Evaluation method for eye diseases

Abstract

Provided is a new eye disease onset risk evaluation method with which it is possible to evaluate the risk of eye disease onset even in a pre-disease stage. Also provided is a new method with which it is possible to evaluate the progress of a late-stage eye disease.　One example of this invention is an eye disease onset risk or late-stage eye disease progress evaluation method for evaluating an eye disease through molecular dynamics analysis of the anterior chamber environment, the method including a step for using mass spectrometry (MS) to measure the concentration level of metabolites in collected anterior aqueous humor.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the benefit of Japanese Patent Application No. 2020 - 99804, filed with the Japan Patent Office on June 9, 2020. The entire disclosure of the Japanese application (specification, claims, drawings, abstract), for all purposes, is incorporated herein by reference as if fully set forth herein.

[0002] The present invention belongs to the technical field related to the medical evaluation of eye diseases, particularly eye diseases caused by disruption of the anterior chamber microenvironment, and further, corneal endothelial dysfunction and diseases generally referred to as glaucoma that are pathologically related. In this technical field, the present invention is characterized by a comprehensive molecular dynamics analysis technique that becomes possible for the first time by, in addition to examining the molecular dynamics of the anterior eye chamber microenvironment, particularly proteinaceous molecules, nucleic acid - based molecules, extracellular secretory particles, etc. that appear in the aqueous humor of the anterior chamber, according to disease states, and also measuring the concentration levels of metabolites by mass spectrometry (MS). The present invention relates to a method for evaluating the onset risk and progression of eye diseases related to disruption of the anterior chamber microenvironment, particularly a method for evaluating the onset risk of eye diseases or a method for evaluating the progression of the disease state of eye diseases.

Background Art

[0003] In addition to cancer, circulatory disorders, and cognitive dysfunction, providing appropriate medical treatment for sensory organ disorders is a fundamental issue both at home and abroad, including Japan, which is a super - aged society. Eighty percent of sensory information enters through vision and forms the basis of an individual's dignity. There is no doubt that environmental factors, in addition to genetic factors, are involved in age - related eye diseases, particularly glaucoma diseases. In fact, environmental factors are known to affect retinal ganglion cells / fiber column bands / intraocular pressure, etc. as glaucoma disease states. The inventors of the present invention have independently conducted gene research related to the onset of various glaucoma subtypes and also participated in an international consortium. However, except for LOXL1, the relative risk is low, the pathological mechanism has not been elucidated, and the function of the genes identified as risk factors and their correspondence with clinical subtypes have not been clarified. That is, their usefulness has not been proven at all.

[0004] Therefore, the present inventors have turned their attention to all environmental factors including aging, and assuming the existence of "environmental factors" that disrupt the anterior chamber tissue homeostasis in addition to genetic factors, they have been challenging for many years the analysis of the molecular dynamics of the anterior chamber microenvironment and its identification, as well as the analysis of the functional network formation among the anterior eye tissues.

[0005] There are many eye diseases caused by the disruption of the anterior chamber microenvironment. Typical examples include corneal endothelial disorders, glaucoma, and cataracts. Any of these may occur secondarily to diseases that cause disruption of the blood-aqueous barrier, such as trauma, ophthalmic surgery, and uveitis.

[0006] Based on such a concept, the present inventors have been vigorously promoting research for many years to examine the molecular dynamics of anterior chamber microenvironment factors in terms of disease type characteristics, molecular dynamics of proteinaceous molecules, nucleic acid molecules, extracellular secretory particles, exosomes, metabolites, etc. that appear in the aqueous humor, for three disease types of corneal endothelial disorder, glaucoma, and cataract, and have found a technique for selecting molecular dynamics specific to disease types and pathological conditions, thus completing the present invention.

[0007] The reason for focusing on the anterior chamber microenvironment factors is that the analysis of only factors that affect the whole body like risk genes cannot clarify the function of pathological exacerbation factors locally. As body fluids applicable to the evaluation of the onset risk or disease progression, multiple body fluids such as blood, urine, saliva, and sweat are known, and in the ophthalmic field, tears, etc. are also among the targets. However, in diseases where the local pathological condition is exacerbated or controlled, the local molecular dynamics are not necessarily projected onto the systemic body fluids. In the field related to the present invention, although there are only a few prior findings regarding blood and tears, none of them have reached the level of practical usefulness confirmation.

[0008] The inventors have been conducting clinical research on regenerative medicine using cultured human corneal endothelial cells with characteristics identical to those of in-vivo tissues for many years and have confirmed clinical POC (Proof of Concept). Cultured human corneal endothelial cells with various cell characteristics can be obtained according to various culture environments. It can also be regarded as a simulation model of the influence of culture environment factors on cell characteristics and cell degeneration in vitro. The endothelial tissue microenvironment of the living body, especially the anterior chamber microenvironment, is as diverse as the culture environment, and results have been obtained that are presumed to cause aging, degeneration, and transformation of the cells constituting the human corneal endothelial tissue. Bullous keratopathy, which is corneal opacity due to corneal endothelial tissue dysfunction, is accompanied by severe visual impairment and causes long-term difficulties in daily life. Although it is a refractory disease, the cause and pathology of bullous keratopathy are still unclear and there is no drug treatment method. In Japan, approximately 800 corneal transplants are performed annually on patients with bullous keratopathy, but only the dysfunctional tissue is replaced with donor tissue, and a continuous decrease in endothelial cells (late-stage tissue failure) occurs after the transplantation. Whether the dysfunctional pathology of the tissue is homogeneous or diverse, and what the cause of late-stage tissue failure is, remain unclear.

[0009] Against such a background, in order to provide optimal corneal transplantation and regenerative medicine for patients with bullous keratopathy, research has been intensively conducted aiming to establish an innovative molecular evaluation method and utilize it as an indicator for medical standardization by analyzing the anterior chamber tissue microenvironment of patients with bullous keratopathy, especially microRNA (miRNA), exosomes, extracellular vesicles (EV), cytokines related to SASP (senescence-associated secretory phenotype) in the aqueous humor, and various metabolites measurable by mass spectrometry. This is research towards establishing an evaluation method for the onset risk and progression pathology of eye diseases related to the breakdown of the anterior chamber microenvironment characterized by comprehensive molecular dynamics analysis technology, particularly an evaluation method for the onset risk of glaucoma or an evaluation method for the progression of glaucoma pathology.

[0010] As shown in Figure 2, the argument focusing on the analysis of these molecular dynamics is the reason for considering that these molecular groups form a network among the anterior chamber tissues. Through the analysis of the network among the anterior chamber tissues, for the first time, it has become possible to provide an evaluation technique for the risk of onset or disease progression targeting the molecular dynamics of the anterior chamber environment, which is directly related to eye diseases, particularly eye diseases caused by the breakdown of the anterior chamber microenvironment, and furthermore, the pathological conditions of the disease generally called glaucoma.

[0011] As one of the local anterior chamber microenvironment factors dealt with in the present invention, one end of the tissue network related to the molecular dynamics of the aqueous humor in the anterior chamber is introduced.

[0012] The movement of substances with large molecular weights from plasma to aqueous humor is restricted by the blood-aqueous barrier controlled by the non-pigmented epithelial cells of the ciliary body and the endothelial cells of the iris blood vessels. In fact, while the protein concentration in plasma is 6 g / dL, the protein concentration in the aqueous humor of the anterior chamber is as low as 25 mg / dL. However, when the blood-aqueous barrier is damaged due to trauma, inflammation, etc., proteins with large molecular weights such as immunoglobulins and β-lipoproteins also leak and appear in the aqueous humor of the anterior chamber. Thus, external stresses such as trauma and inflammation are one of the local factors that disrupt the anterior chamber microenvironment. The noise caused by such disturbing factors must be fully considered in establishing the evaluation technique aimed at in the present invention.

[0013] This clearly shows that the molecular dynamics of the aqueous humor in the anterior chamber also change and break down reversibly and irreversibly in eye inflammatory diseases including cataracts, and it is necessary to pay attention to the selection of the control aqueous humor in the anterior chamber to be used for the selection of candidate molecular groups to be used as the molecular dynamics analysis and evaluation index for the target diseases.

[0014] In the present invention, from this perspective, that is, in the molecular dynamics analysis of aqueous humor and the selection of a candidate molecular group to be used as an evaluation index, the inventors intended to exclude non-specific leakage molecules associated with inflammation, and thus selected cataract patients instead of healthy individuals as the control. As a result, different from previous reports, the present invention is the first to achieve an invention with disease specificity and practicality. In cataract patients, it has been confirmed that not only cytokines as proteinaceous molecules, but also the dynamics of, for example, miRNAs and metabolites in the aqueous humor are different from those in patients with corneal endothelial degeneration (Figs. 3 to 5).

[0015] Degeneration of corneal endothelial cells (factors such as aging and oxidative stress) → changes in the molecular species and amounts produced by the cells → degeneration and cell death of trabecular meshwork cells in the vicinity → reduced trabecular meshwork function → increased intraocular pressure → glaucoma. There may be fluctuations in the molecular dynamics of the anterior chamber microenvironment through this pathway (see Fig. 6). Clinically, it is presumed that many cases of glaucoma that develop after corneal transplantation are steroid glaucoma for the following reasons. (1) When only full-thickness corneal transplantation was available in the past, long-term use of steroids was required after the surgery, and the incidence of increased intraocular pressure was high. (2) In Descemet stripping automated endothelial keratoplasty (DSAEK), the postoperative steroid dosage was reduced and the duration was shortened, and the incidence of increased intraocular pressure decreased significantly. (3) By combining oral administration of an immunosuppressant (cyclosporine), the steroid dosage was reduced, and the incidence of increased intraocular pressure decreased. (4) Trabeculotomy, which is effective for steroid glaucoma, is often effective for increased intraocular pressure after corneal transplantation.

[0016] However, in glaucoma / corneal endothelial disorder secondary to the exfoliation syndrome, changes in the aqueous humor composition due to the breakdown of the blood-aqueous barrier are considered to be a common pathological condition for both diseases. The mechanisms of increased intraocular pressure → corneal endothelial disorder and corneal endothelial disorder → glaucoma (increased intraocular pressure) are not necessarily accompanied by simultaneous occurrence of corneal endothelial disorder and glaucoma in patients with the exfoliation syndrome. At present, this cannot be explained from clinical observations, and basic molecular dynamics analysis is required (the present invention also provides some solutions to this problem).

