ophthalmic diseases. (8) Individuals whose total score on the dry eye questionnaire (DEQ-5 Japanese version) in the screening examination is 6 or less. (9) Persons who meet the following criteria in the screening test 1) The tear film breakup time (BUT) is ≥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) Individuals with co-existing autoimmune diseases, including atopic dermatitis. (2) Persons with severe eye impairment due to Stevens-Johnson syndrome or ocular pemphigoid (3) Persons with a history of ophthalmic surgery [including corneal refractive surgery (LASIK) and punctal plug insertion] (4) Glaucoma patients (currently undergoing treatment) (5) Individuals with concomitant anterior segment diseases other than dry eye. (6) Persons with complications from infectious diseases (7) Persons who are unable to discontinue or are expected to discontinue the use of eye drops (all prescription and over-the-counter eye drops except artificial tears) from the time of the screening test until the end of the study period. (8) Persons who are unable to stop wearing contact lenses one week prior to each visit (9) Other persons whom the principal investigator or other relevant personnel deem unsuitable for this study (Cancellation Criteria) The principal investigator or other research personnel discontinued the study for the subject in question if any of the following conditions were met. (1) If a participant requests to discontinue participation in the study (2) In other cases where the principal investigator or other relevant personnel deem it inappropriate. (Study Design) Partial occlusion * Intergroup comparison test (prospective observational study) * : For the photographers of confocal laser scanning microscopy and the personnel in charge of image analysis, the condition of the subjects was concealed during the implementation.

[0053] (Study Process) According to the following procedures, dry eye patients and healthy adult volunteers were enrolled in this study, and various observations and examinations were conducted (Figure 1).

[0054] (1) Visit 1 The responsible physician for the study implementation and the co - responsible physicians for the study implementation at the medical institution (hereinafter referred to as the responsible physician for the study implementation, etc.) fully explained the content of this study and other matters related to the study to dry eye patients or healthy adults using an explanatory document, and obtained written consent for their voluntary participation in this study.

[0055] (2) Visit 2 The responsible physician for the study implementation, etc. conducted a questionnaire survey, self - reported symptoms [using the Dry Eye Questionnaire 5 (DEQ - 5 Japanese version)], and ophthalmological examinations (BUT test, corneal epithelial damage) as screening tests for all subjects who had given written consent, and confirmed the eligibility of the subjects. The background of the subjects is shown in Figure 2.

[0056] Note that Visit 2 can also be conducted on the same day as Visit 1.

[0057] (3) Visit 3 After Visit 2, the principal investigators had the subjects return to the participating medical institution for tear collection (twice for each eye), a medical interview, subjective symptoms (using a questionnaire on dry eye (DEQ-5 Japanese version, Ocular Surface Disease Index; OSDI Japanese version), a questionnaire on neuropathic ocular pain (NOP), and a visual analog scale (VAS) for dry eye symptoms), and ophthalmic examinations.

[0058] Furthermore, tear fluid samples should be collected at least one hour apart, and ophthalmic examinations should be performed at least one hour after the second tear fluid sample collection. The order in which the medical interview and the subjective symptoms / questionnaire survey are conducted does not matter.

[0059] (4) Visit 4 After Visit 3, the principal investigator and other researchers had the subjects return to the participating medical institution for a confocal laser scanning microscope examination.

