JP7202609B2 - Diagnostic biomarkers for optic neuropathy - Google Patents

Description

The present invention provides a method for determining the risk of developing optic nerve damage and/or the presence or absence of developing optic nerve damage; a method for determining the severity of optic nerve damage; The present invention relates to a biomarker for determining the presence or absence of a biomarker for determining the severity of optic neuropathy, and a method for screening a preventive agent for the onset of optic neuropathy and/or a therapeutic agent for optic neuropathy.

The retina is composed of 10 layers: inner limiting membrane, nerve fiber layer, ganglion cell layer, inner reticular layer, inner nuclear layer, outer reticular layer, outer nuclear layer, outer limiting membrane, photoreceptor layer, and retinal pigment epithelium layer. It is a tissue with a thickness of 0.1 to 0.5 mm, and contains retinal nerve cells such as photoreceptor cells, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. Retinal neurons play an important role in the reception and transmission of visual information, such as converting light stimuli into electrical signals and transmitting them to the brain. Visual information received from the eye is converted into electrical signals by photoreceptor cells, and transmitted to ganglion cells after passing through horizontal cells, bipolar cells, and amacrine cells. The electrical signal is then transmitted to the brain via a bundle of optic nerve fibers (optic nerve) containing the axons of ganglion cells.

When the optic nerve is damaged for some reason, homeostasis of the optic nerve cannot be maintained, and the transmission of visual information to the brain is hindered, resulting in visual field disorders such as blindness and constriction of the visual field. The main causes of optic nerve damage are increased intraocular pressure (high intraocular pressure; IOP), impaired retinal blood circulation/retinal ischemia, and increased excitatory amino acids. It is known that the glutamate signal cascade is activated in association with these pathologies, the axons of retinal ganglion cells (RGC) are damaged, RGC cell death (apoptosis) is induced, and the optic nerve is damaged. is considered to receive

Glaucoma, which is one of the major causes of blindness in the world, is known as a typical example of optic neuropathy (Non-Patent Document 1). Since glaucoma has no subjective symptoms, early detection is difficult, and it is a disease that leads to blindness, so early diagnosis is very important. In the past, the diagnosis of glaucoma has focused on fundus examination, and after the patient has been given a mydriatic drug to dilate the pupils before the examination, the doctor directly observes the retina with a fundus scope or fundus camera. are doing. However, the current situation is that there is a possibility that dilation of the pupil by a mydriatic will impede the flow of aqueous humor, increase the intraocular pressure, and further worsen the condition. In addition, direct observation of the retina by a doctor is not necessarily an objective test method, and since the doctor has to deal with each individual patient, it is difficult to apply it to mass examinations that handle a large number of subjects.

As described above, the existing examination methods have not a few problems. A low-burden, high-throughput assay method is desired.

A test method using biomarkers is one of the objective and high-throughput methods. So far, a method for diagnosing glaucoma before the onset of glaucoma using mutation of the myocillin (MYOC) gene as an index (Patent Document 1), and a method related to the fact that mutation of the optineurin gene (OPTN) is a causative gene of glaucoma. (Non-Patent Document 2) and that there are genetic polymorphisms associated with the progression of glaucoma (Patent Documents 2 to 4). However, glaucoma that can be diagnosed is limited to those caused by these genes. In addition, in these documents, verification is performed using samples derived from patients who developed glaucoma, and it is not known whether the risk of developing glaucoma can be determined. Further, a method for detecting glaucoma in glaucoma patients using a protein whose expression fluctuates as an index is disclosed (Patent Document 5). However, even in this document, the verification is performed using samples derived from patients who developed glaucoma, and it is unknown whether the risk of developing glaucoma can be determined.

On the other hand, axonopathy (injury) induces retinal ganglion cell (RGC) death, which is central to the pathology of glaucoma, but RGC numbers are maintained for several days after axonopathy. It is expected that the expression of mRNA of a gene directly involved in RGC death fluctuates during the period when the number of RGCs is maintained (Non-Patent Document 3). Recently, the present inventors have found a gene that can determine the risk of developing optic nerve damage using an ONC model mouse subjected to axonal damage treatment by optic nerve crush (ONC) (Patent Document 6).

Japanese Patent Application Laid-Open No. 2002-306165 JP 2010-115194 A JP 2013-150628 A JP 2015-180219 A JP 2009-244125 A JP 2015-142540 A

Kwon, Y.H. et al, New England Journal of Medicine 2009, 360(11):1113-1124. Rezaie, T. et al, Science 2002, 295(5557):1077-1079 Fischer, D. et al, The Journal of neuroscience 2004, 24(40):8726-8740

An object of the present invention is to provide a biomarker for diagnosing optic neuropathy that can accurately determine the risk of developing optic neuropathy, the presence or absence of the onset, and the severity of optic neuropathy.

As described above, the present inventors have found a gene that can determine the risk of developing optic neuropathy using ONC model mice that have undergone axonal injury treatment with ONC. This time, the present inventors used liquid chromatograph (LC)-mass spectrometry (MS) and mass spectrometry techniques using imaging MS (IMS) to study ONC model mice and normal We analyzed metabolites with fluctuating concentrations in biological samples with mice. As a result, the following metabolite groups [A group] to [D group] were found to be biomarkers capable of accurately determining the risk of developing optic nerve damage and the severity of optic nerve damage. In addition, by mass spectrometry using biological samples derived from glaucoma patients who developed optic nerve damage, the metabolite groups of [E group] to [J group] below can be used to accurately determine the presence or absence of optic nerve damage. I also confirmed that it was a marker. The present invention has been completed based on these findings.
[Group A]
L-acetylcarnitine, 9-hexadecenoylcarnitine, hydroxybutyrylcarnitine, triacylglycerol (62:12), L-octanoylcarnitine, asparagine-aspartic acid dipeptide, m-aminobenzoic acid [Group B]
phosphatidylcholine (38:7), phosphatidylglycerol (40:6), lysophosphatidylcholine (18:2), phosphatidylglycerol (32:0), S-adenosylmethionine, phosphatidylethanolamine (42:8), phosphatidylglycerol (38 : 6)
[Group C]
Phosphatidylcholine (46:6), sphingomyelin (d18:0/24:0), 8,8-diethoxy-2,6-dimethyl-2-octanol, glycerophosphocholine, β-lactosylceramide (d18:1/22 :0), phosphatidylcholine (44:9)
[Group D]
Monoacylglycerides (18:0/0:0/0:0), D-glucose, guanosine, adenine, adenosine, glutathione, S-formylglutathione, methionine-histidine dipeptide [E group]
3-sulfolactic acid, dimethylsulfone, ribonic acid, arabinonic acid, xylonic acid, L-hydroxyglutaric acid, hydroxycitric acid, glucaric acid, galactaric acid, octenoylcarnitine, betaine, L-acetylcarnitine, methylmalonylcarnitine, L- Carnitine, N,N-diethylethanolamine, L-cystine [Group F]
pyroglutamine, lysophosphatidylcholine (18:2) sn-1, lysophosphatidylethanolamine (18:2), lysophosphatidylcholine (18:2) sn-2, androsterone sulfate, testosterone sulfate, phenol sulfate, lactic acid, D-glucose , hydroperoxylinoleic acid, phosphatidylcholine (34:2), thiomorpholine-3-carboxylic acid, lysophosphatidylcholine (18:2), L-carnitine [G group]
Methyladenosine, succinyladenosine, dodecanoylcarnitine, O-(7-carboxyheptanoyl)carnitine, O-(8-carboxyoctanoyl)carnitine, O-(11-carboxyundecanoyl)carnitine, maleic acid, fumaric acid [ Group H]
Dichloromethane, glutathione, L-tyrosine, L-phenylalanine, 3-mercaptolactic acid, uridine, taurine, aminobutyric acid [Group I]
lysophosphatidylcholine (18:0), methyl palmitate, isopropyl myristate, lysophosphatidylethanolamine (22:6), lysophosphatidylcholine (17:0) sn-1, lysophosphatidylcholine (14:0), lysophosphatidylcholine (17:0) 0) sn-2, lysophosphatidylcholine (15:0), fatty acids, p-cresol sulfate, indoxyl sulfate, hydroxybutyric acid, hydroxybenzoic acid, N-phenylacetylglutamine, aconitic acid, pyridoxal, lysophosphatidylcholine (16:0) , phosphatidylcholine (36:2), phosphatidylcholine (38:4), phosphatidylcholine (40:6), proline betaine, ceramide (d18:1/16:0)
[J Group]
Citric acid (the group of metabolites from [A group] to [J group] may be collectively referred to hereinafter as "the metabolite group")