[0017] The mechanism by which glaucoma is complicated with exfoliation syndrome involves deposition of exfoliated substances on the zonular tissue, production of exfoliated substances in the zonular tissue, and degeneration of the iris pigment epithelium. Pigment granules released from the cells reach the zonular fibers along the flow of aqueous humor, and the dysfunction of zonular fiber cells due to phagocytosis of the pigment by the zonular fiber cells is considered to be involved. In addition, abnormal thickening of Descemet's membrane and production of exfoliated substances in corneal endothelial cells are considered to be involved in corneal endothelial disorders (cell reduction and cell polymorphism) associated with exfoliation syndrome. However, at present, no findings at a level immediately applicable to the evaluation technology of the onset risk or disease progression have been obtained.

[0018] Related to the present invention, another point to note is the background factors of the patients from whom specimens are collected. Along with background factors such as age, gender, and comorbidities, the quality of medical interventions used in diseases related to anterior chamber tissue dysfunction is also important.

[0019] As medications predominantly used commonly / individually in the three types of glaucoma, for the purpose of mainly reducing intraocular pressure, topical medications include sympatholytic agents, prostaglandin agents, carbonic anhydrase inhibitors, parasympathomimetic agents, sympathomimetic agents, Rho kinase inhibitor oral medications, and as oral medications, carbonic anhydrase inhibitors (administered together with potassium L-aspartate to prevent hypokalemia), etc. are used. As oral medications for the purpose of nerve activation, methylcobalamin (a type of vitamin B12), etc. are used. In corneal transplantation related to corneal endothelial dysfunction, steroids, etc. are used. Paying as much attention as possible to the similarities and differences of these background factors to depict the molecular dynamics deviation of the disease-specific anterior chamber environment is one of the essential elements of the present invention.

[0020] Glaucoma is an eye disease in which the retinal nerve fiber layer and optic nerve are damaged due to retinal ganglion cell death or their axonal disorders, resulting in a narrowed visual field. Glaucoma is a neurodegenerative disease with irreversible progression, and it often progresses gradually without subjective symptoms. The progression of glaucoma leads to blindness through the "extremely early stage with unidentifiable changes" (pre-disease stage), "early stage without subjective symptoms", and "functional impairment stage". Therefore, early detection, early treatment, and management are important. Glaucoma is the leading cause of secondary blindness in Japan (25%, 50,000 people), and its prevalence is estimated to be about 5% in people aged 40 and over. It is also one of the major causes of blindness worldwide. The cause of glaucoma includes an increase in the pressure inside the eyeball (intraocular pressure), but there is also glaucoma without an increase in intraocular pressure. Therefore, it is difficult to accurately evaluate whether a person has glaucoma based solely on intraocular pressure measurement, although it is clearly a risk factor. And because intraocular pressure measurement is quantitative, it is widely used in the evaluation of glaucoma.

[0021] Glaucoma includes primary open-angle glaucoma (POAG), primary angle-closure glaucoma, secondary glaucoma, developmental glaucoma, etc. Primary open-angle glaucoma with normal intraocular pressure (below 20 mmHg) is called normal tension glaucoma (NTG), and primary open-angle glaucoma with intraocular pressure exceeding 20 mmHg is called primary open-angle glaucoma in the narrow sense. More than 70% of Japanese glaucoma patients are NTG, but because their intraocular pressure is normal, this type of glaucoma cannot be distinguished by intraocular pressure examination alone. There is also a refractory glaucoma called pseudoexfoliative glaucoma (PEG) or pseudoexfoliation glaucoma / exfoliation syndrome (PEX), in which pseudoexfoliative substances are deposited on the trabecular meshwork, iris, and anterior lens capsule (pseudoexfoliation syndrome).

[0022] The disease types in which a large deviation from the normal profiles of cytokines, miRNAs, exosomes, metabolites, etc. in the aqueous humor is assumed are, in order, exfoliation glaucoma / exfoliation syndrome > primary open-angle glaucoma in the narrow sense > normal tension glaucoma. Also, the molecular dynamics in the aqueous humor and the glaucoma pathology are assumed to involve two pathways. That is, they are roughly classified into those related to increased intraocular pressure and those related to visual field impairment, and it is considered that there are differences in the deviation of the molecular dynamics in the anterior chamber microenvironment. Since normal tension glaucoma is greatly involved in the pathology due to the vulnerability of the optic nerve, normal tension glaucoma specimens are considered suitable for the selection of candidate molecules for the risk assessment of pre-disease glaucoma onset, and are applied to the present invention.

[0023] As a method for evaluating the risk of glaucoma onset or disease progression, in addition to intraocular pressure measurement, optical coherence tomography (OCT) for taking fundus photographs can be mentioned. However, OCT cannot be used for evaluation at the stage of "extremely early stage where changes have not been identified" or for determination of progression at an extremely advanced stage.

[0024] In addition to intraocular pressure, factors involved in glaucoma are said to include insufficient blood flow in the optic nerve, neurotoxicity, depletion of neurotrophic factors, oxidative stress, etc. Therefore, it is speculated that these factors are reflected systemically, and it has been studied whether analysis of metabolites in blood can be used as an evaluation tool for the risk of glaucoma onset or disease progression (Non-Patent Documents 1 and 2). In Non-Patent Document 1, using LC / MS, metabolites in plasma were analyzed, and it was found that there were significant differences in palmitoylcarnitine, sphingolipids, vitamin D-related compounds, steroid precursors, etc. between normal subjects and those with POAG. In Non-Patent Document 2, using LC / MS, 18 discriminant metabolites belonging to carbohydrates, acylcarnitines, phosphatidylcholine, amino acids, and the polyamine family have been clarified.

[0025] There is a debate as to whether glaucoma is a local disease or a systemic disease. In the present invention, however, the target is a condition that is limited locally and not projected systemically, that is, a condition that is limited to and manifested in the microenvironment of the anterior chamber of the eye. As evidence that glaucoma is a systemic disease, (1) diabetes and hypertension are risk factors for glaucoma. (2) Hypotension is also a risk factor for glaucoma (related to reduced ocular perfusion pressure). (3) Age is a risk factor for glaucoma. (4) A decrease in BDNF in the tears of normal-tension glaucoma patients is known. Conversely, as evidence that glaucoma is a local disease, (1) Ocular connective tissue factors such as high intraocular pressure, myopia, and a thin cornea, and optic disc hemorrhage are risk factors for glaucoma. (2) The characteristic finding of glaucoma, the optic disc cupping, results from the compression, stretching, and bending of the connective tissue (lamina cribrosa) of the optic disc in response to intraocular pressure. In addition to simple compression by intraocular pressure, the collapse and remodeling of the lamina cribrosa are closely involved in the changes in the lamina cribrosa. As a result, the pores of the lamina cribrosa through which the axons of retinal ganglion cells pass are deformed, resulting in axonal transport disorders. This axonal transport disorder is thought to lead to retinal ganglion cell death. Based on these concepts, the present invention is different from those in which systemic fluctuations are observed and fluctuations are manifested in blood and other body fluids such as urine and saliva. "Discovery and application of factors that disrupt the microenvironment of the anterior chamber of the eye, which exhibit specific molecular dynamics in the microenvironment of the anterior chamber tissue of the eye" In particular, in addition to assaying the molecular dynamics of proteinaceous molecules, nucleic acid-based molecules, extracellular secretory particles, etc. that appear in the aqueous humor of the anterior chamber by disease state, by also measuring the concentration levels of metabolites, for the first time, a comprehensive molecular dynamics analysis technique has been completed, which is a unique technique for detecting disruptions in the microenvironment of the anterior chamber of the eye, focusing on the detection technology.

[0026] Also, in Non-Patent Document 3, the inflammatory cytokines in the aqueous humor of the anterior chamber were analyzed, and their relevance to open-angle glaucoma and the like was shown. Specifically, in POAG or PEG, it was shown that the levels of interleukin (IL)-1α, IL-2, IL-4, IL-8, IL-23, and CCL2 were significantly increased, and the increase in IL-8 was suggested to be an important risk factor for both the detection and management of glaucoma.

Prior Art Documents

Non-Patent Documents

[0027] [Non-Patent Document 1] Burgess LG, et al., Invest Ophthalmol Vis Sci., 2015;56:5020-5028. [Non-Patent Document 2] Leruez S, et al., Invest Ophthalmol Vis Sci., 2018;59:4355-4361. [Non-Patent Document 3] Ikuyo Chono, et al., Scientific Reports, (2018)8:14533. [Summary of the Invention] [Problems to be Solved by the Invention]

[0028] For eye diseases caused by disruption of the anterior chamber microenvironment, particularly corneal endothelial dysfunction including bullous keratopathy and Fuchs corneal dystrophy, and diseases generally referred to as glaucoma that are pathologically related, in addition to examining the molecular dynamics of the anterior chamber microenvironment, particularly proteinaceous molecules, nucleic acid-based molecules, extracellular secretory particles, etc. that appear in the aqueous humor of the anterior chamber, by disease state, and also measuring the concentration levels of metabolites, for the first time, a comprehensive molecular dynamics analysis technique is characterized. It is an object of the present invention to provide a practical evaluation technique for the onset risk and progression of eye diseases related to disruption of the anterior chamber microenvironment.

[0029] Bullous keratopathy, which is corneal opacity due to corneal endothelial dysfunction, is a refractory disease accompanied by severe visual impairment, but its cause and pathological condition are still unclear, and the scientific evaluation method for indication determination is insufficient. The present inventors have established, for the first time in the world, regenerative medicine called tissue reconstruction by injecting cultured human corneal endothelial cells with homogenized quality as innovative medicine applicable to this pathological condition, and have confirmed good long-term prognosis (Shigeru Kinoshita, et al., New Engl. J Med. 2018;378:995-1003.). However, stratification of the target patient groups for corneal transplantation and this regenerative medicine has not been achieved. In order to widely provide the optimal corneal transplantation and regenerative medicine for bullous keratopathy patients as standard medical treatment at home and abroad, it is important to establish and standardize a scientific molecular evaluation method for the environmental factors of the transplantation site of the target patients and to establish a comprehensive registry format. The diversity of the anterior chamber tissue microenvironmental factors in bullous keratopathy patients was exemplified and verified using the cytokine profile as a model (Figure 1).

[0030] Regarding other eye diseases involving the anterior chamber microenvironment, similar problems are to be solved in glaucoma diseases. In particular, it is an issue to provide an evaluation technique capable of predicting future onset at the pre-disease stage, and to create a technique for identifying the progression of the pathological condition of patients in the end-stage pathological condition with a high risk of blindness that cannot be determined by the current optical coherence tomography technique. In addition, it is also included in the purpose to provide a technique capable of molecularly evaluating the differences among the three types of glaucoma, and to provide an evaluation technique for a new onset risk or pathological condition progression by clarifying the bidirectional causal relationship between corneal endothelial tissue degeneration and visual field impairment, and between increased intraocular pressure and the progression of the disease. This is also essential for providing the basic technology for innovative drug discovery.