[0060] (Evaluation criteria) (1) Primary endpoints Corneal sensory nerve parameters based on confocal laser scanning microscope images: [Corneal Nerve Fibre Length (CNFL), Corneal Nerve Fibre Density (CNFD), Corneal Nerve Branch Density (CNBD), Corneal Nerve Fibre Total Branch Density (CTBD), Corneal Nerve Fibre Area (CNFA), Corneal Nerve Fibre Width (CNFW)] (2) Secondary evaluation items 1) Parameters related to neurodegeneration of corneal sensory nerves based on confocal laser scanning microscope images [Nerve Fibre Tortuosity (NFT)] 2) Comprehensive comparative quantification of tear protein using iTRAQ (Example 2) 3) Tear film breakdown time (BUT) 4) Schirmer Test Method I 5) Fluorescein staining score for corneal and conjunctival epithelial damage 6) Corneal sensation 7) Subjective symptoms [Dry Eye Questionnaire 5 (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 neurogenic eye pain. (1) Questionnaire regarding dry eye (DEQ-5 Japanese version) (Implementation period: Visit 2 and 3) (2) Questionnaire regarding dry eye (OSDI Japanese version) (Implementation period: Visit 3) (3) Questionnaire regarding neurogenic eye pain (NOP) (Implementation period: Visit 3) (4) VAS for dry eye symptoms (blurred vision, photophobia, eye irritation) (Implementation period: Visit 3) (Tear fluid collection) The principal investigator and other researchers performed tear collection from both eyes twice each, for a total of four times, during Visit 3, following the procedure below, for both iTRAQ and oxidative stress marker measurement. The second tear collection (oxidative stress marker) was performed at least one hour after the first tear collection (iTRAQ). The results of the iTRAQ will be described in detail in Example 2. (1) 50 μL of physiological saline was instilled into the lower palpebral conjunctival sac using a sterile micropipette. (2) Without blinking, move the eyeballs four times from side to side and up and down to mix the administered saline solution with the tears. (3) The sample was collected from the lower eyelid conjunctival sac into a low-adsorption container using a micropipette that had been used to administer saline eye drops. (4) The collected samples were promptly frozen and stored at -80°C. (5) Subsequently, the frozen samples were sent to each testing laboratory under frozen conditions using dry ice.

[0061] (Ophthalmic examination) The principal investigator and other researchers performed the following ophthalmic examinations.

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

[0063] (2) Corneal and conjunctival epithelial disorders (Period of implementation: Visit 2 and 3) According to the 2006 dry eye diagnostic criteria (Jun Shimazaki, Dry Eye Research Society, 2006 Dry Eye Diagnostic Criteria), the cornea and conjunctiva were divided into three fractions (temporal bulbar conjunctiva, cornea, and nasal bulbar conjunctiva), and a staining score (fluorescein) of 0 to 3 was assigned to each fraction. These scores were then totaled.

[0064] (3) Corneal sensation examination (Performance timing: Visit 3) The cornea was divided into five sections (central, superior, inferior, temporal, and nasal), and corneal sensation values ​​for each section were recorded using a Cochet-Bonnet corneal sensory meter. Pain tolerance in the central cornea was also measured using the Cochet-Bonnet corneal sensory meter.

[0065] (4) Schirmer Test (Implementation period: Visit 3) The measurement was performed using Method I, which does not involve topical anesthesia.

[0066] (Confocal laser scanning microscopy) In Visit 4, a Rostock corneal module was attached to a Heidelberg-Retina tomograph to image the corneal trigeminal nerve beneath the corneal epithelium. The subject's medical condition was kept confidential from the confocal laser scanning microscope operator during the procedure.

[0067] (cancel) The principal investigator or other research personnel discontinued the study for the subject if any of the following conditions were met: (1) If a participant requests to discontinue participation in the study (2) In other cases where the principal investigator or other relevant personnel deem it inappropriate. (Adverse events) (Definition of adverse events) An adverse event is defined as any undesirable or unintended injury or illness, or its symptoms, resulting from tear collection, testing, etc., regardless of whether or not it is causally related to the study conducted.

[0068] (Collection and recording of adverse events) If an adverse event occurred, the implementing medical institution took appropriate action as necessary, and recorded the details of the symptoms / findings (diagnosis), severity, date of onset, date of disappearance, whether or not treatment was performed (and the details of the treatment), and outcome in the case record.

[0069] (Eyes to be evaluated) For the analysis of each evaluation item excluding subjective symptoms, the primary evaluation target was, in principle, the eye with the shorter BUT in Visit 3 for dry eye patients, and the eye with the longer BUT for healthy adults. If the BUT of both eyes was the same, the right eye was used for evaluation.