That is, the present invention is as follows.
[1] A method for determining the risk of developing an optic nerve disorder, or determining the presence or absence of such a disorder, comprising the following steps (a-1) to (c-1).
(a-1) one or more metabolites selected from the following [Group I], [Group A] to [Group H], and [Group J] in a biological sample collected from a subject measuring the concentration;
[Group I]
fatty acid, LPC (18:0), methyl palmitate, isopropyl myristate, LPE (22:6), LPC (17:0) sn-1, LPC (14:0), LPC (17:0) sn-2 , LPC (15:0), p-cresol sulfate, indoxyl sulfate, hydroxybutyric acid, hydroxybenzoic acid, N-phenylacetylglutamine, aconitic acid, pyridoxal, LPC (16:0), PC (36:2), PC (38:4), PC (40:6), proline betaine, ceramide (d18:1/16:0)
[Group A]
L-Acetylcarnitine, 9-Hexadecenoylcarnitine, Hydroxybutyrylcarnitine, Triacylglycerol (62:12), L-Octanoylcarnitine, Asparagine-Aspartic Acid Dipeptide , m-Aminobenzoic acid
[Group B]
Phosphatidylcholine (PC) (38:7), Phosphatidylglycerol (PG) (40:6), Lysophosphatidylcholine (lysoPC; LPC) (18:2), PG (32:0), S-adenosylmethionine (Adenosylmethionine), Phosphatidylethanolamine (PE) (42:8), PG (38:6)
[Group C]
PC (46:6), Sphingomyelin (SM) (d18:0/24:0), 8,8-diethoxy-2,6-dimethyl-2-octanol, Glycerophosphocholine (GPC), β - Lactosylceramide (d18:1/22:0), PC (44:9)
[Group D]
Monoacylglyceride (18:0/0:0/0:0), D-Glucose, Guanosine, Adenine, Adenosine, Glutathione, S-Formylglutathione, Methionine-Histidine Dipeptide [Group E]
3-sulfolactic acid, dimethylsulfone, ribonic acid, arabinonic acid, xylonic acid, L-hydroxyglutaric acid, hydroxycitric acid, glucaric acid, galactaric acid, octenoylcarnitine, betaine, L-acetylcarnitine, methylmalonylcarnitine, L- Carnitine, N,N-diethylethanolamine, L-cystine [Group F]
pyroglutamine, LPC (18:2) sn-1, lysophosphatidylethanolamine (lysoPE; LPE) (18:2), LPC (18:2) sn-2, androsterone sulfate, testosterone sulfate, phenol sulfate, lactic acid, D-glucose, hydroperoxylinoleic acid, PC (34:2), thiomorpholine-3-carboxylic acid, LPC (18:2), L-carnitine [G group]
Methyladenosine, succinyladenosine, dodecanoylcarnitine, O-(7-carboxyheptanoyl)carnitine, O-(8-carboxyoctanoyl)carnitine, O-(11-carboxyundecanoyl)carnitine, maleic acid, fumaric acid [ Group H]
Dichloromethane, glutathione, L-tyrosine, L-phenylalanine, 3-mercaptolactic acid, uridine, taurine, aminobutyric acid [J group]
citric acid (b-1) comparing the concentration of the metabolite measured in step (a-1) with the concentration of said metabolite in a control subject who does not develop optic neuropathy;
(c-1) if the metabolites measured in step (a-1) are [A group], [B group], or [E group] to [G group] metabolites, step (a-1) When the concentration of the metabolite measured in is higher than the concentration of the metabolite in the control subject, the subject is assessed as having a high risk of developing optic neuropathy or a high possibility of developing optic neuropathy. ,
If the metabolite measured in step (a-1) is a [I group], [C group], [D group], [H group], or [J group] metabolite, step (a-1) When the concentration of the metabolite measured in is lower than the concentration of the metabolite in the control subject, the subject is assessed as having a high risk of developing optic neuropathy or likely to have developed optic neuropathy. process;
[2] A method for determining the severity of optic nerve damage, comprising the following steps (a-2) to (c-2).
(a-2) Measuring the concentration of one or more metabolites selected from the following [Group A] to [Group D] in a biological sample collected from a subject suffering from optic neuropathy process;
[Group A]
L-Acetylcarnitine, 9-Hexadecenoylcarnitine, Hydroxybutyrylcarnitine, Triacylglycerol (62:12), L-Octanoylcarnitine, Asparagine-Aspartic Acid Dipeptide , m-Aminobenzoic acid
[Group B]
Phosphatidylcholine (PC) (38:7), Phosphatidylglycerol (PG) (40:6), Lysophosphatidylcholine (lysoPC; LPC) (18:2), PG (32:0), S-adenosylmethionine (Adenosylmethionine), Phosphatidylethanolamine (PE) (42:8), PG (38:6)
[Group C]
PC (46:6), Sphingomyelin (SM) (d18:0/24:0), 8,8-diethoxy-2,6-dimethyl-2-octanol, Glycerophosphocholine (GPC), β - Lactosylceramide (d18:1/22:0), PC (44:9)
[Group D]
Monoacylglyceride (18:0/0:0/0:0), D-glucose, guanosine, adenine, adenosine, glutathione, S-formylglutathione, methionine-histidine dipeptide (b-2) step comparing the concentration of the metabolite measured in (a-2) with the concentration of the metabolite in a control subject developing optic neuropathy;
(c-2) When the metabolite measured in step (a-2) is a [A group] or [D group] metabolite, the concentration of the metabolite measured in step (a-2) is higher than the control When the concentration of the metabolite is higher than that in the subject, the subject evaluates that the optic neuropathy is more likely to be severe than the control subject,
If the metabolite measured in step (a-2) is a [B group] or [C group] metabolite, the concentration of the metabolite measured in step (a-2) is equal to that of the metabolite in the control subject. assessing that the subject is more likely to have a more severe optic neuropathy than the control when the concentration is lower than the concentration;
[3] The determination method according to [1] or [2] above, wherein the biological sample is a blood sample, a urine sample, an anterior aqueous humor sample, or a retinal sample.
[4] The above [1] to [3], wherein in step (a-1), fatty acid or L-acetylcarnitine is measured, and in step (a-2), L-acetylcarnitine is measured. The determination method according to any one of the above.
[5] The fatty acid is one or more fatty acids selected from the group consisting of oleic acid, palmitic acid, linoleic acid, stearic acid, docosahexaenoic acid, myristic acid, and arachidonic acid [ 4].
[6] Risk of developing optic neuropathy characterized by consisting of one or two or more metabolites selected from the following [I group], [A group] to [H group], and [J group], Or a biomarker for determining the presence or absence of onset,
If the metabolite is a [A group], [B group], or [E group] to [G group] metabolite, from those who are at high risk of developing optic neuropathy or who have developed optic neuropathy the concentration of the metabolite in the collected biological sample is higher than the concentration of the metabolite in a control subject who does not develop optic neuropathy;
If the metabolite is a [I group], [C group], [D group], [H group], or [J group] metabolite, the subject has a high risk of developing optic nerve damage, or develops optic nerve damage. the concentration of the metabolite in a biological sample collected from a subject with optic neuropathy is lower than the concentration of the metabolite in a control subject who does not develop optic neuropathy;
The biomarker for determination.
[Group I]
fatty acid, LPC (18:0), methyl palmitate, isopropyl myristate, LPE (22:6), LPC (17:0) sn-1, LPC (14:0), LPC (17:0) sn-2 , LPC (15:0), p-cresol sulfate, indoxyl sulfate, hydroxybutyric acid, hydroxybenzoic acid, N-phenylacetylglutamine, aconitic acid, pyridoxal, LPC (16:0), PC (36:2), PC (38:4), PC (40:6), proline betaine, ceramide (d18:1/16:0)
[Group A]
L-Acetylcarnitine, 9-Hexadecenoylcarnitine, Hydroxybutyrylcarnitine, Triacylglycerol (62:12), L-Octanoylcarnitine, Asparagine-Aspartic Acid Dipeptide , m-Aminobenzoic acid
[Group B]
Phosphatidylcholine (PC) (38:7), Phosphatidylglycerol (PG) (40:6), Lysophosphatidylcholine (lysoPC; LPC) (18:2), PG (32:0), S-adenosylmethionine (Adenosylmethionine), Phosphatidylethanolamine (PE) (42:8), PG (38:6)
[Group C]
PC (46:6), Sphingomyelin (SM) (d18:0/24:0), 8,8-diethoxy-2,6-dimethyl-2-octanol, Glycerophosphocholine (GPC), β - Lactosylceramide (d18:1/22:0), PC (44:9)
[Group D]
Monoacylglyceride (18:0/0:0/0:0), D-Glucose, Guanosine, Adenine, Adenosine, Glutathione, S-Formylglutathione, Methionine-Histidine Dipeptide [Group E]
3-sulfolactic acid, dimethylsulfone, ribonic acid, arabinonic acid, xylonic acid, L-hydroxyglutaric acid, hydroxycitric acid, glucaric acid, galactaric acid, octenoylcarnitine, betaine, L-acetylcarnitine, methylmalonylcarnitine, L- Carnitine, N,N-diethylethanolamine, L-cystine [Group F]
pyroglutamine, LPC (18:2) sn-1, lysophosphatidylethanolamine (lysoPE; LPE) (18:2), LPC (18:2) sn-2, androsterone sulfate, testosterone sulfate, phenol sulfate, lactic acid, D-glucose, hydroperoxylinoleic acid, PC (34:2), thiomorpholine-3-carboxylic acid, LPC (18:2), L-carnitine [G group]
Methyladenosine, succinyladenosine, dodecanoylcarnitine, O-(7-carboxyheptanoyl)carnitine, O-(8-carboxyoctanoyl)carnitine, O-(11-carboxyundecanoyl)carnitine, maleic acid, fumaric acid [ Group H]
Dichloromethane, glutathione, L-tyrosine, L-phenylalanine, 3-mercaptolactic acid, uridine, taurine, aminobutyric acid [J group]
Citric acid [7] A biomarker for determining the severity of optic neuropathy characterized by comprising one or more metabolites selected from [A group] to [D group] below,
If the metabolite is a [Group A] or [Group D] metabolite, the concentration of said metabolite in a biological sample collected from a subject with high severity of optic nerve damage is low in severity of optic nerve damage. higher than the concentration of the metabolite in
When the metabolite is a [B group] or [C group] metabolite, the concentration of said metabolite in a biological sample collected from a subject with high severity of optic nerve damage is low in severity of optic nerve damage. lower than the concentration of said metabolite in
The biomarker for determination.
[Group A]
L-Acetylcarnitine, 9-Hexadecenoylcarnitine, Hydroxybutyrylcarnitine, Triacylglycerol (62:12), L-Octanoylcarnitine, Asparagine-Aspartic Acid Dipeptide , m-Aminobenzoic acid
[Group B]
Phosphatidylcholine (PC) (38:7), Phosphatidylglycerol (PG) (40:6), Lysophosphatidylcholine (lysoPC; LPC) (18:2), PG (32:0), S-adenosylmethionine (Adenosylmethionine), Phosphatidylethanolamine (PE) (42:8), PG (38:6)
[Group C]
PC (46:6), Sphingomyelin (SM) (d18:0/24:0), 8,8-diethoxy-2,6-dimethyl-2-octanol, Glycerophosphocholine (GPC), β - Lactosylceramide (d18:1/22:0), PC (44:9)
[Group D]
Monoacylglyceride (18:0/0:0/0:0), D-glucose, guanosine, adenine, adenosine, glutathione, S-formylglutathione, methionine-histidine dipeptide [8] biological sample , a blood sample, a urine sample, an anterior aqueous humor sample, or a retinal sample.
[9] The biomarker for determination according to any one of [6] to [8] above, wherein the metabolite is fatty acid or L-acetylcarnitine.
[10] The above [ 9].
[11] A screening method for an agent for preventing the onset of optic neuropathy or a therapeutic agent for optic neuropathy, comprising the following steps (A) to (C).
(A) 1 selected from the following [Group I], [Group A] to [Group H], and [Group J] in a biological sample collected from a non-human animal with optic neuropathy administered with a test substance or measuring the concentration of two or more metabolites;
[Group I]
fatty acid, LPC (18:0), methyl palmitate, isopropyl myristate, LPE (22:6), LPC (17:0) sn-1, LPC (14:0), LPC (17:0) sn-2 , LPC (15:0), p-cresol sulfate, indoxyl sulfate, hydroxybutyric acid, hydroxybenzoic acid, N-phenylacetylglutamine, aconitic acid, pyridoxal, LPC (16:0), PC (36:2), PC (38:4), PC (40:6), proline betaine, ceramide (d18:1/16:0)
[Group A]
L-Acetylcarnitine, 9-Hexadecenoylcarnitine, Hydroxybutyrylcarnitine, Triacylglycerol (62:12), L-Octanoylcarnitine, Asparagine-Aspartic Acid Dipeptide , m-Aminobenzoic acid
[Group B]
Phosphatidylcholine (PC) (38:7), Phosphatidylglycerol (PG) (40:6), Lysophosphatidylcholine (lysoPC; LPC) (18:2), PG (32:0), S-adenosylmethionine (Adenosylmethionine), Phosphatidylethanolamine (PE) (42:8), PG (38:6)
[Group C]
PC (46:6), Sphingomyelin (SM) (d18:0/24:0), 8,8-diethoxy-2,6-dimethyl-2-octanol, Glycerophosphocholine (GPC), β - Lactosylceramide (d18:1/22:0), PC (44:9)
[Group D]
Monoacylglyceride (18:0/0:0/0:0), D-Glucose, Guanosine, Adenine, Adenosine, Glutathione, S-Formylglutathione, Methionine-Histidine Dipeptide [Group E]
3-sulfolactic acid, dimethylsulfone, ribonic acid, arabinonic acid, xylonic acid, L-hydroxyglutaric acid, hydroxycitric acid, glucaric acid, galactaric acid, octenoylcarnitine, betaine, L-acetylcarnitine, methylmalonylcarnitine, L- Carnitine, N,N-diethylethanolamine, L-cystine [Group F]
pyroglutamine, LPC (18:2) sn-1, lysophosphatidylethanolamine (lysoPE; LPE) (18:2), LPC (18:2) sn-2, androsterone sulfate, testosterone sulfate, phenol sulfate, lactic acid, D-glucose, hydroperoxylinoleic acid, PC (34:2), thiomorpholine-3-carboxylic acid, LPC (18:2), L-carnitine [G group]
Methyladenosine, succinyladenosine, dodecanoylcarnitine, O-(7-carboxyheptanoyl)carnitine, O-(8-carboxyoctanoyl)carnitine, O-(11-carboxyundecanoyl)carnitine, maleic acid, fumaric acid [ Group H]
Dichloromethane, glutathione, L-tyrosine, L-phenylalanine, 3-mercaptolactic acid, uridine, taurine, aminobutyric acid [J group]
citric acid (B) comparing the concentration of the metabolite measured in step (A) with the concentration of the metabolite in non-human animals with optic neuropathy to which the test substance is not administered;
(C) If the metabolites measured in step (A) are [A group], [B group], or [E group] to [G group] metabolites, the metabolites measured in step (A) When the concentration is lower than the concentration of metabolites in non-human animals with optic neuropathy to which the test substance is not administered, the test substance is evaluated to be effective in preventing the onset of optic neuropathy or treating optic neuropathy,
If the metabolite measured in step (A) is a [I group], [C group], [D group], [H group], or [J group] metabolite, the metabolism measured in step (A) When the concentration of the substance is higher than the concentration of the metabolite in non-human animals with optic neuropathy to which the test substance is not administered, the test substance is evaluated to be effective in preventing the onset of optic neuropathy or treating optic neuropathy. the step of
[12] The screening method of [11] above, wherein the biological sample is a blood sample, urine sample, anterior aqueous humor sample, or retinal sample.
[13] The screening method of [11] or [12] above, wherein in step (A), fatty acid or L-acetylcarnitine is measured.
[14] The above [ 13].