[0031] Based on the original concept that the predisposing factors of the anterior chamber tissue dysfunction are related to the molecular composition of cytokines, miRNAs, exosomes, metabolites, etc. in the anterior chamber tissue microenvironment, and the natural inflammation including the cell senescence diffusion pathway and the complement system mediated by these molecular groups. The expression of common miRNAs (miR34a, miR378) is decreased in both human corneal endothelial dysfunction tissues and subpopulations of phase transitions of cultured human corneal endothelial cells. These miRNAs control the production of SASP-related cytokines such as IL6, IL8, and VEGF through mitochondrial degeneration and gene expression of epigenetic mechanisms, etc. Basic data on the molecular dynamics of the anterior chamber microenvironment supporting the probability of the new concept applied in the present invention are being accumulated (proteome analysis, transcriptome analysis). Instead of the conventional methods targeting gene expression, it provides an invention that also leads to narrowing down the target molecular species related to the pathological conditions essential for elucidating the molecular pathology and drug discovery from a new perspective targeting metabolic reprogramming and mitochondrial function.

[0032] Glaucoma is said to require early detection, early treatment, and management. However, for example, it is difficult to evaluate at the "extremely early stage with no identified changes". Also, as described above, existing optical methods (fundus photography, OCT) cannot evaluate at the stage of "extremely early stage with no identified changes". Currently, the only quantitative risk factor available for clinical evaluation is intraocular pressure, and intraocular pressure measurement is important for evaluating the onset and progression of glaucoma. However, intraocular pressure measurement is ineffective for normal-tension glaucoma (NTG), which is common among glaucoma patients.

[0033] Also, in the "dysfunction stage", the progression of visual field defects is determined by visual field tests. However, many tests are required until the results are confirmed, and for example, it places a heavy burden on the elderly. In addition, for end-stage glaucoma patients, due to poor fixation, the reproducibility of conventional visual field tests is low, making it difficult to evaluate the progression. Therefore, there is a need for new clinical evaluation methods in addition to or other than optical methods and intraocular pressure measurement.

[0034] The present invention mainly aims to provide a new method for evaluating the risk of developing glaucoma even at the stage of "pre-disease" such as the "very early stage of undetermined change", and also to provide a new method capable of evaluating the progression of the disease state at the stage of end-stage glaucoma such as the "dysfunction stage".

Means for Solving the Problems

[0035] As a result of intensive analysis of the molecular dynamics of the anterior chamber environment, the inventors of the present invention have found that the above problems can be solved by analyzing the concentrations of microRNA (miRNA), exosomes, extracellular vesicles (EV), SASP-related cytokines, and in addition, metabolites measurable by a mass spectrometer that appear in the aqueous humor, and have completed the present invention. Examples of the present invention can include the following aspects.

[0036] [1] An evaluation method for eye diseases by analyzing the molecular dynamics of the anterior chamber environment, including the step of measuring the concentration level of metabolites that appear in the collected aqueous humor by mass spectrometry (MS), an evaluation method for the risk of developing eye diseases or the progression of end-stage eye diseases. [2] The evaluation method according to [1] above, wherein the eye disease is glaucoma. [3] The evaluation method according to [1] or [2] above, wherein the collected aqueous humor is from a subject in the very early stage of undetermined disease state at the pre-disease stage who has not developed an eye disease. [4] The evaluation method according to any one of [1] to [3] above, wherein the mass spectrometry (MS) is gas chromatography-mass spectrometry (GC-MS) and / or liquid chromatography-mass spectrometry (LC-MS). [5] The evaluation method according to any one of [1] to [4] above, wherein the metabolite is included in extracellular vesicles (EV) in the aqueous humor. [6] The evaluation method according to any one of [1] to [5] above, further including the step of selecting those showing differences from the molecular dynamics in tears or blood. [7] The evaluation method according to any one of the above [2] to [6], wherein the glaucoma is primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), or exfoliation glaucoma / exfoliation syndrome (PEG). [8] The evaluation method according to any one of the above [2] to [7], further comprising a step of discriminating the specific pathological conditions of three disease types of primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma / exfoliation syndrome (PEG). [9] The evaluation method according to any one of the above [2] to [8], wherein the metabolite is selected from those that are common or not common to the fluctuations in corneal endothelial dysfunction patients.

[10] The evaluation method according to any one of the above [2] to [9], wherein the metabolite is at least one selected from the group consisting of sugars or polyols, substances produced in the citric acid cycle, acylcarnitines, polyamines, amino acids, and cAMP.

[11] The evaluation method according to

[10] above, wherein the sugar or polyol is arabinonic acid, myo-inositol, or fructose, the substance produced in the citric acid cycle is citric acid or isocitric acid, the acylcarnitine is carnitine, isobutyryl carnitine (C4), or propionyl carnitine, the polyamine is spermidine, and the amino acid is asy-dimethylarginine, quinolinic acid, cysteine, or 3-methylhistidine.

[12] The evaluation method according to

[10] or

[11] above, wherein the sugar or polyol is arabinonic acid or myo-inositol, the acyl carnitine is isobutyryl carnitine (C4), and the amino acid is cysteine.

[13] The metabolite is at least one selected from the group consisting of 2-aminoadipic acid, mannose, GSH (glutathione), alanine, spermine, asparagine, choline, glutamine, glutamic acid, pyroglutamic acid, acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynurenic acid, kynurenine, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabitol, S-adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetyl carnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol. The evaluation method according to any one of [2] to [9] above.

[14] The evaluation method according to any one of [2] to [9] above, wherein the glaucoma is normal tension glaucoma (NTG), and the metabolite is at least one selected from the group consisting of 2-aminoadipic acid or mannose, or arabinonic acid, myo-inositol, cAMP, fructose, asymmetric dimethylarginine, citric acid, quinolinic acid, cysteine, spermidine, carnitine, isobutyryl carnitine (C4), 3-methylhistidine, propionyl carnitine, and isocitric acid.

[15] The glaucoma is exfoliative glaucoma / exfoliation syndrome (PEG), and the metabolite is selected from the group consisting of acetylcholine, xanthine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynurenic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabinitol, adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetylcarnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol, and the evaluation method according to any one of the above [2] to [9].

[16] The evaluation method according to any one of the above [1] to

[15] , wherein the evaluation is to determine the occurrence of retinal ganglion cell degeneration, or the susceptibility to degeneration or cell death of retinal ganglion cells before that, or the vulnerability of retinal ganglion cells.

[17] The evaluation method according to any one of the above [1] to

[16] , including the step of considering the results of comprehensive proteome analysis of the molecular dynamics of proteinaceous molecules included in extracellular microparticles (EV) in the aqueous humor.

[18] The evaluation method according to any one of the above [1] to

[17] , including the step of considering the findings in proteome analysis and / or gene expression analysis using clinical specimens.

[19] The evaluation method according to any one of the above [1] to

[18] , including the step of considering the information on factors in tears, blood, and / or other body fluids.

[0037]

[20] A marker for evaluating the risk of onset of eye diseases or the progression of end-stage eye diseases, and the marker is used for the evaluation method according to any one of the above [1] to

[19] .

[21] The marker for evaluation according to

[20] above, which is at least one selected from the group consisting of sugars or polyols, products in the citric acid cycle, acylcarnitines, polyamines, amino acids, and cAMP.

[22] The sugar or polyol is arabinonic acid, myo-inositol, or fructose; the product in the citric acid cycle is citric acid or isocitric acid; the acylcarnitine is carnitine, isobutyryl carnitine (C4), or propionyl carnitine; the polyamine is spermidine; and the amino acid is asy-dimethylarginine, quinolinic acid, cysteine, or 3-methylhistidine. The marker for evaluation according to

[21] above.

[23] The sugar or polyol is arabinonic acid or myo-inositol; the acylcarnitine is isobutyryl carnitine (C4); and the amino acid is cysteine. The marker for evaluation according to

[21] or

[22] above.

[24] At least one selected from the group consisting of 2-aminoadipic acid, mannose, arabinonic acid, myo-inositol, cAMP, fructose, asy-dimethylarginine, citric acid, quinolinic acid, cysteine, spermidine, carnitine, isobutyryl carnitine (C4), 3-methylhistidine, propionyl carnitine, GSH (glutathione), alanine, spermine, asparagine, choline, glutamine, glutamic acid, pyroglutamic acid, acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynureninic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabitol, adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetyl carnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol, the marker for evaluation according to

[20] above.

[0038]

[25] An evaluation system for the risk of onset of an eye disease or the progression of an end-stage eye disease, comprising means for collecting an aqueous humor specimen and means for analyzing the specimen by mass spectrometry (MS), the evaluation system for use in the evaluation method according to any one of [1] to

[19] above.

[26] The evaluation system according to

[25] above, wherein the eye disease is glaucoma.

[27] The evaluation system according to

[25] above, wherein the eye disease is corneal endothelial dysfunction.

Advantages of the Invention

[0039] According to the present invention, it is possible to evaluate the risk of developing glaucoma even at the stage of latent disease, for example, at the very early stage where changes have not been identified. Further, it is possible to evaluate the vulnerability of retinal ganglion cells to degeneration and cell death, that is, the progression state of end-stage glaucoma, at the stage of "dysfunction period".

[0040] By subjecting miRNAs, exosomes, SASP-related cytokines, and metabolites present in the microenvironment of the anterior chamber to molecular evaluation methods as highly reproducible assay techniques for biomarkers in trace amounts, it is of great significance to provide a technique for differentiating the pathological states of the intractable disease, bullous keratopathy. In particular, removing molecular species that leak into the aqueous humor due to non-specific inflammation, etc., tracking glaucoma disease-selective fluctuations, and considering the interaction of proteinaceous molecules, miRNAs, metabolites, and exosome-containing molecules as molecules related to network construction in the microenvironment of the anterior chamber tissue, and making full use of biostatistical methods such as Lasso analysis, the technique of the present invention, which also includes a method used for pathological evaluation as a plurality of molecular species aggregates, is not limited to only the assay of glaucoma risk factors at the latent disease stage and the determination of glaucoma progression in the "dysfunction period" close to blindness, but is widely applicable to diseases resulting from network disruption in the microenvironment of the anterior chamber tissue, opening up a great prospect for future medicine.