[0070] Furthermore, eyes that were not the primary focus of the analysis were sometimes included in the evaluation for purposes such as confirming the reproducibility of the analysis results or conducting exploratory analyses.

[0071] (Data handling standards) In this study, missing data was not imputed. Furthermore, when abnormal values ​​were observed in oxidative stress marker measurements, etc., the cause was investigated, and, in principle, all measured data was used for analysis, except for abnormal values ​​with clear justification.

[0072] (Analysis method) (Analysis of primary endpoints) Summary statistics were calculated for corneal sensory nerve parameters [Corneal Nerve Fibre Length (CNFL), Corneal Nerve Fibre Density (CNFD), Corneal Nerve Branch Density (CNBD), Corneal Nerve Fibre Total Branch Density (CTBD), Corneal Nerve Fibre Area (CNFA), Corneal Nerve Fibre Width (CNFW)] based on confocal laser scanning microscope images, for both dry eye patients and healthy adults, and comparisons were made between the two groups. The significance level for hypothesis testing was set at a two-sided 0.05.

[0073] (Analysis of secondary endpoints) (1) Summary statistics were calculated for the parameter [Nerve Fibre Tortuosity (NFT)] related to neurodegeneration of corneal sensory nerves based on confocal laser scanning microscope images, for both dry eye patients and healthy adults, and comparisons were made between the two groups.

[0074] (2) The differences in each protein in tears quantified by iTRAQ were examined between dry eye patients and healthy adults (Example 2).

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

[0076] (4) For the Schirmer Test I, summary statistics were calculated for each group of dry eye patients and healthy adults, and comparisons were made between the two groups.

[0077] (5) Summary statistics were calculated for the fluorescein staining score of corneal and conjunctival epithelial disorders for each group, including dry eye patients and healthy adults, and comparisons were made between the two groups.

[0078] (6) Regarding corneal sensation, summary statistics were calculated for each group of dry eye patients and healthy adults, and comparisons were made between the two groups.

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

[0080] (Additional considerations) In addition to analyzing the primary and secondary endpoints, we conducted exploratory analyses of the relationship between the primary and secondary endpoints and the subjects' condition (dry eye / healthy adult) using appropriate methods such as logistic regression. We also examined the relationships between the primary and secondary endpoints.

[0081] (result) Figure 3 summarizes the results of the questionnaire regarding subjective symptoms. Regarding each question related to neurogenic eye pain (NOP), healthy individuals (16 people) did not meet any of the criteria. On the other hand, among dry eye patients, not all patients met any of the criteria; some patients did not meet any criteria. Therefore, it was suggested that dry eye may exist in two forms: one related to neurological abnormalities and one not related to neurological abnormalities.

[0082] Figure 4 summarizes the VAS results for dry eye symptoms (blurred vision, photophobia, and eye irritation). As shown, VAS scores were significantly higher in patients with dry eye.

[0083] Figure 5 summarizes the results of the ophthalmic examination. BUT and pain tolerance were significantly higher in dry eye patients compared to healthy controls, but there were no significant differences between dry eye patients and healthy controls in corneal and conjunctival epithelial damage, corneal sensory examination, and Schirmer test.

[0084] Figure 6 shows the results of each nerve parameter measured by confocal laser scanning microscopy. The following parameters were measured in this examination: Corneal nerve fiber density (CNFD: 1 mm) 2 (Number of fibers per unit area) Corneal nerve branching density (CNBD: 1 mm²) 2 (Number of branching points on the main fiber per unit area) Corneal nerve fiber length (CNFL: 1 mm) 2 (Total length of nerves per area) Total branching density of corneal nerve fibers (CTBD: 1 mm²) 2 (Total number of branching points per area) Corneal nerve fiber region (CNFA: 1 mm) 2 Total area of ​​nerve fibers per unit (mm²) 2 )) Corneal nerve fiber width (CNFW: 1 mm) 2 Average nerve fiber width per unit area (mm) Nerve tortuosity (mean curvature of the main nerve trunk in the cornea) It was revealed that CNBD and CTBD were significantly higher in dry eye patients compared to healthy individuals. Furthermore, a very high correlation was shown between these parameters and BUT (Figure 7).