As another embodiment of the present invention,
The risk of developing optic nerve damage, or the development of optic nerve damage, including the step (a-1) of measuring the concentration of one or more metabolites selected from the metabolite group in a biological sample collected from a subject A method for determining the presence or absence of;
Severity of optic neuropathy, including the step (a-2) of measuring the concentration of one or more metabolites selected from the subject metabolite group in a biological sample collected from a subject developing optic neuropathy a method of determining the degree;
A biomarker for determining the risk of developing optic nerve damage, determining the presence or absence of developing optic nerve damage, or determining the severity of optic nerve damage, consisting of one or more metabolites selected from the metabolite group; ,
Onset of optic neuropathy, including the step (A) of measuring the concentration of one or more metabolites selected from the present metabolite group in a biological sample collected from an optic neuropathy non-human animal to which a test substance is administered A method for screening a prophylactic agent or a therapeutic agent for optic neuropathy,
A method for collecting data for diagnosing the risk of developing an optic neuropathy, or the presence or absence of the onset, including the above steps (a-1) to (c-1);
Including the above steps (a-1) to (c-1), and in step (c-1), the risk of developing optic nerve damage is high, or the possibility of developing optic nerve damage is evaluated ( Treatment to prevent the onset of optic neuropathy, or treatment to treat optic neuropathy (for example, eye drops of ocular hypotensive agents, nerve regeneration agents that promote regeneration or plasticity of RGC axons, etc.) for subjects who have been diagnosed) A method for diagnosing the risk of developing optic neuropathy, or the presence or absence of the onset, optionally including the step (p-1) of applying;
A method for collecting data for diagnosing the severity of optic nerve damage, including the above steps (a-2) to (c-2); and
Including the above steps (a-2) to (c-2), and furthermore, in the step (c-2), the subject evaluated (diagnosed) with a high possibility of aggravated optic nerve damage Diagnosis of the severity of optic neuropathy, optionally including a step (p-2) of administering treatment to treat the disorder (e.g. eye drops of an ocular hypotensive agent, a nerve regeneration agent that promotes regeneration or plasticity of RGC axons, etc.) method; or
One or two or more metabolites selected from the present metabolite group for use as a biomarker in a method for determining (diagnosing) the risk of developing optic neuropathy or the presence or absence of onset, wherein the metabolite is [A group], [B group], or [E group] to [G group] metabolites, biological samples collected from those who are at high risk of developing optic nerve damage or who have developed optic nerve damage. The concentration of the metabolite in the metabolite is higher than the concentration of the metabolite in controls who do not develop optic neuropathy, and the metabolite is [C group], [D group], or [H group] to [J group] If it is a metabolite of a metabolite that is low compared to the concentration of the metabolite; and
One or two or more metabolites selected from the present metabolite group for use as biomarkers in a method for determining (diagnosing) the severity of optic neuropathy, wherein the metabolite is [Group A] or [D group], the concentration of the metabolite in a biological sample collected from a subject with high severity of optic nerve damage is higher than the concentration of the metabolite in a subject with low severity of optic nerve damage. ,
When the metabolite is a [B group] or [C group] metabolite, the concentration of said metabolite in a biological sample collected from a subject with high severity of optic nerve damage is low in severity of optic nerve damage. said metabolite that is lower than the concentration of said metabolite in
A method for determining the effectiveness of a preventive or therapeutic agent (drug) for optic neuropathy, or a screening method for a candidate preventive agent (drug) or therapeutic agent (drug) for optic neuropathy, comprising the above steps (A) to (C). ;
can be mentioned.

According to the determination method or determination biomarker of the present invention, the risk of developing optic neuropathy, the presence or absence of the onset, and the severity of optic neuropathy can be determined with high accuracy. Appropriate treatment can be received in the post-early stage, which leads to prevention of onset of optic nerve damage and early treatment of optic nerve damage. In addition, the screening method of the present invention contributes to the development of prophylactic (protective) or therapeutic agents (drugs) for optic neuropathy.