Brief Description of the Drawings

[0041]

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Mode for Carrying Out the Invention

[0042] The present invention will be described in detail below. 1. Evaluation Method and Evaluation Marker According to the Present Invention The evaluation method according to the present invention (hereinafter referred to as the "present invention evaluation method") is a method for evaluating the risk of onset of eye diseases or the progression of advanced eye diseases by molecular dynamics analysis of the anterior chamber environment, and includes a step of measuring the concentration level of metabolites appearing in the collected aqueous humor by mass spectrometry (MS). Further, the evaluation marker according to the present invention (hereinafter referred to as the "present invention evaluation marker") is an evaluation marker for the risk of onset of eye diseases or the progression of advanced eye diseases, and is a marker for use in the present invention evaluation method. The present invention evaluation method is directed to eye diseases caused by the breakdown of the anterior chamber microenvironment, particularly corneal endothelial dysfunction including bullous keratopathy, Fuchs corneal dystrophy, etc., and diseases generally referred to as glaucoma that are pathologically related. In addition to testing the molecular dynamics of protein molecules, nucleic acid molecules, extracellular secretory particles, etc. appearing in the anterior chamber environment, particularly in the aqueous humor, according to the disease state, the concentration level of metabolites is also measured, and for the first time, a comprehensive molecular dynamics analysis technique is used to practically evaluate the risk of onset of eye diseases related to the breakdown of the anterior chamber environment and the progression of the disease state. It is a method that can be used, and includes a comprehensive molecular dynamics analysis technique for testing the molecular dynamics of protein molecules, nucleic acid molecules, extracellular secretory particles, etc. appearing in the anterior chamber microenvironment, particularly in the collected aqueous humor, according to the disease state, and also measuring the concentration level of metabolites. As shown in the test examples described below, the concentration levels of proteinaceous molecules, nucleic acid-based molecules, extracellular secretory particles, and metabolites that appear in the aqueous humor collected from patients with corneal transplantation, patients targeted for regenerative medicine by injection of cultured human corneal endothelial cells, cataract patients, normal-tension glaucoma (NTG) patients, primary open-angle glaucoma (POAG) patients, or exfoliative glaucoma (PEG) patients are measured by multiplex bead assay, enzyme immunoassay (ELISA), miRNA chip, gas chromatograph mass spectrometer (GC-MS), or liquid chromatograph mass spectrometer (LC-MS), and compared with other diseases using the cataract group as a control.

[0043] By assaying cytokines, which are one of the environmental factors in the aqueous humor of patients with bullous keratopathy, it was revealed that (1) there is diversity in the microenvironment profiles in the aqueous humor among patients, (2) this microenvironment profile can be differentiated according to the causative diseases of patients with bullous keratopathy (Figs. 7 to 9), and (3) it was confirmed that the diversity is not due to gender or age differences. (4) Three SASP-related cytokines in the aqueous humor that correlate with the maintenance of corneal endothelial cell density after corneal transplantation were identified, and a significant correlation with the in vivo effect was recognized biostatistically. It was demonstrated for the first time in the world that the long-term prognosis of the transplantation outcome after corneal transplantation can be determined by assaying the aqueous humor before surgery, that is, the possibility that molecular species in the aqueous humor are related to the breakdown of the functions of cells present in the anterior chamber tissue (Fig. 10). In addition, it was followed up whether the diversity of the molecular dynamics of the above aqueous humor is related to the long-term prognosis of corneal transplantation patients. Subsequently, a correlation was recognized with five miRNA molecular species in addition to cytokines (see Fig. 5), and it was verified that this technique has practicality for standard techniques for evaluating the prognosis of corneal transplantation and cell injection regenerative medicine, that is, for assaying the degeneration and non-degeneration of corneal endothelial cells.

[0044] Bullous keratopathy, a representative disease of corneal endothelial tissue dysfunction, is known for a significant decrease in corneal endothelial cell density and the flattening and enlargement of cell shape. In Fuchs corneal endothelial dystrophy, the formation of Guttata, where extracellular matrix components (ECM) are deposited from endothelial cells to the posterior surface of Descemet's membrane, is also known. The molecular mechanism of why this decrease in cell density, cell enlargement, and Guttata formation occur is unknown. Even in cultured human corneal endothelial cells, phase transitions to cells with a morphology different from the small cobblestone-like corneal endothelial cell morphology frequently occur, but the molecular mechanism of this cell phase transition is also unknown. First, by comprehensively analyzing miRNAs whose expression fluctuates in diseased tissues and phase-transition cells, miRNAs with decreased expression in both were selected, and miRNA34a-5p and miRNA378a-3p were selected by validation using quantitative RT-PCR (Figure 11).

[0045] The inventors have a hypothesis that cell competition and cell synchronization play important roles in the exacerbation of the disease state, and as shown in Figure 12, they proposed the idea of whether the exacerbation of the disease state progresses or is suppressed by the paracrine action of exosomes. Analysis was carried out from the perspective of whether miRNA34a-5p and miRNA378a-3p might affect the quantity and quality of exosomes produced by cultured human corneal endothelial cells. It is known that SASP also plays a major role in cell competition and cell synchronization. It was confirmed by the Elisa method that the lower the intracellular expression of miRNA34a-5p and miRNA378a-3p, the more IL8, MCP1, and VEGF, which are SASP, are secreted from the cells (Figure 13), and it was verified that these may age surrounding cells through paracrine action.

[0046] Next, the exosome bias was also investigated. The amount of exosomes secreted by cells with different expression levels of miRNA34a-5p and miRNA378a-3p was examined, and it was confirmed that the amount of exosome-equivalent protein secreted by cells with different expression levels of miRNA34a-5p and miRNA378a-3p scarcely fluctuated, being 220 ng, 240 ng, 200 ng, and 260 ng. On the other hand, significant differences were observed in the types of secreted exosomes assayed using exosome surface markers. As the intracellular miRNA34a-5p and miRNA378a-3p expression levels decreased, the production of exosomes with CD9, CD63, and CD81 surface markers increased. It was elucidated that exosomes secreted by metastatic cells with decreased miRNA34a-5p and miRNA378a-3p were double-positive for CD9 and CD63, differing from non-metastatic cells (Figure 14). Furthermore, as the intracellular miRNA34a-5p and miRNA378a-3p expression levels decreased, miRNA23a, miRNA24, and miRNA184 were identified as miRNAs encapsulated and secreted in exosomes (Figure 15).

[0047] Most importantly, to explore whether there is a possibility of controlling the pathological progression by cell competition and cell synchronization in diseased tissues as well, 68 specimens of aqueous humor before transplantation from corneal transplantation patients and 19 specimens of aqueous humor from cataract patients as a control were used to perform a comprehensive measurement of miRNAs and cytokines in the aqueous humor. Aqueous humor with a large increase in SASP was selected, the amount of miRNA in the aqueous humor was compared with that of cataract patients as a control, and five types of miRNAs that decreased when SASP was high in the aqueous humor were selected (Figure 5). In summary, a decrease in miRNA34a-5p and miRNA378a-3p enhances the secretion of exosomes encapsulating miRNA23a, miRNA24, miRNA184, etc. These exosomes are taken up by surrounding cells paracrinally, and the action of the encapsulated miRNAs enhances SASP, which causes surrounding cells to undergo senescence and degeneration. The existence of such a vicious cycle of disease progression has been revealed. As one of the molecular dynamics of the anterior chamber microenvironment that forms the basis of the present invention, miRNAs secreted from endothelial cells in the form contained in exosomes may be involved in the pathological conditions including glaucoma targeted in the present invention through the disruption of the network between anterior chamber tissues for the first time. This is one of the important factors supporting the probability of the present invention (Fig. 16).

[0048] The inventors have already reported that there is diversity in the metabolites produced by subpopulations of cultured human corneal endothelial cells, and the clinical effects in cell injection regenerative medicine vary corresponding to the types (Junji Hamuro, et al., IOVS, 2020;Vol.61,No.2:1-12). It is considered that cytokines produced and secreted by transplanted cultured cells, soluble miRNAs contained in secreted exosomes, and cytokines and metabolites induced by gene expression → production induction → production induction by these miRNAs change the molecular dynamics of the anterior chamber and affect the clinical effects. This suggests that cytokines, miRNAs, metabolites, etc. produced by corneal endothelial tissue may be more or less related to endothelial dysfunction and glaucoma pathologies even in vivo. It shows the usefulness of the technology for identifying candidate biomarker molecules that specifically exist in the aqueous humor of the anterior chamber and may be related to the pathological conditions, i.e., corneal endothelial cell degeneration and optic nerve cell degeneration.

[0049] Candidate molecular species were also selected for metabolites other than cytokines and soluble miRNAs. This is an advanced analysis of metabolites using GC-MS and LC-MS. In exfoliative glaucoma patients, abnormalities have been observed in the polyamine metabolism system, such as Spermidine and Spermine from Ornithine, which is linked to the urea cycle and the polyamine system. The existence of molecular species common to those detected in cerebrospinal fluid in Parkinson's disease has also been clarified among the candidate molecular species. The procedure for the selection of aqueous humor according to the present invention is shown in the figure (Figure 17).

[0050] As shown in the test examples described below, aqueous humor was collected from patients scheduled for corneal transplantation and patients scheduled for cell injection regenerative medicine surgery as target patients for the depiction of intraocular molecular species in cataract patients for the purpose of excluding non-specific intraocular molecular species and other patients with anterior chamber tissue dysfunction [corneal endothelial dysfunction patients]. In addition, as glaucoma patients with different disease states and types, aqueous humor was also collected from normal tension glaucoma (NTG) patients, primary open angle glaucoma (POAG) patients, or exfoliation glaucoma (PEG) patients. The concentration levels of metabolites appearing in the aqueous humor were measured by a gas chromatograph-mass spectrometer (GC-MS) or a liquid chromatograph-mass spectrometer (LC-MS). Using the cataract group as a control, first, the molecular dynamics of the aqueous humor of patients scheduled for corneal transplantation and patients scheduled for cell injection regenerative medicine surgery were compared with each of the three disease type groups of glaucoma (NTG, POAG, PEG) by the Wilcoxon rank sum test to select molecular species specific to glaucoma disease. Figures 18 to 23 are Volcano Plots showing the LC-MS or GC-MS measurement results of metabolites in the preoperative aqueous humor of glaucoma group, corneal transplantation group, and cell injection group patients with the cataract group as a control. The vertical axis represents -log 10 (p-value), and the horizontal axis represents log2(FC), respectively. The light-colored dots in the figure represent amino acid metabolites, and the dark-colored dots represent other metabolites. Here, the p-value is a statistical significant difference, and FC is the ratio of the average value of the glaucoma group to the average value of the cataract group (Fold Change) (Figures 18 to 23). It was found that there were obvious differences between cataract patients and the other three diseases.