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

[0086] (material and method) Tear samples were taken from 30 eyes of healthy individuals (15 right eyes, 15 left eyes) and 67 eyes of dry eye patients (34 right eyes, 33 left eyes) collected in Example 1 (Figure 1). The tear samples were frozen and stored at -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 curve standard sample solutions and QC samples are prepared in duplicate, while the measurement sample is measured only once.

[0096] [Table 9]

[0097] Preparation of control samples A pool of tear fluid samples from healthy individuals was prepared. The selection of healthy individuals for use in the pool was 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 correlations between detected protein ratios and neuronal parameters (P<0.1). Of the proteins shown in Figure 8, cystatin-S, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, and protein S100-A9 were particularly correlated with both CNBD and CTBD, indicating that these proteins are strongly correlated with neuronal abnormalities.

[0105] Cysteine-S correlates with five parameters: CNFD, CNBD, CNFL, CTBD, and tortuosity, suggesting it is one of the markers reflecting the state of ocular nerve abnormalities (Figure 9). Similarly, profilin-1, protein S100-A8, neutrophil gelatinase-related lipocalin, phospholipid transport protein, and protein S100-A9 also correlate with nerve fiber branching density (of CNBD and CTBD) and nerve fiber length (CNFL), suggesting they are also markers reflecting the state of ocular nerve abnormalities (Figure 10).

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

[0107] (Example 3: Diagnosis of nerve abnormalities in the eye) Tear fluid is collected from the subject, 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 for healthy individuals, the subject can be diagnosed as having or likely to have a neurological abnormality. In addition to cystatin-S, markers identified in Example 2 may be further measured.

[0108] (Example 4: Screening of drugs to treat nerve abnormalities in the eye) Candidate drugs are instilled into dry eye models such as lacrimal gland excision models, forced eye opening models, tear secretion suppression models, corneal flap models, and Sjögren's syndrome models. Tear samples are collected before and after instillation, and the concentrations of cystatin-S in the tears are compared. If the concentration of cystatin-S in the tears after instillation is significantly higher than the concentration of cystatin-S in the tears before instillation, the candidate drug is identified as effective in treating neurological abnormalities in the eye.

[0109] As described above, the present invention has been illustrated using preferred embodiments, but it is understood that the scope of the present invention should be interpreted solely by the claims. Patents, patent applications and documents cited herein should be incorporated herein by reference as if their contents were specifically described herein.

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

[0111] A marker correlated with eye abnormalities (particularly nerve abnormalities and dry eye) is provided. This marker is useful as an indicator for diagnosis and evaluation of drug effectiveness, and can be used in fields such as pharmaceuticals.

Claims

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

2. A method for detecting dry eye in the eye, comprising the step of measuring at least one marker selected from the group consisting of 14-3-3 protein θ, secretory leukocyte protease inhibitor (Antileukoproteinase), galectin-3-binding protein, and any combination thereof in a tear fluid sample obtained from a subject, A method in which the correlation between the amount of the marker and at least one parameter selected from the group consisting of BUT, OSDI, and DEQ5 indicates whether or not the subject has dry eye.

3. A method for detecting dry eye associated with corneal sensory nerve abnormalities in the eye, comprising a step of measuring a marker including at least one marker according to claim 1 and at least one marker according to claim 2, A method for indicating whether a subject has dry eye associated with corneal sensory nerve abnormalities, wherein the correlation between the amount of at least one neuronal parameter selected from the group consisting of CNBD, CTBD, CNFD, CNFL, and tortuosity, and at least one dry eye index selected from the group consisting of BUT, OSDI, and DEQ5, and the amount of the marker.

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