FIG. 1A shows the expression levels of RBPMS (RNA binding protein with multiple splicing) in retinal samples for the CT group and three types of ONC groups (ONC group, 2nd day, ONC group, 4th day, and ONC group, 7th day). is analyzed by an indirect fluorescent antibody method. In the figure, "RBPMS" indicates an RBPMS fluorescence image, "DAPI" indicates a DAPI fluorescence image, and "Merge" indicates an image in which the RBPMS fluorescence image and the DAPI fluorescence image are superimposed. FIG. 1B is a diagram showing the results of measuring the number of RBPMS-positive retinal cells in retinal samples based on RBPMS fluorescence images for the CT group and the three types of ONC groups. "*" and "**" in the figure indicate statistically significant differences (P<0.05 and P<0.01), respectively. FIG. 1C is a diagram showing the results of analysis of the expression level of RBPMS in retinal samples by Western blotting. "NC" in the figure indicates a retinal sample from a right eye with crushed optic nerve, and "S" indicates a retinal sample from a left eye without crushed optic nerve. CT group (“CT” in the figure) and three ONC groups (ONC group on day 2 [ONC2 in the figure], ONC group on day 4 [ONC4 in the figure], and ONC group on day 7 [in the figure ONC7]), the concentration of the metabolite group (group A to D metabolites) in the retinal sample was quantified by mass spectrometry using a liquid chromatograph-mass spectrometer (LC-MS). FIG. 10 is a diagram showing a heat map created by performing hierarchical clustering analysis based on the quantitative results obtained. Four A group metabolites (L-acetylcarnitine, 9-hexadecenoylcarnitine, hydroxybutyrylcarnitine, and triacylglycerol [62:12]) in retinal samples for the CT group and the three ONC groups ) was determined by mass spectrometry using LC-MS. For the CT group and the above three ONC groups, the concentrations of the three A group metabolites (L-octanoylcarnitine, asparagine-aspartic acid dipeptide, and m-aminobenzoic acid) in the retinal samples were measured by LC-MS. It is a figure which shows the result quantified by the used mass spectrometry. Four B group metabolites (PC[38:7], PG[40:6], LPC[18:2], and PG[32:0 ]) is determined by mass spectrometry using LC-MS. Concentrations of three B group metabolites (S-adenosylmethionine, PE[42:8], and PG[38:6]) in retinal samples for the CT group and the three ONC groups were measured by LC- It is a figure which shows the result of having quantified by the mass spectrometry method using MS. Three C-group metabolites (PC[46:6], SM[d18:0/24:0], and 8,8-diethoxy-2, 6-dimethyl-2-octanol) is a diagram showing the results of quantifying the concentration by mass spectrometry using LC-MS. Concentrations of three C group metabolites (GPC, β-lactosylceramide [d18:1/22:0], and PC [44:9]) in retinal samples for the CT group and the three ONC groups is quantified by mass spectrometry using LC-MS. Levels of four D group metabolites (monoacylglycerides [18:0/0:0/0:0], D-glucose, guanosine, and adenine) in retinal samples for the CT group and the three ONC groups FIG. 4 shows the results of concentration quantification by mass spectrometry using LC-MS. Concentrations of four group D metabolites (adenosine, glutathione, S-formylglutathione, and methionine-histidine dipeptide) in retinal samples for the CT group and the above three ONC groups were analyzed by mass spectrometry using LC-MS. It is a figure which shows the result quantified by the method. Three metabolite groups (L-acetylcarnitine [Fig. 11A], PC [38:7] [Fig. 11B], and PG [40:6] [ FIG. 11C]) is a diagram showing the results of analysis of the distribution by mass spectrometry using IMS. For each group in FIGS. 11A-C, the lower panel is an optical microscope image of a retinal sample stained with hematoxylin and eosin (HE), the right panel is an enlarged optical microscope image of the boxed area in the lower panel, and the left panel is an optical microscope image. The figure is an IMS analysis image corresponding to the optical microscope image on the right. FIG. 2 is a diagram showing the results of quantifying the concentration of the subject metabolite group (L-acetylcarnitine) in the anterior aqueous humor samples of a healthy group and a glaucoma group by mass spectrometry using LC-MS. The L-acetylcarnitine concentration level on the vertical axis indicates the peak intensity of L-acetylcarnitine. “***” in the figure indicates that there is a statistically significant difference (P=0.0001352). FIG. 13A is a diagram showing the results of quantifying the concentration of the present metabolite group (3-sulfolactic acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “***” in FIG. 13A indicates a statistically significant difference (P=0.0003). FIG. 13B is a diagram showing the results of quantifying the concentration of the subject metabolite group (3-sulfolactic acid) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. “*” and “***” in FIG. 13B indicate statistically significant differences (P=0.0242 and P<0.0001) by the Steel-Dwass test, respectively. Each black circle (●) in the figure indicates each subject (same below). The 3-sulfolactic acid concentration level on the vertical axis in the figure indicates the peak intensity of 3-sulfolactic acid. FIG. 14A is a diagram showing the results of quantifying the concentration of the present metabolite group (dimethylsulfone) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 14B is a diagram showing the results of quantifying the concentration of the subject metabolite group (dimethylsulfone) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. . "###" and "***" in the figure indicate that there is a statistically significant difference (P=0.0004 and P<0.0001) by the Steel-Dwass test, respectively. The dimethylsulfone concentration level on the vertical axis in the figure indicates the peak intensity of dimethylsulfone. FIG. 15A shows the results of quantifying the concentration of this metabolite group (ribonic acid, arabinonic acid, or xylonic acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 4 is a diagram showing; FIG. 15B shows the concentration of this metabolite group (ribonic acid, arabinonic acid, or xylonic acid) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group, quantified by mass spectrometry using LC-MS. FIG. 10 is a diagram showing the results of the experiment. "***" in the figure indicates that there is a statistically significant difference (P<0.0001) by the Steel-Dwass test. The concentration level of ribonic acid, arabinonic acid, or xylonic acid on the vertical axis in the figure indicates the peak intensity of ribonic acid, arabinonic acid, or xylonic acid. FIG. 16A is a diagram showing the results of quantifying the concentration of this metabolite group (L-hydroxyglutaric acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. . FIG. 16B shows the results of quantifying the concentrations of this metabolite group (L-hydroxyglutarate) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. It is a diagram. "*" and "***" in the figure indicate that there is a statistically significant difference (P=0.0450 and P<0.0001) by the Steel-Dwass test, respectively. The L-hydroxyglutaric acid concentration level on the vertical axis in the figure indicates the peak intensity of L-hydroxyglutaric acid. FIG. 17A is a diagram showing the results of quantifying the concentration of the present metabolite group (hydroxycitric acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 17B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (hydroxycitric acid) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. "**" and "***" in the figure indicate that there is a statistically significant difference (P = 0.0135 and P < 0.0001) by the Steel-Dwass test, respectively. The hydroxycitric acid concentration level on the vertical axis in the figure indicates the peak intensity of hydroxycitric acid. FIG. 18A is a diagram showing the results of quantifying the concentration of the subject metabolite group (glucaric acid or galactaric acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. . FIG. 18B shows the results of quantifying the concentration of the subject metabolite group (glucaric acid or galactaric acid) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. It is a diagram. "*" and "***" in the figure indicate statistically significant differences (P=0.0111 and P<0.0001) by the Steel-Dwass test, respectively. The glucaric acid or galactaric acid concentration level on the vertical axis in the figure indicates the peak intensity of glucaric acid or galactaric acid. FIG. 19A is a diagram showing the results of quantifying the concentration of this metabolite group (octenoylcarnitine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 19A indicates a statistically significant difference (P=0.0284). FIG. 19B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (octenoylcarnitine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. “*” and “**” in FIG. 19B indicate that there is a statistically significant difference (P=0.0282 and P=0.0084) by the Steel-Dwass test, respectively. The octenoylcarnitine concentration level on the vertical axis in the figure indicates the peak intensity of octenoylcarnitine. FIG. 20A is a diagram showing the results of quantifying the concentration of the subject metabolite group (betaine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 19B is a diagram showing the results of quantifying the concentration of the subject metabolite group (betaine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The betaine concentration level on the vertical axis in the figure indicates the peak intensity of betaine. FIG. 21A is a diagram showing the results of quantifying the concentrations of this metabolite group (L-acetylcarnitine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “***” in FIG. 21A indicates a statistically significant difference (P=0.0003). FIG. 21B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (L-acetylcarnitine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. “*” and “***” in FIG. 21B indicate statistically significant differences (P=0.0289 and P<0.0001) by Tukey-Kramer test, respectively. The L-acetylcarnitine concentration level on the vertical axis in the figure indicates the peak intensity of L-acetylcarnitine. FIG. 22A is a diagram showing the results of quantifying the concentrations of this metabolite group (methylmalonylcarnitine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 22A indicates a statistically significant difference (P=0.01). FIG. 22B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (methylmalonylcarnitine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. “***” in FIG. 22B indicates that there is a statistically significant difference (P=0.0006) by the Steel-Dwass test. The methylmalonylcarnitine concentration level on the vertical axis in the figure indicates the peak intensity of methylmalonylcarnitine. FIG. 23A is a diagram showing the results of quantifying the concentrations of this metabolite group (L-carnitine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 23A indicates a statistically significant difference (P=0.0023). FIG. 23B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (L-carnitine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. “*” and “***” in FIG. 23B indicate statistically significant differences (P=0.0196 and P=0.0004) by Tukey-Kramer test, respectively. The L-carnitine concentration level on the vertical axis in the figure indicates the peak intensity of L-carnitine. FIG. 24A is a diagram showing the results of quantifying the concentrations of the subject metabolite group (N,N-diethylethanolamine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. is. FIG. 24B shows the results of quantifying the concentration of this metabolite group (N,N-diethylethanolamine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. It is a figure which shows. "***" in the figure indicates that there is a statistically significant difference (P<0.0001) by the Steel-Dwass test. The N,N-diethylethanolamine concentration level on the vertical axis in the figure indicates the peak intensity of N,N-diethylethanolamine. FIG. 25A is a diagram showing the results of quantifying the concentrations of this metabolite group (L-cystine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 25B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (L-cystine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. "*" and "***" in the figure indicate statistically significant differences (P=0.0346 and P<0.0001) by the Steel-Dwass test, respectively. The L-cystine concentration level on the vertical axis in the figure indicates the peak intensity of L-cystine. FIG. 26A is a diagram showing the results of quantifying the concentrations of this metabolite group (dichloromethane) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 26B is a diagram showing the results of quantifying the concentration of the subject metabolite group (dichloromethane) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “*” in FIG. 26B indicates that there is a statistically significant difference (P=0.0495) by the Steel-Dwass test. The dichloromethane concentration level on the vertical axis in the figure indicates the peak intensity of dichloromethane. FIG. 27A is a diagram showing the results of quantifying the concentration of this metabolite group (glutathione) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 27B is a diagram showing the results of quantifying the concentration of this metabolite group (glutathione) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “###” and “***” in the figure indicate statistically significant difference (P=0.0005 and P<0.0001) by Tukey-Kramer test, respectively. The glutathione concentration level on the vertical axis in the figure indicates the peak intensity of glutathione. FIG. 28A is a diagram showing the results of quantifying the concentration of the subject metabolite group (L-tyrosine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 28B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (L-tyrosine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. The L-tyrosine concentration level on the vertical axis in the figure indicates the peak intensity of L-tyrosine. FIG. 29A is a diagram showing the results of quantifying the concentrations of this metabolite group (L-phenylalanine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 29B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (L-phenylalanine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. The L-phenylalanine concentration level on the vertical axis in the figure indicates the peak intensity of L-phenylalanine. FIG. 30A is a diagram showing the results of quantifying the concentration of the present metabolite group (3-mercaptolactic acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “***” in FIG. 30A indicates a statistically significant difference (P<0.0001). FIG. 30B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (3-mercaptolactic acid) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. “##” and “**” in FIG. 30B indicate that there is a statistically significant difference (P=0.0027 and P=0.0016) by the Steel-Dwass test, respectively. The 3-mercaptolactic acid concentration level on the vertical axis in the figure indicates the peak intensity of 3-mercaptolactic acid. FIG. 31A is a diagram showing the results of quantifying the concentration of the present metabolite group (uridine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 31B is a diagram showing the results of quantifying the concentration of the present metabolite group (uridine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “**” and “***” in the figure indicate statistically significant differences (P=0.0029 and P<0.0001) by Tukey-Kramer test, respectively. The uridine concentration level on the vertical axis in the figure indicates the peak intensity of uridine. FIG. 32A is a diagram showing the results of quantifying the concentrations of the subject metabolite group (taurine) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 32A indicates a statistically significant difference (P=0.0018). FIG. 32B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (taurine) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “*” and “***” in FIG. 32B indicate statistically significant differences (P=0.0443 and P=0.0011) by Tukey-Kramer test, respectively. The taurine concentration level on the vertical axis in the figure indicates the peak intensity of taurine. FIG. 33A is a diagram showing the results of quantifying the concentration of the subject metabolite group (aminobutyric acid) in the anterior aqueous humor samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. “***” in FIG. 33A indicates a statistically significant difference (P<0.0001). FIG. 33B is a diagram showing the results of quantifying the concentration of the subject metabolite group (aminobutyric acid) in the anterior aqueous humor samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. . “***” in FIG. 33B indicates that there is a statistically significant difference (P<0.0001) by the Steel-Dwass test. The aminobutyric acid concentration level on the vertical axis in the figure indicates the peak intensity of aminobutyric acid. FIG. 34A is a diagram showing the results of quantifying the concentrations of the subject metabolite group (pyroglutamine) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 34B is a diagram showing the results of quantifying the concentration of the subject metabolite group (pyroglutamine) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “#” and “*” in FIG. 34B indicate statistically significant differences (P=0.015 and P=0.0231) by Steel-Dwass test, respectively. The pyroglutamine concentration level on the vertical axis in the figure indicates the peak intensity of pyroglutamine. FIG. 35A is a diagram showing the results of quantifying the concentration of this metabolite group (LPC (18:2) sn-1) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. is. “*” in FIG. 35A indicates a statistically significant difference (P=0.0383). Figure 35B shows the results of quantifying the concentration of the subject metabolite group (LPC (18:2) sn-1) in plasma samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. It is a figure which shows. The LPC(18:2) sn-1 concentration level on the vertical axis in the figure indicates the peak intensity of LPC(18:2) sn-1. FIG. 36A is a diagram showing the results of quantifying the concentration of the present metabolite group (LPE (18:2)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 36B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (LPE (18:2)) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPE (18:2) concentration level on the vertical axis in the figure indicates the peak intensity of LPE (18:2). FIG. 37A is a diagram showing the results of quantifying the concentration of the present metabolite group (LPC(18:2)sn-2) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. is. Figure 37B shows the results of quantifying the concentration of the subject metabolite group (LPC (18:2) sn-2) in plasma samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. It is a figure which shows. The LPC(18:2) sn-2 concentration level on the vertical axis in the figure indicates the peak intensity of LPC(18:2) sn-2. FIG. 38A is a diagram showing the results of quantifying the concentrations of the present metabolite group (androsterone sulfate) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 38B is a diagram showing the results of quantifying the concentration of the subject metabolite group (androsterone sulfate) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The androsterone sulfate concentration level on the vertical axis in the figure indicates the peak intensity of androsterone sulfate. FIG. 39A is a diagram showing the results of quantifying the concentration of the subject metabolite group (testosterone sulfate) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 39B is a diagram showing the results of quantifying the concentration of the present metabolite group (testosterone sulfate) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The testosterone sulfate concentration level on the vertical axis in the figure indicates the peak intensity of testosterone sulfate. FIG. 40A is a diagram showing the results of quantifying the concentration of the subject metabolite group (phenol sulfate) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 40B is a diagram showing the results of quantifying the concentration of the subject metabolite group (phenol sulfate) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The phenol-sulfuric acid concentration level on the vertical axis in the figure indicates the peak intensity of phenol-sulfuric acid. FIG. 41A is a diagram showing the results of quantifying the concentration of the subject metabolite group (lactic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 41B is a diagram showing the results of quantifying the concentration of the subject metabolite group (lactic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The lactic acid concentration level on the vertical axis in the figure indicates the peak intensity of lactic acid. FIG. 42A is a diagram showing the results of quantifying the concentration of the subject metabolite group (D-glucose) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 42B is a diagram showing the results of quantifying the concentration of the present metabolite group (D-glucose) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The D-glucose concentration level on the vertical axis in the figure indicates the peak intensity of D-glucose. FIG. 43A is a diagram showing the results of quantifying the concentration of the subject metabolite group (hydroperoxylinoleic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 43B is a diagram showing the results of quantifying the concentrations of this metabolite group (hydroperoxylinoleic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. . The hydroperoxylinoleic acid concentration level on the vertical axis in the figure indicates the peak intensity of hydroperoxylinoleic acid. FIG. 44A is a diagram showing the results of quantifying the concentration of the present metabolite group (PC(34:2)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 44B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (PC(34:2)) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The PC(34:2) concentration level on the vertical axis in the figure indicates the peak intensity of PC(34:2). FIG. 45A is a diagram showing the results of quantifying the concentration of the subject metabolite group (thiomorpholine-3-carboxylic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. . FIG. 45B shows the results of quantifying the concentrations of this metabolite group (thiomorpholine-3-carboxylic acid) in plasma samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. It is a diagram. The thiomorpholine-3-carboxylic acid concentration level on the vertical axis in the figure indicates the peak intensity of thiomorpholine-3-carboxylic acid. FIG. 46A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC (18:2)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 46B is a diagram showing the results of quantifying the concentration of the present metabolite group (LPC (18:2)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPC (18:2) concentration level on the vertical axis in the figure indicates the peak intensity of LPC (18:2). FIG. 47A is a diagram showing the results of quantifying the concentration of the subject metabolite group (L-carnitine) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 47B is a diagram showing the results of quantifying the concentration of the present metabolite group (L-carnitine) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The L-carnitine concentration level on the vertical axis in the figure indicates the peak intensity of L-carnitine. FIG. 48A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC (18:0)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 48B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (LPC(18:0)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPC (18:0) concentration level on the vertical axis in the figure indicates the peak intensity of LPC (18:0). FIG. 49A is a diagram showing the results of quantifying the concentrations of the subject metabolite group (methyl palmitate or isopropyl myristate) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. . “***” in FIG. 49A indicates that there is a statistically significant difference (P<0.0001). Figure 49B shows the results of quantifying the concentration of the subject metabolite group (methyl palmitate or isopropyl myristate) in plasma samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. It is a diagram. “***” in FIG. 49B indicates that there is a statistically significant difference (P<0.0001) by the Tukey-Kramer test. The methyl palmitate or isopropyl myristate concentration level on the vertical axis in the figure indicates the peak intensity of methyl palmitate or isopropyl myristate. FIG. 50A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPE (22:6)) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 50B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (LPE (22:6)) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPE (22:6) concentration level on the vertical axis in the figure indicates the peak intensity of LPE (22:6). FIG. 51A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC(17:0)sn-1) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. is. Figure 51B shows the results of quantifying the concentration of the subject metabolite group (LPC (17:0) sn-1) in plasma samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. It is a figure which shows. The LPC(17:0) sn-1 concentration level on the vertical axis in the figure indicates the peak intensity of LPC(17:0) sn-1. FIG. 52A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC (14:0)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 52B is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC(14:0)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPC(14:0) concentration level on the vertical axis in the figure indicates the peak intensity of LPC(14:0). FIG. 53A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC(17:0)sn-2) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. is. Figure 53B shows the results of quantifying the concentration of the subject metabolite group (LPC (17:0) sn-2) in plasma samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. It is a figure which shows. The LPC(17:0)sn-2 concentration level on the vertical axis in the figure indicates the peak intensity of LPC(17:0)sn-2. FIG. 54A is a diagram showing the results of quantifying the concentration of the subject metabolite group (LPC (15:0)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 54A indicates a statistically significant difference (P=0.0497). FIG. 54B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (LPC(15:0)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPC (15:0) concentration level on the vertical axis in the figure indicates the peak intensity of LPC (15:0). FIG. 55A is a diagram showing the results of quantifying the concentration of the subject metabolite group (oleic acid) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 55A indicates a statistically significant difference (P=0.007). FIG. 55B is a diagram showing the results of quantifying the concentration of the present metabolite group (oleic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “*” in FIG. 55B indicates that there is a statistically significant difference (P=0.0101) by the Tukey-Kramer test. The oleic acid concentration level on the vertical axis in the figure indicates the peak intensity of oleic acid. FIG. 56A is a diagram showing the results of quantifying the concentration of the subject metabolite group (p-cresol sulfate) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 56B is a diagram showing the results of quantifying the concentration of the subject metabolite group (p-cresol sulfate) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. . The p-cresol sulfuric acid concentration level on the vertical axis in the figure indicates the peak intensity of p-cresol sulfuric acid. FIG. 57A is a diagram showing the results of quantifying the concentration of the present metabolite group (palmitic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 57A indicates a statistically significant difference (P=0.0093). FIG. 57B is a diagram showing the results of quantifying the concentration of the subject metabolite group (palmitic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “*” in FIG. 57B indicates that there is a statistically significant difference (P=0.0251) by the Tukey-Kramer test. The palmitic acid concentration level on the vertical axis in the figure indicates the peak intensity of palmitic acid. FIG. 58A is a diagram showing the results of quantifying the concentration of the subject metabolite group (linoleic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 58B is a diagram showing the results of quantifying the concentration of the present metabolite group (linoleic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The linoleic acid concentration level on the vertical axis in the figure indicates the peak intensity of linoleic acid. FIG. 59A is a diagram showing the results of quantifying the concentration of the subject metabolite group (stearic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 59A indicates that there is a statistically significant difference (P=0.0063). FIG. 59B is a diagram showing the results of quantifying the concentration of the present metabolite group (stearic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “**” in FIG. 59B indicates that there is a statistically significant difference (P=0.0083) by the Tukey-Kramer test. The stearic acid concentration level on the vertical axis in the figure indicates the peak intensity of stearic acid. FIG. 60A is a diagram showing the results of quantifying the concentration of the subject metabolite group (indoxyl sulfate) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 60B is a diagram showing the results of quantifying the concentration of the present metabolite group (indoxyl sulfate) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The indoxyl sulfuric acid concentration level on the vertical axis in the figure indicates the peak intensity of indoxyl sulfuric acid. FIG. 61A is a diagram showing the results of quantifying the concentration of the subject metabolite group (hydroxybutyric acid) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 61A indicates a statistically significant difference (P=0.0209). FIG. 61B is a diagram showing the results of quantifying the concentration of the subject metabolite group (hydroxybutyric acid) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “*” in FIG. 61B indicates that there is a statistically significant difference (P=0.026) by the Steel-Dwass test. The hydroxybutyric acid concentration level on the vertical axis in the figure indicates the peak intensity of hydroxybutyric acid. FIG. 62A is a diagram showing the results of quantifying the concentration of the subject metabolite group (docosahexaenoic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 62A indicates a statistically significant difference (P=0.0209). FIG. 62B is a diagram showing the results of quantifying the concentration of the present metabolite group (docosahexaenoic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The docosahexaenoic acid concentration level on the vertical axis in the figure indicates the peak intensity of docosahexaenoic acid. FIG. 63A is a diagram showing the results of quantifying the concentration of the present metabolite group (myristic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 63A indicates a statistically significant difference (P=0.0086). FIG. 63B is a diagram showing the results of quantifying the concentration of the subject metabolite group (myristic acid) in plasma samples of the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. “*” in FIG. 63B indicates that there is a statistically significant difference (P=0.0159) by the Steel-Dwass test. The myristic acid concentration level on the vertical axis in the figure indicates the peak intensity of myristic acid. FIG. 64A is a diagram showing the results of quantifying the concentration of the subject metabolite group (hydroxybenzoic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 64B is a diagram showing the results of quantifying the concentrations of this metabolite group (hydroxybenzoic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The hydroxybenzoic acid concentration level on the vertical axis in the figure indicates the peak intensity of hydroxybenzoic acid. FIG. 65A is a diagram showing the results of quantifying the concentrations of the subject metabolite group (N-phenylacetylglutamine) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 65B is a diagram showing the results of quantifying the concentration of the subject metabolite group (N-phenylacetylglutamine) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. The N-phenylacetylglutamine concentration level on the vertical axis in the figure indicates the peak intensity of N-phenylacetylglutamine. FIG. 66A is a diagram showing the results of quantifying the concentration of the subject metabolite group (arachidonic acid) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 66A indicates a statistically significant difference (P=0.0089). FIG. 66B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (arachidonic acid) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “*” in FIG. 66B indicates that there is a statistically significant difference (P=0.0293) by the Steel-Dwass test. The arachidonic acid concentration level on the vertical axis in the figure indicates the peak intensity of arachidonic acid. FIG. 67A is a diagram showing the results of quantifying the concentration of the present metabolite group (aconitic acid) in plasma samples of the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 67B is a diagram showing the results of quantifying the concentration of the subject metabolite group (aconitic acid) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The aconitic acid concentration level in the figure indicates the peak intensity of aconitic acid. FIG. 68A is a diagram showing the results of quantifying the concentration of the subject metabolite group (pyridoxal) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 68B is a diagram showing the results of quantifying the concentration of the subject metabolite group (pyridoxal) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The pyridoxal concentration level on the vertical axis in the figure indicates the peak intensity of pyridoxal. FIG. 69A is a diagram showing the results of quantifying the concentration of the present metabolite group (LPC (16:0)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 69B is a diagram showing the results of quantifying the concentration of the present metabolite group (LPC(16:0)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The LPC (16:0) concentration level on the vertical axis in the figure indicates the peak intensity of LPC (16:0). FIG. 70A is a diagram showing the results of quantifying the concentration of the subject metabolite group (PC(36:2)) in plasma samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. FIG. 70B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (PC(36:2)) in plasma samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The PC (36:2) concentration level on the vertical axis in the figure indicates the peak intensity of PC (36:2). FIG. 71A is a diagram showing the results of quantifying the concentration of the subject metabolite group (PC(38:4)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 71A indicates a statistically significant difference (P=0.0472). FIG. 71B is a diagram showing the results of quantifying the concentration of the present metabolite group (PC(38:4)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The PC(38:4) concentration level on the vertical axis in the figure indicates the peak intensity of PC(38:4). FIG. 72A is a diagram showing the results of quantifying the concentration of the subject metabolite group (PC (40:6)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 72B is a diagram showing the results of quantifying the concentrations of the subject metabolite group (PC (40:6)) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. is. The PC (40:6) concentration level on the vertical axis in the figure indicates the peak intensity of PC (40:6). FIG. 73A is a diagram showing the results of quantifying the concentration of the subject metabolite group (proline betaine) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 73B is a diagram showing the results of quantifying the concentration of the subject metabolite group (proline betaine) in plasma samples of the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The proline betaine concentration level on the vertical axis in the figure indicates the peak intensity of proline betaine. FIG. 74A shows the results of quantifying the concentration of the subject metabolite group (ceramide (d18:1/16:0)) in plasma samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. It is a diagram. FIG. 74B shows the concentrations of the metabolite group (ceramide (d18:1/16:0)) in plasma samples of the healthy group, NTG group, and POAG group, which were quantified by mass spectrometry using LC-MS. It is a figure which shows a result. The ceramide (d18:1/16:0) concentration level on the vertical axis in the figure indicates the peak intensity of ceramide (d18:1/16:0). FIG. 75A is a diagram showing the results of quantifying the concentrations of this metabolite group (methyladenosine) in urine samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 75A indicates a statistically significant difference (P=0.0018). FIG. 75B is a diagram showing the results of quantifying the concentration of the subject metabolite group (methyladenosine) in urine samples for the healthy group, NTG group and POAG group by mass spectrometry using LC-MS. “***” in FIG. 75B indicates that there is a statistically significant difference (P=0.0007) by the Tukey-Kramer test. The methyladenosine concentration level on the vertical axis in the figure indicates the peak intensity of methyladenosine. FIG. 76A is a diagram showing the results of quantifying the concentration of the subject metabolite group (succinyladenosine) in urine samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. “**” in FIG. 76A indicates a statistically significant difference (P=0.0078). FIG. 76B is a diagram showing the results of quantifying the concentrations of this metabolite group (succinyladenosine) in urine samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. “**” in FIG. 76B indicates that there is a statistically significant difference (P=0.0028) by the Steel-Dwass test. The succinyladenosine concentration level on the vertical axis in the figure indicates the peak intensity of succinyladenosine. FIG. 77A is a diagram showing the results of quantifying the concentration of the subject metabolite group (dodecanoylcarnitine) in urine samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 77A indicates a statistically significant difference (P=0.0123). FIG. 77B is a diagram showing the results of quantifying the concentrations of this metabolite group (dodecanoylcarnitine) in urine samples for the healthy group, NTG group, and POAG group by mass spectrometry using LC-MS. The dodecanoylcarnitine concentration level on the vertical axis in the figure indicates the peak intensity of dodecanoylcarnitine. FIG. 78A shows the results of quantifying the concentrations of this metabolite group (O-(7-carboxyheptanoyl)carnitine) in urine samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. It is a diagram. “*” in the figure indicates that there is a statistically significant difference (P=0.0204). FIG. 78B shows the concentration of this metabolite group (O-(7-carboxyheptanoyl)carnitine) in urine samples for the healthy group, NTG group, and POAG group, quantified by mass spectrometry using LC-MS. It is a figure which shows a result. The O-(7-carboxyheptanoyl)carnitine concentration level on the vertical axis in the figure indicates the peak intensity of O-(7-carboxyheptanoyl)carnitine. FIG. 79A shows the results of quantifying the concentrations of this metabolite group (O-(8-carboxyoctanoyl)carnitine) in urine samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. It is a diagram. “**” in FIG. 79A indicates a statistically significant difference (P=0.0035). FIG. 80B shows the concentration of this metabolite group (O-(8-carboxyoctanoyl)carnitine) in urine samples for the healthy group, NTG group, and POAG group, quantified by mass spectrometry using LC-MS. It is a figure which shows a result. “*” in FIG. 80B indicates that there is a statistically significant difference (P=0.0105) by the Steel-Dwass test. The O-(8-carboxyoctanoyl)carnitine concentration level on the vertical axis in the figure indicates the peak intensity of O-(8-carboxyoctanoyl)carnitine. Figure 80A shows the results of quantifying the concentration of the subject metabolite group (O-(11-carboxyundecanoyl)carnitine) in urine samples for the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 4 is a diagram showing; “**” in FIG. 80A indicates a statistically significant difference (P=0.0091). FIG. 80B shows the concentrations of this metabolite group (O-(11-carboxyundecanoyl)carnitine) in urine samples for the healthy group, NTG group, and POAG group, quantified by mass spectrometry using LC-MS. FIG. 10 is a diagram showing the results of the experiment. The O-(11-carboxyundecanoyl)carnitine concentration level on the vertical axis in the figure indicates the peak intensity of O-(11-carboxyundecanoyl)carnitine. FIG. 81A is a diagram showing the results of quantifying the concentration of the subject metabolite group (maleic acid or fumaric acid) in urine samples from a healthy group and a glaucoma group by mass spectrometry using LC-MS. “*” in FIG. 81A indicates statistically significant difference (P=0.0458). FIG. 81B is a diagram showing the results of quantifying the concentration of the present metabolite group (maleic acid or fumaric acid) in urine samples for the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. be. The maleic acid or fumaric acid concentration level on the vertical axis in the figure indicates the peak intensity of maleic acid or fumaric acid. FIG. 82A is a diagram showing the results of quantifying the concentration of the present metabolite group (citric acid) in urine samples from the healthy group and the glaucoma group by mass spectrometry using LC-MS. FIG. 82B is a diagram showing the results of quantifying the concentration of the present metabolite group (citric acid) in urine samples from the healthy group, the NTG group, and the POAG group by mass spectrometry using LC-MS. The citric acid concentration level on the vertical axis in the figure indicates the peak intensity of citric acid.