[0051] One of the major pillars forming the framework of the present invention may exclude not only molecular species in the aqueous humor of cataract patients but also molecular species that appear due to corneal endothelial dysfunction when selecting molecular species related to glaucoma pathology in the aqueous humor. For this purpose, first, cluster analysis shown in FIG. 24 was performed to examine the differences in metabolites among six diseases. On the right side of the figure, it was shown by principal component analysis that the aqueous humor metabolites of glaucoma patients were separated from those of cataract patients, and results suggesting that polyamine compounds and carnitine-related molecular species were involved in the divergence were obtained (FIG. 24).

[0052] Among these metabolites, there are various ones, such as metabolites common to patients scheduled for corneal transplantation, patients scheduled for cell injection regenerative medicine surgery, and one glaucoma patient each from three disease type groups with different pathologies, those existing alone in each disease, and those common to specific two diseases. In order to exclude molecular species selectively contained in the aqueous humor of corneal endothelial dysfunction patients, further analysis using Venn diagrams as shown in FIGS. 25 to 29 was performed. Metabolites contained alone or in common are also described in the figures (a biostatistical analysis method called Cross Validation). The numbers in the Venn diagrams indicate the number of metabolites. In this statistical process, only those showing an increase or decrease and having a p-value <0.05 indicating a significant difference were analyzed for cataract patients (FIGS. 25 to 29). In the comparison with the exfoliative glaucoma group, metabolites detected by both LC-MS and GC-MS and those detected alone were described, but for NTG patients and POAG patients, only metabolites detected by both LC-MS and GC-MS were described. As is clear from FIGS. 25, 28, and 29, among the 80 detected metabolites in the exfoliative glaucoma group, 15 were specific to this disease type, 5 out of a total of 21 in NTG, and 9 out of a total of 48 in POAG. This is considered to indicate one aspect of the usefulness of the method for selecting disease state-selective metabolites applied for the first time in the present invention.

[0053] When this method is used only for the three types of glaucoma, there are metabolites common to both the NTG group and the POAG group, metabolites common to both the NTG group and the PEG group, metabolites common to both the POG A group and the PEG group, and metabolites common to all three of the NTG group, the POAG group, and the PEG group. This relationship can also be represented by a Venn diagram such as Fig. 30. The metabolites that vary commonly among the three disease types are 15 types (with duplication due to differences in measurement methods) in the central part of the Venn diagram. Specifically, they are arabinonic acid, myo-inositol, cAMP, fructose, asy-dimethylarginine, citric acid, quinolinic acid, cysteine, spermidine, carnitine, isobutyryl carnitine (C4), 3-methylhistidine, propionyl carnitine, and isocitric acid. These are considered to be in a stage where it is difficult to judge the degree of progression by optical methods. As a result, statistically significant differences (p value < 0.05) were observed in 22 types (with duplication due to differences in measurement methods) in comparison with the NTG group, 54 types (with duplication due to differences in measurement methods) in comparison with the POAG group, and 83 types of metabolites (with duplication due to differences in measurement methods) in comparison with the PEG group.

[0054] Among the metabolites that vary commonly among the three disease types, myo-inositol, fructose, citric acid, cysteine, carnitine, and propionylcarnitine have been reported as molecules that increase in the retinal tissue in the medium to long term in a rat optic nerve injury model (Agudo-Barriuso, M., IOVS. 54, 4249-11 (2013)). The rat optic nerve injury model used in this report does not give any suggestion regarding the progression of the disease state of human glaucoma patients, and there is no clear consensus in the academic community on at which stage of the progression of glaucoma the cell death of retinal ganglion occurs, nor is it suggested in this report, and the significance of the increase has not been studied either. Regarding carnitine included in this report, in a recent report of an optic nerve injury model using mice, it is described as a molecule that increases in the retinal tissue early, contrary to this report (Sato, K., Sci Rep., 8, 11930-13 (2018)). These things cast doubt on the significance of the metabolites described in both reports. It truly speaks to the indispensability of verification with human specimens for the claim of usefulness in glaucoma disease state evaluation.

[0055] Also, as shown in the test examples described later, two metabolites in NTG, eight metabolites in POAG, and 44 metabolites in PEG (with duplicates due to differences in measurement methods) variably changed specifically in relation to each pathological condition. The metabolites showing such specific changes were 2-aminoadipic acid, mannose, GSH (glutathione), alanine, spermine, asparagine, choline, glutamine, glutamic acid, pyroglutamic acid, acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynurenic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabinitol, adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetylcarnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol. Among these, the two metabolites showing specific changes in NTG were specifically 2-aminoadipic acid and mannose. The eight metabolites showing specific changes in POAG were specifically GSH (glutathione), alanine, spermine, asparagine, choline, glutamine, glutamic acid, and pyroglutamic acid.Forty-four types that showed unique variations with PEG (with duplication due to differences in measurement methods) are specifically acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, quinurenic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabinitol, S-adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetylcarnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol. These metabolites can be used as markers for differentiating the disease type of glaucoma (markers for evaluation in the present invention). Each of the above metabolites may be included in extracellular vesicles (EVs) in the aqueous humor.

[0056] Therefore, by collecting aqueous humor from a subject (patient) and measuring the concentration levels of metabolites that appear in the collected aqueous humor by mass spectrometry (MS), the risk of glaucoma onset in the pre-disease state can be evaluated, and the progression of end-stage glaucoma can also be evaluated. The mass spectrometry that can be used in the present invention includes any technique that enables measurement of the molecular weight (i.e., mass) or mass variable corresponding to a metabolite. The component technologies constituting the mass spectrometry include sample introduction, ionization, mass separation, ion detection, recording, etc. Further, as an apparatus for specifically realizing the mass spectrometry, for example, a mass spectrometer can be mentioned. The mass spectrometer can be an apparatus corresponding to each component technology of the above mass spectrometry and including a sample introduction unit, an ionization unit, a mass separation unit, a detection unit, a recording unit, etc.

[0057] As the ionization method in the above mass spectrometry method, an electron ionization (EI) method, a chemical ionization (CI) method, a field desorption (FD) ionization method, a fast atom bombardment (FAB) method, a liquid secondary ion (LSI) method, a matrix-assisted laser desorption / ionization (MALDI) method, an electrospray ionization (ESI) method, an atmospheric pressure chemical ionization (APCI) method, etc. can be used.

[0058] When using tandem mass spectrometry, a collision-induced dissociation (CID) method in which inert gas molecules such as a rare gas (for example, He, Ne, Ar; preferably, Ar) or N2 are collided with the charged particles generated by the first ionization can be further applied.

[0059] The above mass spectrometry method can be combined with other analytical methods such as chromatography, capillary electrophoresis (CE), etc. Examples of such analytical methods include chromatography-mass spectrometry such as gas chromatography / mass spectrometry (GC-MS), liquid chromatography / mass spectrometry (LC-MS), supercritical fluid chromatography / mass spectrometry (SFC-MS), and capillary electrophoresis / mass spectrometry (CE-MS). Among these, chromatography-mass spectrometry is preferred, and GC-MS and / or LC-MS are more preferred.

[0060] In chromatography-mass spectrometry, total ion chromatogram (TIC) scan and / or selected ion monitoring (SIM) analysis, or TIC scan and / or multiple reaction monitoring (MRM) analysis can be performed. Here, MRM analysis is also called selected reaction monitoring (SRM) analysis and can be performed in tandem mass spectrometry. When analyzing a mixture of a large number of metabolites (metabolome), individual metabolites can be identified using the scan mode. Then, for the identified individual metabolites, high-precision quantitative analysis can be performed in the SIM mode or MRM mode by specifying the mass of the metabolite. Among these, from the perspective of high-sensitivity analysis, MRM is preferred.

[0061] Specific examples of the evaluation in the evaluation method of the present invention include determining the occurrence of retinal ganglion cell degeneration, determining the susceptibility of retinal ganglion cells to degeneration or cell death before that, and determining the vulnerability of retinal ganglion cells.

[0062] In order to provide an evaluation technique for the risk of onset or disease progression that is practically useful, as described above, only the comparison between two groups selected as described above and using logistic regression analysis and the selection of metabolites using a Venn diagram alone result in the selection of multiple candidate substances, and it is considered insufficient to depict the breakdown of the network structure in the anterior chamber tissue microenvironment. Therefore, as a further technique in the present invention, a biostatistical method capable of selecting the characteristics between groups as molecular species groups was applied. This method can be applied not only to the analysis within each of the protein molecular species (such as cytokines and complement system proteins related to natural inflammation), miRNAs (which are often contained in exosomes), metabolites, etc. present in the anterior chamber tissue microenvironment, but also to the integrated analysis including these different molecular groups.

[0063] Here, embodiments of metabolites will be exemplified. The purpose is to extract combinations of a plurality of disease discrimination marker candidates (metabolites) related to three types of glaucoma (POAG, NTG, and PEG). For all metabolites measured by the above-mentioned each platform, selection of metabolites was carried out using LASSO (least absolute shrinkage and selection operator), which is one of the machine learning methods, and a set of models (combinations of a plurality of metabolites and their weights) was extracted. The number of specimens is 28 cases of POAG, 24 cases of NTG, 29 cases of PEG, and 31 cases of cataract patients. The results are shown in FIGS. 31 to 38. As a progression evaluation marker (marker for evaluation of the present invention) of end-stage glaucoma in which cell death of retinal ganglion, which is one of the objects of the present invention, can occur, there is a possibility of application of the Lasso analysis result in glaucoma versus cataract. That is, as factors that increase, there are 19 types including Arabinonic acid, Cysteine, N6-Acetyllysine, Creatine, 3-Aminoisobutyric acid, Xanthosine, Carnitine, Indoleacetic acid, Riboflavin, GSH, Isocitric acid, Glutamic acid, Lysine, Argininosuccinic acid, Asparagine, 2-Aminoadipic acid, Butyrylcarnitine (C4), Quinolinic acid, Gluconic acid, and as factors that decrease, there are 12 types including N-Acetyl-Asp-Glu, Lactic acid, Threonine, Putrescine, cAMP, Creatine, GSH, Isocitric acid, Glutamic acid, Lysine, Asparagine, 2-Aminoadipic acid, Quinolinic acid, a total of 31 types. However, as common ones among the three disease types, those that increase are limited to Cysteine only, and those that decrease are limited to cAMP only.

[0064] Next, for the evaluation of the preliminary army of early glaucoma risks that cannot be detected by current evaluation techniques, the results of this analysis in comparison with the NTG group are utilized. Even in the Lasso analysis with a limited population of 31 cases in the cataract group and 24 cases in the NTG group, a total of 8 "combinations" of Creatine, Quinolinic acid, Isocitric acid, myo-Inositol, Myristic acid, Fructose, 2-Deoxytetronic acid, and Mannose were selected, and the discrimination rate of this combination was 76.4%, the sensitivity was 66.7%, and the specificity was 83.9%.

[0065] Thus, significant results can be obtained even in the analysis of each of the same molecular species such as the metabolites found in the aqueous humor. However, it is also possible to apply biostatistical methods that can discriminate the characteristics between groups as molecular species groups to different molecular species groups. That is, this method uses a biostatistical method that can select more significant molecular species from among molecular species groups such as protein molecular species (cytokines, complement system proteins related to natural inflammation, etc.), miRNAs (often contained in exosomes), and metabolites present in the microenvironment of the anterior chamber tissue. Here, embodiments will be exemplified for metabolites and miRNAs that vary in the aqueous humor.

[0066] As described above, in degenerated human corneal endothelial cells, which are a simulation model of corneal endothelial dysfunction patient tissues, miRNA34a-5p and miRNA378a-3p are decreased, and exosomes embedding miRNA23a, miRNA24, miRNA184, etc. are enhanced in secretion, are paracrinally taken up by surrounding cells, and the SASP is enhanced by the action of the embedded miRNAs. The existence of a vicious cycle of disease progression in which surrounding cells undergo senescence and degeneration due to this SASP (cell senescence-related secretory phenotype) has been revealed. As one of the molecular dynamics of the anterior chamber microenvironment that forms the basis of the present invention, miRNAs secreted from endothelial cells in the form contained in exosomes may be involved in the pathological conditions including glaucoma targeted in the present invention through the breakdown of the network between anterior chamber tissues (see Figure 16).

[0067] According to this concept, aqueous humor was extracted from patients with corneal endothelial dysfunction (bullous keratopathy) who had undergone mass spectrometry analysis and miRNA analysis (n = 29). Regression coefficients were calculated using Lasso (Least absolute shrinkage and selection operator) with 2565 miRNAs as explanatory variables and 212 metabolites detected in the aqueous humor by mass spectrometry as target variables, and miRNAs related to the pathogenesis of corneal endothelial dysfunction and metabolites correlated with their families were extracted (Figure 46). That is, (1) metabolites correlated with SASP (senescence-associated secretory phenotype)-suppressive miRNAs in aqueous humor: miR-145-5p, miR-302e, miR-1246, miR-3607-3p; (2) metabolites correlated with miRNAs highly expressed in the culture supernatant of degenerated cells of corneal endothelial cells: miR-23a-3p, miR-24-3p, miR-92b-5p, miR-184; (3) metabolites correlated with miRNAs lowly expressed in the same degenerated cells: miR-34a-5p, miR-378a-3p, miR-29a-3p, miR-29b-3p, miR-184 were extracted, and the molecular species shown in Figure 47 were selected.

[0068] Among these, three metabolites, 2-Hydroxybutyric acid, 3-Hydroxyisobutyric acid, and 4-Hydroxybenzoic acid, were selected as metabolites correlated with the clinical increase shown in Figure 25. These are called aliphatic hydroxy acids, are biosynthesized from keto acids, are abundantly contained in the important energy-metabolic pathway TCA cycle of mitochondria, and precisely control mitochondrial function.

[0069] In this way, the usefulness of the method according to the present invention has been shown, and this usefulness will be further strengthened by further improving the population size. Further improvement as an evaluation technique requires carrying out this analysis including protein molecular species (such as cytokines and complement system proteins related to natural inflammation), miRNAs (which are often contained in exosomes), metabolites, etc. present in the microenvironment of the anterior chamber tissue, the analysis results of which have been described in detail above. Such a method is also included in the present invention. Accordingly, there is provided a method for evaluating the onset risk and progression of eye diseases related to the disruption of the microenvironment of the anterior chamber in eye diseases, particularly eye diseases caused by the disruption of the microenvironment of the anterior chamber.

[0070] For all metabolites targeted for extracting combinations of multiple disease discrimination marker candidates (metabolites) related to the three types of glaucoma (POAG, NTG, and PEG) introduced here, selection of metabolites was carried out using LASSO (least absolute shrinkage and selection operator), one of the machine learning methods, in the plasma of patients from whom aqueous humor specimens were collected (this is a limited test with 20 cases of cataract control, 7 cases of POAG, 9 cases of NTG, and 10 cases of PEG). As a result, different from the results in the above-mentioned aqueous humor, in the LC-MS test for glaucoma vs. cataract, Glutamic acid, Anthranilic acid, and Taurine increased, while Indolepyruvic acid, 4-Hydroxyproline, and Trigonelline decreased, and completely different test results from those of aqueous humor were obtained. For what is common to the three disease types, neither increasing nor decreasing ones could be selected. This is interpreted as indicating that there is scientific rationality in the test of local aqueous humor in glaucoma compared to systemic body fluids.

[0071] In addition to the method described here, the inventors of the present invention have obtained a suitable population for screening patients at extremely early glaucoma risk as the patients to be analyzed. This can be utilized to establish the originality and practical usefulness of the present invention.

[0072] The inventors collected glaucoma specimens from among the patients treated at the glaucoma outpatient clinic, and recruited volunteers as normal controls, who underwent the same number of detailed examinations and diagnoses as those at the glaucoma outpatient clinic. As a result, they were able to collect more than 4,400 glaucoma cases and more than 2,400 normal controls. In addition to DNA and RNA extraction and preservation from these specimens, immortalized cell lines were established from them using Epstein-Barr virus, and these specimens have become semi-permanent biological resources. By utilizing these specimens, the inventors reported, for the first time in the world, the genes of primary open-angle glaucoma (Proc Natl Acad Sci U S A. 2009), and identified, ahead of the world, that CDKN2BAS-1 is a gene specifically involved in primary open-angle glaucoma rather than normal-tension glaucoma (PLoS One. 2012), and also identified a novel gene specific to Japanese people in exfoliation glaucoma (Sci Rep. 2014, PLoS One. 2012). In addition, the inventors have identified various glaucoma disease type genes such as primary open-angle glaucoma (Hum Mol Genet. 2015.), exfoliation glaucoma (Nat Genet. 2015, Nat. Genet., 2017), and primary angle-closure glaucoma (Nat Genet. 2016). Thus, although susceptibility genes related to the onset of glaucoma have been identified, most of them have a low relative risk, and there are almost no genes that have been associated with the onset mechanism and disease state aggravation. This fact clearly shows the limitation of seeking risk factors in single factors such as genes, that is, it does not meet practical usefulness, and tells the practical usefulness of the present invention, which for the first time has been completed and depicts disease type-specific molecules in comparison with related diseases related to the breakdown of the anterior chamber environment. In addition, in the present invention, by comprehensively overlooking three factors: protein molecules including cytokines related to the functional network formation between anterior eye tissues, nucleic acid molecules such as miRNAs that control the acquired expression (epigenetic expression) of genes, and metabolites whose production is controlled by the involvement of these molecular groups, a new technique for assaying the breakdown of the anterior chamber environment at the molecular level is also disclosed, and the combination of these two leads to the provision of a new and practical technique for evaluating the onset risk or disease progression.

[0073] There are various environmental factors as risk factors for the onset of glaucoma. With aging, the number of retinal ganglion cells decreases, intraocular pressure increases in the cold, oxidative stress causes dysfunctions in the zonular fibers and changes in the composition of the aqueous humor, deteriorating the aqueous humor dynamics and causing an increase in intraocular pressure, and also causing damage to axons and retinal ganglion cells. The morbidity of glaucoma increases with aging, and it is impossible with current technologies to guarantee a lifetime of normalcy without symptoms developing in the future. There is a need for "normal controls who have maintained normalcy without symptoms for a long time; a glaucoma onset-resistant group", and the inventors have an advantage in having such a patient group. By comparing normal controls with an age-corrected glaucoma group, it is possible for the first time to estimate the onset resistance throughout life. The present invention also includes a technique of using cases that have maintained normalcy for 10 years as controls. By using cases that have maintained normalcy for 10 years, which became possible only with these technological accumulations, and applying the technology of the present invention, it leads to an epoch-making evaluation technique for projecting the breakdown of the anterior chamber environment at the pre-disease stage.

[0074] Thus, in addition to the comprehensive analysis of cytokines, miRNAs, and metabolites in the aqueous humor, which is the evaluation method technology of the present invention, by providing a molecular evaluation technology for disease type stratification that combines information from conventional diagnosis and treatment including optical methods and genomic information, it realizes appropriate medical treatment for patients with eye diseases caused by the breakdown of the anterior chamber microenvironment, particularly diseases generally referred to as glaucoma, and contributes to the establishment of an international standard evaluation method for onset risk or disease progression.

[0075] In addition, the evaluation method of the present invention can perform a more accurate evaluation by combining other analysis results, findings, information, etc. Such analysis results, etc. include, for example, · Results of molecular dynamics analysis of metabolites by comprehensive metabolome analysis (metabolomics) in patients with corneal endothelial dysfunction · Results of analysis of protein molecular dynamics by comprehensive proteome analysis (proteomics) · Findings in gene expression analysis using proteomics and / or clinical specimens · Information related to factors in tears, blood, and / or other body fluids and comprehensive medical information analysis such as these are also included in the present invention.

[0076] 2. Evaluation system according to the present invention The evaluation system according to the present invention (hereinafter referred to as "the present invention evaluation system") is an evaluation system for the risk of onset of eye diseases or the progression of advanced eye diseases, comprising means for collecting aqueous humor specimens (specimen collection means) and means for analyzing the specimens by mass spectrometry (MS) (analysis means), and is an evaluation system for use in the evaluation method of the present invention.

[0077] Examples of the specimen collection means included in the present invention evaluation system include a pipette.

[0078] Examples of the analysis means included in the present invention evaluation system include a mass spectrometer. The mass spectrometer can be an apparatus including a sample introduction unit, an ionization unit, a mass separation unit, a detection unit, a recording unit, etc.

[0079] As the mass separation unit, a double focusing mass spectrometer (magnetic field type mass spectrometer), a quadrupole type mass spectrometer (Quadrupole Mass Spectrometer: Q MS), an ion trap (Ion Trap: IT) mass spectrometer, a time-of-flight (Time-Of-Flight: TOF) mass spectrometer, a Fourier transform ion cyclotron resonance (Fourier Transform-Ion Cyclotron Resonance: FT-ICR) mass spectrometer, etc. can be used.