As a method for determining the risk of developing optic neuropathy or the presence or absence of the development of optic neuropathy of the present invention, one or two or more selected from the following [A group] to [J group] in a biological sample collected from a subject. Step (a-1) of measuring and optionally quantifying the concentration of metabolites (hereinafter sometimes referred to as "metabolite concentration in the subject"); Step (b-1) of comparing with the concentration of the metabolite in the control (hereinafter sometimes referred to as "concentration of the metabolite in the control"); A group], [B group], or [E group] to [G group] metabolites, when the "metabolite concentration in the subject" is higher than the "metabolite concentration in the control", the subject has a high risk of developing optic neuropathy, or is likely to have developed optic neuropathy, and the metabolites measured in step (a-1) are the following [C group], [D group] , or [H group] to [J group] metabolites, when the "metabolite concentration in the subject" is lower than the "metabolite concentration in the control", the subject is at risk of developing optic neuropathy or high possibility of developing optic neuropathy (c-1); There is no particular limitation as long as it is
In addition, as a method for determining the severity of optic nerve damage according to the present invention, one or Step (a-2) of measuring the concentration of two or more metabolites (hereinafter sometimes referred to as "metabolite concentration in optic nerve impaired subject") and quantifying if necessary; "metabolite concentration in optic nerve impaired subject ” with the concentration of the metabolite in a control subject developing optic nerve damage (hereinafter sometimes referred to as “metabolite concentration in a control subject with optic nerve damage”); and step (a If the metabolites measured in -2) are the following [A group] or [D group] metabolites, the "concentration of metabolites in optic neuropathy subjects" is higher than the "concentration of metabolites in optic neuropathy controls" When it is high, the subject evaluates that the optic neuropathy is likely to be more severe than the control subject, and the metabolite measured in step (a-2) is [B group] or [C group] metabolism If it is a substance, when the "metabolite concentration in the optic neuropathy subject" is lower than the "metabolite concentration in the optic neuropathy control subject", the subject may have more severe optic neuropathy than the control subject. It is not particularly limited as long as it includes the steps (a-2) to (c-2) of the step evaluated as high (hereinafter sometimes referred to as "determination method 2") (determination method 1 and determination Method 2 may be collectively referred to hereinafter as the “determination method”). This determination method is a method for assisting a doctor in diagnosing the risk of developing optic nerve damage or diagnosing the presence or absence of the onset of optic nerve damage, and does not include a diagnostic act by a doctor.
[Group A]
L-acetylcarnitine (HMDB ID number; HMDB0000201), 9-hexadecenoylcarnitine (HMDB ID number; HMDB0013207), hydroxybutyrylcarnitine (HMDB ID number; HMDB0013127), triacylglycerol (62:12), L- Octanoylcarnitine (HMDB ID number; HMDB0000791), asparagine-aspartic acid dipeptide (HMDB ID number; HMDB0028727), m-aminobenzoic acid (HMDB ID number; HMDB0001891)
[Group B]
PC (38:7) (CHEBI ID No.; CHEBI:64498), PG (40:6), LPC (18:2) (HMDB ID No.; HMDB0010386), PG (32:0) (HMDB ID No.; HMDB0010570) , S-adenosylmethionine (HMDB ID number; HMDB0001185), PE (42:8), PG (38:6)
[Group C]
PC (46:6), SM (d18:0/24:0) (HMDB ID number; HMDB0012094), 8,8-diethoxy-2,6-dimethyl-2-octanol (HMDB ID number; HMDB0034557), GPC ( HMDB ID number; HMDB0000086), β-lactosylceramide (d18:1/22:0) (HMDB ID number; HMDB0011594), PC (44:9)
[Group D]
monoacylglyceride (18:0/0:0/0:0) (HMDB ID number; HMDB0011131), D-glucose (HMDB ID number; HMDB0000122), guanosine (HMDB ID number; HMDB0000133), adenine (HMDB ID number; HMDB0000034), adenosine (HMDB ID number; HMDB0000050), glutathione (HMDB ID number; HMDB0000125), S-formylglutathione (HMDB ID number; HMDB0001550), methionine-histidine dipeptide (HMDB ID number; HMDB0028975)
[Group E]
3-Sulfolactic acid (HMDB ID number; HMDB0060176), Dimethyl sulfone (HMDB0004983), Ribonic acid (HMDB ID number; HMDB0000867), Arabinonic acid (HMDB ID number HMDB0000539), Xylonate (HMDB ID number; HMDB0060256), L-Hydroxyglutaric acid (HMDB ID number; HMDB0000694), Hydroxycitric acid (HMDB ID number; HMDB0031159), glucar Glucaric acid (HMDB ID number; HMDB0000663), Galactaric acid (HMDB ID number; HMDB0000639), Octenoylcarnitine (HMDB ID number; HMDB0013324), Betaine (HMDB ID number; HMDB0000043), L-Acetylcarnitine (HMDB ID number; HMDB0000201), Methylmalonylcarnitine (HMDB ID number; HMDB0013133), L-Carnitine (HMDB ID number; HMDB0000062), N,N- Diethylethanolamine (HMDB ID number; HMDB0033971), L-cystine (HMDB ID number; HMDB0000192)
[Group F]
Pyroglutamine (HMDB ID number; HMDB0000079), LPC (18:2) sn-1 (HMDB ID number; HMDB0010386), LPE (18:2) (HMDB ID number; HMDB0011507), LPC (18:2) sn-2 (HMDB ID number; HMDB0010386), Androsterone sulfate (HMDB ID number; HMDB0002759), Testosterone sulfate (HMDB ID number; HMDB0002833), Phenol sulphate (HMDB ID number; HMDB0060015), Lactic acid (HMDB ID number; HMDB0000190), D-glucose (HMDB ID number; HMDB0000122), Hydroperoxylinoleic acid (HMDB ID number; HMDB0004706), PC (34:2) (HMDB ID No.; HMDB0007880), Thiomorpholine-3-carboxylate (HMDB ID No.; HMDB0059611), LPC (18:2) (HMDB ID No.; HMDB0010386), L-Carnitine ( HMDB ID number; HMDB0000062)
[Group G]
Methyladenosine (HMDB ID number; HMDB0003331), Succinyladenosine (HMDB ID number; HMDB0000912), Dodecanoylcarnitine (HMDB ID number; HMDB0002250), O-(7-carboxyheptanoyl ]) Carnitine, O-(8-carboxyoctanoyl) carnitine, O-(11-carboxyundecanoyl) carnitine, Maleic acid (HMDB ID number; HMDB0000176), fumaric acid ( Fumaric acid) (HMDB0000134)
[Group H]
Dichloromethane (HMDB ID number; HMDB0031548), glutathione (GSH; Glutathione) (HMDB ID number; HMDB0000125), L-Tyrosine (HMDB ID number; HMDB0000158), L-phenylalanine (HMDB ID number HMDB0000159), 3-Mercaptolactic acid (HMDB ID number; HMDB0002127), Uridine (HMDB ID number; HMDB0000296), Taurine (HMDB ID number; HMDB0000251), Aminobutyric acid (HMDB ID number; HMDB0000112)
[Group I]
LPC (18:0) (HMDB ID No.; HMDB0010384), Methyl palmitic acid (HMDB ID No.; HMDB0061859), Isopropyl myristate (HMDB ID No.; HMDB0040392), LPE (22:6) ) (HMDB ID number; HMDB0011496), LPC (17:0) sn-1 (HMDB ID number; HMDB0012108), LPC (14:0) (HMDB ID number; HMDB0010379), LPC (17:0) sn-2 ( HMDB ID number; HMDB0012108), LPC (15:0) (HMDB ID number; HMDB0010381), fatty acids (e.g. oleic acid, palmitic acid, linoleic acid, stearic acid, docosahexaenoic acid, Myristic acid, arachidonic acid, stearic acid, alpha-linolenic acid, gamma-linolenic acid, docosapentaenoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosatetraenoic acid, stearidonic acid), p-Cresol sulfate (HMDB ID number; HMDB0011635), Indoxyl sulfate (HMDB ID number; HMDB0000682), Hydroxybutyric acid (HMDB ID number; HMDB0000008), Hydroxybenzoic acid (HMDB ID number; HMDB0000500), N-phenyl Phenylacetylglutamine (HMDB ID number; HMDB0006344), Aconitic acid (HMDB ID number; HMDB0000072), Pyridoxal (HMDB ID number; HMDB0001545), LPC (16:0) (HMDB ID number; HMDB0010382), PC (36:2) (HMDB ID No.; HMDB0008331), PC (38:4) (HMDB ID No.; HMDB0008626), PC (40:6) (HMDB ID No.; HMDB0008727), Proline Betaine ) (HMDB ID number; HMDB0004827), ceramide (Ceramide) (d18:1/16:0) (HMDB ID number; HMDB0004949)
[J Group]
Citric acid (HMDB ID number; HMDB0000094)