[0080] The mass separation unit may be a single mass spectrometer or a tandem mass spectrometer. There is no particular limitation on the mode of the tandem mass spectrometer. For example, a triple quadrupole type mass spectrometer, a quadrupole-time-of-flight (Q-TOF) mass spectrometer, an ion trap-time-of-flight (IT-TOF) mass spectrometer, a tandem TOF mass spectrometer can be used. Among these, a triple quadrupole type mass spectrometer is preferred.

[0081] The above analysis means can be combined with other analysis means such as a chromatograph, a capillary electrophoresis (CE) apparatus, etc. Examples of such analysis means include chromatograph mass spectrometers such as gas chromatograph / mass spectrometer (GC-MS), liquid chromatograph / mass spectrometer (LC-MS), supercritical fluid chromatograph / mass spectrometer (SFC-MS), and capillary electrophoresis / mass spectrometer (CE-MS). Among these, chromatograph mass spectrometers are preferred, and GC-MS and / or LC-MS are more preferred.

Examples

[0082] Test examples or examples are shown below to explain the present invention, but the present invention is not limited by these examples at all.

[0083] Test Example Comprehensive Metabolite Analysis Using aqueous humor collected from 31 cataract patients, 81 glaucoma patients, and 62 patients with corneal endothelial disorders, comprehensive metabolite analysis (metabolome analysis) by GC-MS and LC-MS was performed. The aqueous humor was collected from each patient in an amount of 100 μL using a dedicated pipette. The breakdown of the glaucoma patient group (81 patients) is 24 patients with normal-tension glaucoma (NTG), 28 patients with primary open-angle glaucoma (POAG), and 29 patients with exfoliative glaucoma (PEG). The glaucoma specimens were collected during surgery from glaucoma cases in which visual field impairment became apparent. The breakdown of the corneal endothelial disorder group (62 patients) is 41 patients who underwent corneal endothelial transplantation (corneal transplantation group) and 21 patients who received cell injection by corneal endothelial regenerative medicine (cultured human corneal endothelial cell injection therapy) (cell injection group).

[0084] (1) Measurement by gas chromatography-mass spectrometer <Sample preparation> First, 720 μL of an internal standard solution (2-isopropylmalic acid 0.1 mg / mL) was added to 30 mL of methanol and mixed well (methanol containing internal standard). 30 μL of aqueous humor sample was placed in a tube, 154 μL of methanol containing internal standard was added, and immediately stirred strongly with a tabletop mixer. After mixing at 37 °C and 1200 rpm for 10 minutes using a thermostatic shaker, centrifugation was performed, and only the supernatant was collected at 120 μL in a new microtube to remove proteins.

[0085] Next, 72 μL of 1% acetic acid and 96 μL of chloroform were added to the obtained supernatant, stirred for 15 seconds with a tabletop mixer, and then centrifuged again. Thereafter, 60 μL of the supernatant was collected in a new microtube to extract hydrophilic metabolites. Then, for the obtained supernatant, the solvent was vaporized using a centrifugal evaporator, and then dried by freeze-drying to remove moisture. Finally, 40 μL of methoxyamine pyridine solution (20 mg / mL) was added to the sample after freeze-drying for oxime derivatization (shaken at 37 °C for 30 minutes), and then 20 μL of MSTFA was added for trimethylsilylation (shaken at 37 °C for 30 minutes), and this was used as the sample.

[0086] <Measuring device> For the above sample, GC-MS analysis was performed using a single quadrupole gas chromatograph-mass spectrometer (GCMS-QP2010Ultra, manufactured by Shimadzu Corporation) to collect data. The analysis was performed under the following conditions.

[0087] <Measurement conditions> GC section: The sample injection volume was set to 1 μL, and a DB-5 (30 m × 0.25 mm I.D., df = 1 μm, capillary column manufactured by Agilent Technologies) was used as the column. The column oven temperature was maintained at 80 °C for 2.5 minutes from the start of measurement, and then reached 280 °C at a temperature increase rate of 12.5 °C / min and was maintained at the reached temperature for 4.5 minutes. The vaporization chamber temperature was set to 280 °C, and the linear velocity of the carrier gas (He) was set to 39 cm / sec. MS section : The interface temperature and the ion source temperature were set to 200 °C and 250 °C, respectively. Quantitative analysis was performed in the scan mode.

[0088] (2) Measurement by liquid chromatography mass spectrometer <Sample preparation> The aqueous humor of the specimen was dispensed into a microtube, and an aqueous solution of 2-isopropylmalic acid was added thereto. The process of further removing proteins is the same as that during GC-MS analysis. Next, the process of extracting hydrophilic metabolites is also the same as that during GC-MS analysis. And, similarly, the solvent was vaporized using a centrifugal evaporator, and then dried by freeze-drying to remove moisture. Finally, 25 μL of 0.1% aqueous formic acid solution was added to the specimen after freeze-drying and stirred well, and this was dispensed into a vial as the sample.

[0089] <Measuring device> For the above sample, LC-MS analysis was performed using a high-performance liquid chromatograph (Nexera, manufactured by Shimadzu Corporation) as the LC part and a triple quadrupole mass spectrometer (LCMS-8040, manufactured by Shimadzu Corporation) as the MS part, and data was collected. The analysis was performed under the following conditions.

[0090] <Measurement conditions> LC section: The sample injection volume was set to 4 μL, and a Shim-pack GIST C18-AQ (250 mm I.D. × 2.1 mm L, 3 μm, a reverse-phase column manufactured by Shimadzu GLC) was used as the column. The column oven temperature was set to 40 °C, and the flow rate of the mobile phase was set to 0.2 mL / min. Two types of mobile phases, A (a mixed solvent with a volume ratio of formic acid: water = 1:1000) and B (a mixed solvent with a volume ratio of formic acid: acetonitrile = 1:1000), were used, and the mixing ratio of the two mobile phases was changed over time to perform gradient analysis. The time program for the gradient analysis is as follows. 0% B (0 - 3.0 min) → 60% B (15.0 min) → 95% B (15.01 min) → 95% B (15.01 - 20.0 min) → 0% B (20.1 - 25.0 min)

[0091] MS section : ESI (Positive / Negative) was used as the ionization method. Compounds were identified in the scan mode, and quantitative analysis was performed in the MRM mode. The set values for each analysis are as follows. Nebulizer gas flow rate: 3 L / min Drying gas flow rate: 15 L / min DL temperature: 250 °C Heat block temperature: 400 °C

[0092] (3) Analysis of measurement results (3-1) Analysis of glaucoma group data with the cataract group as the control In the analysis by GC-MS, 96 types of metabolites were identified, and in the analysis by LC-MS, 114 types of metabolites were identified, and their peak area values were calculated. The value obtained by dividing the peak area value of each metabolite by the peak area value of the internal standard substance (2-isopropylmalic acid) added in a certain amount to each sample was adopted as a semi-quantitative value reflecting the concentration in the aqueous humor and compared among each group.

[0093] Using the cataract group as a control, pairwise comparisons were made between each of the three glaucoma subtypes (NTG, POAG, PEG) and the control group using the Wilcoxon rank sum test. As a result, statistically significant differences (p-value < 0.05) were observed in 22 metabolites when compared with the NTG group, 54 metabolites when compared with the POAG group, and 83 metabolites when compared with the PEG group. Among these, 15 metabolites showed consistent variation across all three subtypes. Table 1 shows the list of 15 metabolites, the ratio of the mean value (Fold Change: FC) compared with the control group, and the p-value. Additionally, 2 metabolites were identified as uniquely varying in the NTG group, 8 metabolites in the POAG group, and 44 metabolites in the PEG group compared with the control group. Table 2 shows the FC and p-value for these metabolite lists compared with the control group. These metabolites can be used as glaucoma subtype discriminant markers (markers for the evaluation of the present invention).

[0094] [Table 1]

[0095] [Table 2]

[0096] (3-2) Performance evaluation of metabolites selected considering corneal endothelial disorder group data as markers for evaluating the onset risk or disease progression of glaucoma Among the 15 metabolites found to vary consistently across the three glaucoma subtypes, four metabolites, namely arabinonic acid, myo-inositol, cAMP, and fructose, were identified as having differences when compared with the corneal endothelial disorder group (corneal transplantation specimens and cell injection specimens). The marker performance of these four metabolites was evaluated when considering all glaucoma cases as positive regardless of the cause and cataract cases as negative. The results are shown in Figures 39 - 42.

[0097] Figures 39 to 42 show the results of quantifying the amount of each metabolic product in the aqueous humor of the corneal endothelial disorder group (corneal transplant group and cell injection group), cataract group, and glaucoma group by GC-MS or LC-MS. The figures below show the ROC (Receiver Operating Characteristic) curves when each metabolic product is used as a marker for evaluating glaucoma. The AUC (Area Under Curve) value is shown in each figure.

[0098] These results show that regardless of which metabolite was used as a marker, a relatively high true positive rate was achieved even when the false positive rate was small. In addition, the AUC values ​​were high, and in particular, arabinonic acid showed the best value of 0.809.

[0099] (3-3) Evaluation of marker performance by multiple logistic regression analysis Among the 15 metabolites found to be commonly altered in the three types of glaucoma, In order to find a combination that can accurately discriminate between glaucoma groups, a prediction model using multiple logistic regression analysis was examined. The optimal metabolites were selected using the variable addition and subtraction method, and arabinonic acid, cAMP, cysteine, spermidine, and isobutyrylcarnitine (C4) were selected. The regression coefficients of the prediction model created using these five metabolites are shown in Table 3. The marker performance was evaluated by ROC analysis when cataracts were negative and all glaucoma, regardless of cause, were positive, resulting in good results of AUC of 0.931, sensitivity of 0.84, and specificity of 0.94 (Figure 43).

[0100] [Table 3]

[0101] Furthermore, the Pearson product-moment correlation coefficients among these 15 metabolites were shown using a color map (Figure 44). Based on the degree of correlation, the metabolites were classified into sugars and polyols (arabinoic acid, myo-inositol, fructose), the citric acid cycle (citric acid, isocitric acid), amino acids (asy-dimethylarginine, quinolinic acid, cysteine, 3-methylhistidine), polyamines (spermidine), acylcarnitines (carnitine, isobutyryl carnitine (C4), propionyl carnitine), and cAMP. The metabolites selected by the above prediction model were each selected one by one from each group, and the marker performance was maintained even when replaced with metabolites within the group. For example, when arabinoic acid was replaced with myo-inositol, the regression coefficients of the created prediction model are shown in Table 4. The performance of the created prediction model was found to be comparable, with an AUC of 0.924, sensitivity of 0.83, and specificity of 0.94 (Figure 45).