In addition, as a biomarker for determining the risk of onset of optic neuropathy or the presence or absence of onset of optic neuropathy of the present invention, optic neuropathy consisting of one or two or more metabolites selected from the above [A group] to [J group] If the risk of developing or determining (diagnostic) the presence or absence of the onset of the metabolite is a metabolite of the above [A group], [B group], or [E group] ~ [G group] the concentration of said metabolite in a biological sample taken from a subject at high risk of developing optic neuropathy or who has developed optic neuropathy compared to the concentration of said metabolite in a control subject who does not develop optic neuropathy; , is high, and if the metabolite is one of the above [C group], [D group], or [H group] to [J group] metabolites, those who are at high risk of developing optic neuropathy, or who have developed optic neuropathy The concentration of the metabolite in a biological sample collected from a person who has been diagnosed is lower than the concentration of the metabolite in a control subject who does not develop optic neuropathy. 1”) is not particularly limited.
In addition, the biomarker for determining the severity of optic neuropathy of the present invention is composed of one or more metabolites selected from the above [Group A] to [Group D] to determine the severity of optic neuropathy. when the metabolite is a metabolite of the above [A group] or [D group], the concentration of the metabolite in a biological sample collected from a person with high severity of optic neuropathy is When the concentration of the metabolite is higher than that in subjects with low severity of optic neuropathy, and the metabolite is the above [Group B] or [Group C] metabolite, it is collected from a subject with high severity of optic neuropathy. The concentration of the metabolite in the biological sample obtained is lower than the concentration of the metabolite in a person with low severity of optic neuropathy, and the biomarker for determination (hereinafter referred to as "biomarker 2 for determination") There is no particular limitation as long as it is present) (Biomarker 1 for this determination and Biomarker 2 for this determination may be collectively referred to hereinafter as "biomarker for this determination").

In addition, as a screening method for a preventive agent for the onset of optic neuropathy or a therapeutic agent for optic neuropathy of the present invention, the above-mentioned Measure the concentration of one or more metabolites selected from [Group A] to [Group J] (hereinafter sometimes referred to as "concentration of metabolites in test substance-administered animals"), and if necessary Step (A) of quantifying; Step (B) to compare with the metabolites measured in step (A) is [A group], [B group], or [E group] to [G group] metabolites In some cases, when the "concentration of metabolites in animals administered the test substance" is lower than the "concentration of metabolites in animals not administered the test substance", the test substance prevents the onset of optic neuropathy or treats optic neuropathy. If the metabolites measured in step (A) are metabolites of [C group], [D group], or [H group] to [J group], "test substance administration When the "concentration of the metabolite in the animal" is higher than the "concentration of the metabolite in the animal untreated with the test substance", the test substance is evaluated to be effective in preventing the onset of optic neuropathy or treating optic neuropathy. (C); The method is not particularly limited as long as it includes the steps (A) to (C) (hereinafter sometimes referred to as "this screening method").

In the present invention, biological samples include, for example, tears, blood (e.g., plasma, serum), aqueous humor (anterior or posterior aqueous humor), vitreous humor, liquid samples such as urine, retinas containing RGCs, Examples include non-liquid samples such as eye drops and vitreous, and blood samples, urine samples, anterior aqueous humor samples, and retina samples are preferred.

When determining the risk of developing optic neuropathy, the subject in this determination method 1 is usually a subject who has not developed optic neuropathy and who is uncertain whether or not she will develop optic neuropathy in the future. , In addition, when determining the presence or absence of the onset of optic neuropathy, it is usually possible to include subjects for whom it is unknown whether or not they have developed optic neuropathy. A person who has been evaluated (determined) as having a high risk of developing Moreover, as a subject in the present determination method 2, for example, a subject who has developed optic nerve disorder but whose severity is unknown can be mentioned.