[0102]

Table 4

[0103] (3-4) Metabolome analysis considering data of the corneal endothelial disorder group In addition, regarding the amounts of metabolites in the aqueous humor in the PEG group, corneal transplantation group, and cell injection group, the statistical significance (p-value) by the Wilcoxon rank sum test when the cataract group was used as a control, and the ratio (Fold Change: FC) of the average value of each group based on the average value of the cataract group were comprehensively analyzed. The results are shown in Figures 18 to 23 (however, in the figures, the PEG group is denoted as glaucoma). Among these, Figures 18 to 20 are Volcano plots based on measurements by LC-MS, and Figures 21 to 23 are Volcano plots based on measurements by GC-MS. The light-colored points in each figure indicate amino acid metabolites, and the dark-colored points indicate other metabolites. For metabolites within the range of -1.0 < log2(FC) < 1.0 and 1.3 < -log (p-value) 10 (p-value) their names are described in each figure.

[0104] Among these, for example, looking at the PEG group, it can be seen that among the metabolites measured by LC-MS, carnitine shows a significant difference at a high level for the cataract group. It can be seen that reduced glutathione (GSH) is a metabolite that has both a concentration difference and a significant difference with respect to the cataract group. Also, among the metabolites measured by GC-MS, it can be seen that aconitic acid has both a concentration difference and a significant difference.

[0105] (3-5) Extraction of miRNAs involved in the pathogenesis of corneal endothelial dysfunction and metabolites correlated with their families by the Lasso (Least absolute shrinkage and selection operator) regression method. Lasso (Least absolute shrinkage and selection operator) regression was used for the extraction of miRNAs involved in the pathogenesis of corneal endothelial dysfunction and metabolites correlated with their families. This method is one of the feature selection methods for selecting the explanatory variable X necessary to explain the target variable Y using L1 regularization, and the model is selected by minimizing the following formula.

[0106] [Number]

[0107] Here, aqueous humor (n = 29) from patients with corneal endothelial dysfunction (bullous keratopathy) who have undergone mass spectrometry analysis and miRNA analysis is targeted. This analysis is to mathematically obtain a set of metabolites that contribute to the target variable from 2565 miRNAs, which are the explanatory variables, for each of the 212 metabolites that are the target variables. When the analytical data has more than 80% missing values in the metabolite, which is the target variable, it is excluded from the analysis target. For the complementation of other missing values, 0 was substituted for the metabolite and 0.1 was substituted for miRNA. Then, the target variable and the explanatory variables were binarized (0 or 1) with their respective median values, and the parameter λ was determined to be the optimal value by 10-fold cross-validation. Note that by model selection using Lasso regression, the weights (regression coefficients) of the coefficients of the explanatory variables that do not contribute to the target variable become 0, and by reducing the number of explanatory variables simultaneously with model selection, it becomes possible to extract the contributing explanatory variables.

[0108] The regression coefficients of a set of metabolites selected by the model selection using the said analytical method were calculated, and metabolites correlated with miRNAs and their families involved in the pathological condition of corneal endothelial dysfunction were extracted (Figure 46). That is, (1) metabolites correlated with SASP (senescence-associated secretory phenotype)-suppressive miRNAs: miR-145-5p, miR-302e, miR-1246, miR-3607-3p in the aqueous humor, (2) metabolites correlated with miRNAs highly expressed in the culture supernatant of degenerated cells of corneal endothelial cells: miR-23a-3p, miR-24-3p, miR-92b-5p, miR-184, (3) metabolites correlated with miRNAs lowly expressed in the same degenerated cells: miR-34a-5p, miR-378a-3p, miR-29a-3p, miR-29b-3p, miR-184 were extracted, and the molecular species shown in Figure 47 were selected.

Claims

1. A method for measuring the concentration level of metabolites appearing in collected aqueous humor by mass spectrometry (MS) in order to evaluate the risk of glaucoma onset or the progression of end-stage glaucoma through molecular dynamics analysis of the anterior chamber environment, the method including the step of measuring the concentration level of metabolites appearing in the collected aqueous humor by mass spectrometry (MS) in order to discriminate the specific pathologies of three disease types: primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma / exfoliation syndrome (PEG).

2. The method according to claim 1, wherein the collected aqueous humor is from a subject in a very early stage with an unidentified pathological condition at the pre-disease stage without developing an eye disease.

3. The method according to claim 1 or 2, wherein the mass spectrometry (MS) is gas chromatography-mass spectrometry (GC-MS) and / or liquid chromatography-mass spectrometry (LC-MS).

4. The method according to any one of claims 1 to 3, wherein the metabolite is included in extracellular vesicles (EV) in the aqueous humor.

5. The method according to any one of claims 1 to 4, further including the step of selecting those showing differences in molecular dynamics in tears or blood.

6. The method according to any one of claims 1 to 5, wherein the metabolite is selected from those that are common or not common with the variations in corneal endothelial dysfunction patients.

7. The method according to any one of claims 1 to 6, wherein the metabolite is at least one selected from the group consisting of sugars or polyols, products in the citric acid cycle, acylcarnitines, polyamines, amino acids, and cAMP.

8. The method according to claim 7, wherein the sugar or polyol is arabinonic acid, myo-inositol, or fructose; the product substance in the citric acid cycle is citric acid or isocitric acid; the acylcarnitine is carnitine, isobutyryl carnitine (C4), or propionyl carnitine; the polyamine is spermidine; and the amino acid is asym-dimethylarginine, quinolinic acid, cysteine, or 3-methylhistidine.

9. The method according to claim 7 or 8, wherein the sugar or polyol is arabinonic acid or myo-inositol; the acylcarnitine is isobutyryl carnitine (C4); and the amino acid is cysteine.

10. The method according to any one of claims 1 to 6, wherein the metabolite is at least one selected from the group consisting of 2-aminoadipic acid, mannose, GSH (glutathione), alanine, spermidine, asparagine, choline, glutamine, glutamic acid, pyroglutamic acid, acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynurenic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabitol, adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetylcarnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol.

11. The method according to any one of claims 1 to 6, wherein the glaucoma is normal tension glaucoma (NTG), and the metabolite is at least one selected from the group consisting of 2-aminoadipic acid, mannose, arabinonic acid, myo-inositol, cAMP, fructose, asym-dimethylarginine, citric acid, quinolinic acid, cysteine, spermidine, carnitine, isobutyryl carnitine (C4), 3-methylhistidine, propionyl carnitine, and isocitric acid.

12. The method according to any one of claims 1 to 6, wherein the glaucoma is exfoliation glaucoma / exfoliation syndrome (PEG), and the metabolite is acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynurenic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabinitol, adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetyl carnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol.

13. The method according to any one of claims 1 to 12, wherein the evaluation is to determine the occurrence of retinal ganglion cell degeneration, or the susceptibility to degeneration or cell death of retinal ganglion cells before that, or the vulnerability of retinal ganglion cells.

14. The method according to any one of claims 1 to 13, comprising the step of considering the results of comprehensive proteome analysis of the molecular dynamics of proteinaceous molecules contained in extracellular microparticles (EV) in the aqueous humor.

15. The method according to any one of claims 1 to 14, comprising the step of taking into account findings in proteomic analysis and / or gene expression analysis using clinical specimens.

16. The method according to any one of claims 1 to 15, comprising the step of taking into account information on factors in tears, blood, and / or other body fluids.

17. An evaluation marker for use in a method for evaluating the risk of glaucoma onset or progression of end-stage glaucoma, comprising the step of measuring the concentration level of metabolites appearing in the collected aqueous humor by mass spectrometry (MS) and the step of discriminating the specific pathologies of three disease types: primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma / exfoliation syndrome (PEG). The evaluation marker is at least one selected from the group consisting of sugars or polyols, products in the citric acid cycle, acylcarnitines, polyamines, amino acids, and cAMP.

18. The evaluation marker according to claim 17, wherein the sugar or polyol is arabinonic acid, myo-inositol, or fructose, the product in the citric acid cycle is citric acid or isocitric acid, the acylcarnitine is carnitine, isobutyryl carnitine (C4), or propionyl carnitine, the polyamine is spermidine, and the amino acid is asym-dimethylarginine, quinolinic acid, cysteine, or 3-methylhistidine.

19. An evaluation marker for use in a method for evaluating the risk of glaucoma onset or progression of end-stage glaucoma, comprising the step of measuring the concentration level of metabolites appearing in the collected aqueous humor by mass spectrometry (MS) and the step of discriminating the specific pathologies of three disease types: primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliation glaucoma / exfoliation syndrome (PEG). An evaluation marker that is at least one selected from the group consisting of 2-aminoadipic acid, mannose, arabinonic acid, myo-inositol, cAMP, fructose, asym-dimethylarginine, citric acid, quinolinic acid, cysteine, spermidine, carnitine, isobutyryl carnitine (C4), 3-methylhistidine, propionyl carnitine, GSH (glutathione), alanine, spermine, asparagine, choline, glutamine, glutamic acid, pyroglutamic acid, acetylcholine, xanthosine, N-acetylarginine, glycine, 3-aminoisobutyric acid, cystine, kynureninic acid, kynurenic acid, 4-hydroxyproline, pyridoxic acid, isocitric acid, N-acetylglucosamine, GSSG (glutathione disulfide), N'-formylkynurenine, creatinine, N-acetylmethionine, ornithine, citrulline, oleamide, arabitol, adenosylhomocysteine, hippuric acid, transurocanic acid, urea, succinic acid, riboflavin, 2-hydroxyglutaric acid, malic acid, hypotaurine, pipecolic acid, guanidinoacetic acid, acetyl carnosine, 2-oxoglutaric acid, 3-hydroxyisovaleric acid, maltose, uridine, 1,5-anhydro-D-sorbitol, fucose, and 2-aminoethanol.

20. An evaluation system for the risk of glaucoma onset or the progression of end-stage glaucoma, comprising means for collecting an aqueous humor specimen and means for analyzing the specimen by mass spectrometry (MS). An evaluation system for use in an evaluation method for the risk of glaucoma onset or the progression of end-stage glaucoma, comprising a step of measuring, by mass spectrometry (MS), the concentration level of metabolites appearing in the collected aqueous humor, and a step of discriminating the specific pathologies of three disease types: primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and exfoliative glaucoma / exfoliation syndrome (PEG).

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