The non-human animal with optic neuropathy in the screening method of the present invention may be a non-human animal that has spontaneously developed optic neuropathy, or according to the method described in the section [Generation of ONC model mice] in the Examples of the specification of the present application. , it may be a non-human animal that has induced the onset of optic neuropathy. Examples of non-human animals include mice, as well as non-human mammals such as rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats, and dogs.

In the present invention, "developing optic neuropathy" means that the axons of retinal ganglion cells (RGC) are damaged (denatured) due to some cause such as an increase in intraocular pressure, and RGC cell death ( Symptoms or symptoms (e.g., narrowing of the visual field, blindness, etc.) resulting from functional and/or structural abnormalities of the eye (e.g., RGC axon damage and RGC cell death) caused by the induction of apoptosis) It means the state of appearance. The optic nerve disorders include, specifically, glaucoma (primary glaucoma [e.g., primary open angle glaucoma [POAG], normal tension glaucoma [NTG], primary angle closure glaucoma], secondary glaucoma, developmental glaucoma), optic neuropathy caused by poor circulation, ischemic optic neuropathy, retinal vessel occlusion (central retinal artery occlusion, branch retinal artery occlusion, central retinal vein occlusion, branch retinal vein occlusion) disease, etc.), retinitis pigmentosa, Leber's disease, retinopathy of prematurity, retinal detachment, retinal pigment epithelial detachment, macular degeneration, and optic nerve disorders such as diabetic retinopathy. Such glaucoma means an optic nerve disorder in which the aforementioned functional and/or structural abnormalities of the eye can be improved or suppressed, usually by lowering (lowering) intraocular pressure. The "optic neuropathy" of the present invention includes hereditary optic neuropathy and non-hereditary (sporadic) optic neuropathy.

In the Determination Method and the Biomarker for Determination, the concentration of the metabolite group in the biological sample is determined by pre-treating the collected biological sample (sample) and using a publicly known method that can specifically detect the metabolite group. It can be measured using methods such as mass spectrometry. Such a mass spectrometry method converts the metabolite group contained in the biological sample into gaseous ions (ionization) using an ion source, moves them in a vacuum in the analysis unit, uses electromagnetic force, or flies A measurement method using a mass spectrometer that can separate and detect a biological sample ionized by a time lag according to the mass-to-charge ratio. Ionization methods using the ion source include, for example, electron ionization (EI) method, chemical ionization (CI) method, field desorption ionization (FD) method, fast atom bombardment (FAB) method, and matrix-assisted laser desorption. The ionization (MALDI) method and the electrospray ionization (ESI) method can be mentioned. In addition, in the analysis unit, the metabolite group ionized by various ionization methods is separated by an analyzer according to mass. Such analyzers include, for example, a magnetic field mass separator (Sector MS), a quadrupole mass separator (QMS), a time-of-flight mass separator (TOFMS), a Fourier transform ion cyclotron mass separator (FT-ICRMS ) can be mentioned, and a combination thereof may also be used. Tandem mass spectrometry (MS/MS), which combines two or more mass spectrometry methods, can also be used. In addition, by gas chromatograph (GC), liquid chromatograph (LC), high performance liquid chromatograph (HPLC), and ultra high performance liquid chromatograph (UHPLC), the metabolite group contained in the biological sample was separated and separated from contaminants. It can be purified and analyzed. Such LC, HPLC, and UHPLC may include cation or anion exchange chromatographs, normal phase or reverse phase chromatographs, and may be combinations thereof. Imaging MS (IMS) analysis of the present metabolite group on biological tissue can also be performed by performing mass spectrometry directly on a section of the biological sample.

In the present invention, the concentration of the metabolite group in the biological sample may be an absolute value or a relative value. ) can be given as a reference.

In the Determination Method and the Biomarker for Determination, both of the subjects to be compared (subjects and controls; those who are at high risk of developing optic nerve damage or who have developed optic nerve damage and controls; Concentrations of metabolites in subjects with high and less severe optic neuropathy) are used to correspond to each other. Therefore, if one is an absolute value or a relative value, the other is also an absolute value or a relative value, respectively. Of the two to be compared, the concentration of the metabolite in the comparison control may be measured each time based on the prepared biological sample when the present determination method is carried out, or may be measured in advance. Also, the biological samples to be compared are preferably prepared by substantially the same method. Moreover, it is preferable that the method for measuring the concentration of the metabolite is substantially the same for both of the two to be compared.

In addition, in the screening method of the present invention, concentrations of metabolites corresponding to each other are used for both comparisons (a non-human animal with optic neuropathy administered with a test substance and a non-human animal with optic neuropathy not administered with a test substance). Therefore, if one is an absolute value or a relative value, the other is also an absolute value or a relative value, respectively. Of the two to be compared, the concentration of the metabolite in the comparative control may be measured each time based on the biological sample prepared when the present screening method is carried out, or may be measured in advance. Also, the biological samples to be compared are preferably prepared by substantially the same method. Moreover, it is preferable that the method for measuring the concentration of the metabolite is substantially the same for both of the two to be compared.

In step (c-1) of Determination Method 1, the metabolites measured in step (a-1) are the above [A group], [B group], or [E group] to [G group] metabolites. In some cases, when the "concentration of metabolite in subject" is higher than the "concentration of metabolite in control," the subject is at high risk or likely to have developed optic neuropathy. Evaluated (determined), when the "concentration of metabolite in subject" is not higher than the "concentration of metabolite in control", the subject has a low risk of developing optic neuropathy or is likely to have developed optic neuropathy. evaluated as low. In addition, in the step (c-1) of the Determination Method 1, the metabolites measured in the step (a-1) are metabolites of the above [C group], [D group], or [H group] to [J group] substance, when the "concentration of the metabolite in the subject" is lower than the "concentration of the metabolite in the control", the subject has a high risk of developing optic neuropathy or is likely to have developed optic neuropathy. Scored as high and when the "metabolite concentration in the subject" is not lower than the "metabolite concentration in the control", the subject has a low risk of developing optic neuropathy or is likely to have developed optic neuropathy. rated as low.

An arbitrary threshold value (cutoff value) is set to evaluate (determine) whether or not the "concentration of metabolites in the subject" is higher or lower than the "concentration of metabolites in the control". Such thresholds can be, for example, the mean of "concentration of metabolites in controls", "mean + standard deviation (SD)", "mean + 2 SD", "mean + 3 SD", median, "Median+SD", "Median+2SD", "Median+3SD" and the like can be mentioned. In addition, the threshold is determined by sensitivity (percentage of those who develop optic neuropathy later on or those who develop optic neuropathy can be correctly determined as positive) and specificity (those who do not develop optic neuropathy later on, and those who develop optic neuropathy). Statistical analysis software was used based on the data of "concentration of metabolites in subjects" and "concentrations of metabolites in controls" in order to increase the percentage of those who did not do so as correctly judged as negative. It can also be calculated using an ROC (Receiver Operating Characteristic) curve.

As used herein, "a control subject who does not develop optic neuropathy" means a healthy subject who has a low or almost no risk of developing optic neuropathy and has not developed optic neuropathy, more specifically, a biological sample The concentration of one or two or more metabolites selected from the above [A group], [B group], and [E group] to [G group] in the above threshold ("metabolite concentration in the subject" , threshold for evaluating whether it is higher or lower than the "concentration of metabolites in controls"), and / or the above [C group], [D group ], and the concentration of one or more metabolites selected from [H group] to [J group] is higher than the above threshold; means healthy subjects who have not developed optic nerve damage. It should be noted that "a control subject who does not develop optic nerve damage" is preferably a subject who is similar to the subject in terms of age, best corrected visual acuity (BCVA), eye axial length, etc. (subjects with no statistically significant difference).

In the step (c-2) of the Determination Method 2, if the metabolite measured in the step (a-2) is the [A group] or [D group] metabolite, the metabolite concentration in the optic neuropathy subject ”is higher than the “concentration of metabolites in optic neuropathy control subjects”, the subject evaluates (determines) that the optic neuropathy is likely to be more severe (progressed) than the control subjects, and “optic neuropathy When the "metabolite concentration in the subject" is not higher than the "metabolite concentration in the optic neuropathy control," the subject is rated as less likely to have severe optic neuropathy than the control. In addition, in the step (c-2) of the Judgment Method 2, if the metabolites measured in the step (a-2) are [B group] or [C group] metabolites, "Metabolites in optic neuropathy subjects When the "concentration of metabolites in optic neuropathy control subjects" is lower than the "concentration of metabolites in optic neuropathy control subjects," the subject is more likely to have aggravated (progressed) optic neuropathy than the control subjects. When Metabolite Concentration" is not lower than Metabolite Concentration in Optic Neuropathy Controls, subjects are assessed as less likely to have more severe optic neuropathy than controls.

A threshold value (cut-off value) is optional for evaluating (determining) whether or not the "concentration of metabolites in subjects with optic neuropathy" is higher or lower than the "concentration of metabolites in subjects with optic neuropathy." Such thresholds include, for example, the mean value of "concentration of metabolites in optic neuropathy control subjects", "mean value + standard deviation (SD)", "mean value + 2SD", "mean value +3SD", median, "median+SD", "median+2SD", "median+3SD", and the like. In addition, the threshold is set so that the sensitivity (percentage of people with severe optic nerve damage that can be correctly determined as positive) and specificity (percentage of people with non-severe optic nerve damage that can be correctly determined as negative) are high. It can also be calculated using an ROC curve using statistical analysis software based on the data of "concentration of metabolites in subjects with optic nerve damage" and the data of "concentrations of metabolites in control subjects with optic nerve damage".

The above-mentioned "control subject with optic neuropathy" is someone who is similar to the subject in terms of age, best corrected visual acuity (BCVA), axial length, etc. (other person with no statistically significant difference). Although possible, it is preferable to be the same subject as the subject who has developed optic neuropathy. In this case, the "concentration of metabolites in optic neuropathy control subjects" is the concentration of metabolites in a biological sample collected from a subject with optic neuropathy prior to the time the biological sample was collected from the subject. .

In step (C) of the screening method, if the metabolites measured in step (A) are [A group], [B group], or [E group] to [G group] metabolites, When the metabolite concentration in the substance-administered animal is lower than the metabolite concentration in the test substance-unadministered animal, the test substance is evaluated as effective in preventing the onset of optic neuropathy or treating optic neuropathy ( judgment), and when the "concentration of metabolites in animals administered the test substance" is not lower than the "concentration of metabolites in animals untreated with the test substance", the test substance prevents the onset of optic neuropathy or prevents optic nerve damage. Evaluate (determine) that the treatment is not effective. In addition, in step (C) of the screening method, if the metabolites measured in step (A) are [C group], [D group], or [H group] to [J group] metabolites, When the "concentration of metabolites in animals administered the test substance" is higher than the "concentration of metabolites in animals untreated with the test substance", the test substance is considered to be effective in preventing the onset of optic neuropathy or in treating optic neuropathy. Evaluate (judgment), and when the "concentration of metabolites in animals administered the test substance" is not higher than the "concentration of metabolites in animals untreated with the test substance", the test substance prevents the onset of optic neuropathy or affects the optic nerve. Evaluate (determine) that it is not effective in treating the disorder.

An arbitrary threshold is used to evaluate (determine) whether or not the “concentration of metabolites in animals administered the test substance” is lower or higher than the “concentration of metabolites in animals not administered the test substance”. can be set, and such thresholds include, for example, the mean value of "metabolite concentration in test substance-unadministered animals", "mean value + standard deviation (SD)", "mean value + 2SD", "mean value + 3 SD", median, "median + SD", "median + 2 SD", "median + 3 SD", and the like. In addition, the threshold is the sensitivity (percentage of substances that are effective in preventing the onset of optic nerve damage or treating optic nerve damage that can be correctly determined as positive) and specificity (effective in preventing the onset of optic nerve damage or treating optic nerve damage). Based on the data of "concentration of metabolites in animals administered the test substance" and "concentrations of metabolites in animals untreated with the test substance", It can also be calculated using a ROC curve using statistical analysis software.

In the present invention, the one or two or more metabolites selected from the above [A group] to [J group] include, for example, one or two selected from the above [A group] to [J group] , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 species, 20 species, 21 species, 22 species, 23 species, 24 species, 25 species, 26 species, 27 species, 28 species, 29 species, 30 species, 31 species, 32 species, 33 species, 34 species, 35 species, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 species, 70 species, 71 species, 72 species, 73 species, 74 species, 75 species, 76 species, 77 species, 78 species, 79 species, 80 species, 81 species, 82 species, 83 species, 84 species, 85 species, 86 types, 87 types, 88 types, 89 types, 90 types, 91 types, 92 types, 93 types, or 94 types of metabolites can be mentioned, and selected from the above [A group] to [D group] As the one or more metabolites, for example, 1, 2, 3, 4, 5, 6, 7 selected from the above [A group] to [D group], 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27 or 28 metabolites, preferably those containing at least fatty acids or L-acetylcarnitine, such fatty acids such as oleic acid, palmitic acid, linoleic acid, stearic acid Acids, docosahexaenoic acid, myristic acid, and arachidonic acid can be preferably exemplified by one or more fatty acids selected from the group consisting of. In addition, as metabolites containing L-acetylcarnitine, specifically, one type of L-acetylcarnitine; two types of combination of L-acetylcarnitine and PC (38:7); L-acetylcarnitine and PG (40:6); three combinations of L-acetylcarnitine, PC (38:7) and PG (40:6).

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

1. Confirmation that this metabolite group is useful as a biomarker for determining optic neuropathy 1
To identify biomarkers for determining optic neuropathy, mass spectrometry was performed using retinal samples from ONC model mice.

1-1 Materials and methods [model animals]
C57BL/6J mice (male, 8-12 weeks old; manufactured by Clea Japan, Inc.) were used as model animals in this example. All mouse handling and experiments were performed in accordance with the Statement Guidelines for the Use of Animals in Ophthalmic and Vision Research of the American Society for the Study of Ophthalmology (ARVO) and the guidelines of the Tohoku University Graduate School of Medicine and the Animal Care and Use Committee. .

[Preparation of ONC model mouse]
A mixture of ketamine (100 mg/kg) and xylazine (9 mg/kg) was administered intramuscularly to C57BL/6J mice, and the literature "Ryu, M. et al. J Neurosci Res 90, 802-815, doi:10.1002/ jnr.22800 (2012)." and the literature "Fujita, K. et al. Sci Rep 5, 18141, doi:10.1038/srep18141 (2015)." Axonal injury was performed to prepare an ONC model mouse. Briefly, the optic nerve of the right eye was exposed and the optic nerve approximately 2 mm posterior to the globe was crushed with forceps for 5 seconds (ONC group: n=16). After surgery, an antibiotic (levofloxacin)-containing ointment was applied and the mice were kept on a heating pad. As a control, C57BL/6 mice treated without crushing the optic nerve were used (CT group: n=8). Isolated retinas from the CT group and 2 days, 4 days, and 7 days after ONC treatment (ONC group day 2: n=8, ONC group day 4: n=4, and ONC group 7, respectively). On day n = 4), the retinas isolated from each group were analyzed by the methods described in the following items [Indirect fluorescent antibody method], [Western blotting method], and [Preparation of biological samples for LC-MS]. used for

[Indirect fluorescent antibody method]
Mouse retinas were fixed in 4% paraformaldehyde solution according to the method described in the document "Sato, K. et al, J Neurosci. 2013, 33(44):17458-68.", and then frozen sections were prepared. Then, an indirect immunofluorescence method using an anti-RBPMS antibody was performed. Briefly, retinal cryosections were treated with a primary antibody reaction in a solution containing a rabbit anti-RBPMS antibody (diluted 1/200, Abcam), followed by a secondary antibody conjugated with Alexa Fluor 488. Secondary antibody reaction treatment was performed in a solution containing (goat anti-rabbit IgG Alexa Fluor 488, manufactured by Abcam). After that, it was mounted with a mounting medium containing DAPI (4',6-diamidino-2-phenylindole) (manufactured by Vectashield) to prepare a sample for indirect fluorescent antibody method. Fluorescence images were acquired using a fluorescence microscope (“Axiovert 200, Carl Zeiss” or “BZ-9000, Keyence”).

[Western blotting method]
Mouse retinas were homogenized according to the method described in the document “Sato, K. et al, J Neurosci. - separated by PAGE and transferred to PVDF membrane. The membrane was subjected to blocking treatment in Tw-PBS containing 1% skim milk (PBS [Phosphate buffered saline] containing 0.05% Tween 20) for 1 hour at room temperature, followed by rabbit anti-RBPMS antibody (1/ A primary antibody reaction treatment was carried out overnight at 4° C. in a Tw-PBS solution containing 1000-fold dilution (manufactured by Abcam). After that, the membrane was washed with Tw-PBS solution, and HRP (Horseradish peroxidase)-conjugated secondary antibody (used at 1/5000 dilution, HRP-conjugated donkey anti-rabbit IgG [manufactured by Sigma]) was included. Secondary antibody reaction treatment was performed in the Tw-PBS solution for 1 hour at room temperature, and after washing with the Tw-PBS solution, the signal derived from the RBPMS protein was detected using the issued detection reagent (ECL prime, manufactured by GE Healthcare). Detected. In addition, after removing the primary and secondary antibodies by treating the membrane with Restore Western Blot Stripping Buffer (manufactured by Thermo Scientific), mouse anti-β-actin antibody (diluted 1/5000) was used as an internal standard. , Sigma) was similarly performed.

[Reagents for LC-MS]
Methanol, chloroform, and acetonitrile for LC-MS were purchased from Kanto Chemical Co., Ltd., and ammonium formate (1 mol/L1) and formic acid for LC-MS were purchased from Wako Pure Chemical Industries, Ltd. All commercial products were used as chemical standard substances for LC-MS. Specifically, the chemical standard substance phosphatidylcholine (PC) was purchased from Avanti Polar Lipids, and spermine, carnitine, and α-cyano-4-hydroxycinnamic acid (α-cyano-4-hydroxycinnamic acid (CHCA) were used. The included chemical standards were purchased from Sigma-Aldrich, and 9-Aminoacridine (9-AA) was purchased from Merck.

[Preparation of biological sample for LC-MS]
Mouse retinas were placed in 2.0 mL tubes and cryopreserved. 200 μL of methanol containing 0.1% formic acid was added to the thawed retina, and the Lysis and Homogenization System (5000×rpm, 15 seconds, 2Zr beads) (manufactured by Bertin) using a bead homogenizer (Precellys) was used. homogenized. Furthermore, after homogenizing with an ultrasonic cleaner for 10 minutes, centrifugation was performed at 16,400 g for 20 minutes at 4°C. Washed three times with methanol containing 0.1% formic acid. Retinal samples for study quality control (SQC) LC-MS were prepared by withdrawing 30 μL of each sample from the 96-well plate and mixing in a 15 mL tube. Based on such SQC, using a diluent (50% methanol containing 0.1% formic acid), four types of dilution quality control (dQC), that is, 2-fold dilution (d2QC), 4-fold dilution QC (d4QC), 8-fold diluted QC (d8QC), and 16-fold diluted QC (d16QC) were prepared.

[Mass spectrometry using LC-MS]
Two types of LC-MS: ultra high performance liquid chromatograph-quadrupole time of flight mass spectrometry (UHPLC-QTOF) equipped with a reversed-phase (C18) column; /MS) and a liquid chromatograph-Fourier transform type mass spectrometry (LC-FT/MS) equipped with a normal phase (hydrophilic interaction chromatography; HILIC) column. The analytical method was carried out according to the method described in the document "Saigusa, D. et al. PLoS One 11, e0160555, doi: 10.1371/journal.pone.0160555 (2016)."In addition, UHPLC- equipped with a C18 column. The above SQC was used for mass spectrometry using QTOF/MS, and the four types of dQC above were used for mass spectrometry using LC-FT/MS equipped with a HILIC column.

[Data processing]
All data obtained in both positive and negative ion modes, respectively, using the two LC-MS methods described above were processed using Progenesis QI software (Nonlinear Analysis) for peak picking, alignment, and normalization. Dynamics) to yield peak intensities versus retention times (RT) (also called elution time) and mass-to-charge ratio (m/z) (reference “Saigusa, D. et al. PLoS One 11, e0160555, doi:10.1371/journal.pone.0160555 (2016).”).

[Hierarchical clustering analysis]
A heat map was generated for the list of Z-scores for each metabolite for the CT group and the three ONC groups (ONC day 2, ONC group day 4, and ONC group day 7). Z-scores were selected by the Kruskal-Wallis test (p<0.004) when the coefficient of variation (CV) was lower than 30% in each group using the gplots package of the R program (version 3.2.0). . Metabolite phylogenetic trees were generated by the full-linkage clustering method and correlation distance measurements with the amap package (see FIG. 1B). The colored scale bar (see FIG. 1A) varies from blue (left) to white (middle) to red (right) to indicate low, moderate and high intensity metabolites, respectively.

[Statistical analysis]
The intensities of the identified metabolites were imported into SIMCA 13.0 software (Umetrcxs) for multivariate analysis and their relative amounts were analyzed by principal component analysis (PCA) and orthogonal partial least squares discrimination. It was evaluated by analysis (orthogonal partial least square-discriminant analysis; OPLS-DA). P values were calculated by Student's t-test, Welch's t-test, Wilcoxon rank sum test, Tukey-Kramer test, or Steel-Dwass test.

[Preparation of biological sample for IMS]
Two days after ONC treatment, 8 μm sections of mouse retina tissue were prepared using a cryostat (CM3050S; manufactured by Leica Microsystems) and then placed on an indium tin oxide slide glass (manufactured by Matsunami Glass Industry Co., Ltd.). , into a 50 mL plastic tube containing silica gel and coated with matrix. After that, 660 mg of CHCA or 9-AA was attached on a slide glass to a thickness of 1.4 μm using an automatic pretreatment device iMLayer (manufactured by Shimadzu Corporation), and then placed in a box equipped with filter paper. and permeated with 350 μL of water/methanol=95/5 (v/v). Thereafter, the slide glass containing the retinal sample was incubated at 85°C for 3 minutes with CHCA and at 40°C for 3 minutes with 9-AA, followed by drying in a desiccator for 30 minutes. , prepared retinal samples for IMS.

[Mass spectrometry using IMS]
Mass spectrometry was performed on the dried IMS retinal sample immediately using MALDI-IMS (manufactured by Shimadzu Corporation) in positive ion mode and negative ion mode. The obtained data were subjected to image processing using imaging MS solution software (manufactured by Shimadzu Corporation). The site of the retinal sample exposed to laser irradiation was identified by observation with an optical microscope after the retinal sample was HE-stained.

1-2 Results [Indirect fluorescent antibody method and Western blotting method using anti-RBPMS antibody]
In order to confirm the occurrence of optic neuropathy in ONC model mice, the CT group and three types of ONC groups (ONC group, 2nd day, ONC group, 4th day, and ONC group, 7th day) were examined for retinas. The number of retinal cells expressing RBPMS, one of the ganglion cell markers (RBPMS-positive retinal cells) (see FIG. 1B) and the amount of RBPMS expression in the retinal samples (see FIGS. 1A and 1C) were analyzed. As a result, on day 2 of the ONC group, almost the same level of RBPMS as in the CT group was detected, whereas on day 4 of the ONC group, the expression level of RBPMS was lower than in the CT group, and the ONC group On the 7th day, it was shown that the decrease level increased further (see FIG. 1).
This result indicates that the 2nd day of the ONC group was in the very early stage after the onset, and thereafter, the optic nerve disorder became more severe with the passage of time.

[Mass spectrometry using LC-MS]
Next, between the CT group and the above three ONC groups, metabolites with varying concentrations in the retina were analyzed using two types of LC-MS (UHPLC-QTOF/MS and LC-FT/MS). As a result of analysis by mass spectrometry, 28 types of this metabolite group were identified (see Table 1). Hierarchical clustering analysis was performed on these metabolites, and they were classified into four groups (groups A to D) (see Table 1 and FIG. 2B).

Group A and B metabolite concentrations in the retina were higher in the ONC group than in the CT group, and particularly for group A metabolites, increased over day 4 after ONC treatment (Figs. 2B, 3, and 4). 4), and group B metabolites were shown to be elevated 2 days after ONC treatment (see FIGS. 2B, 5, and 6). On the other hand, group C and D metabolite concentrations in the retina were lower in the ONC group than in the CT group, especially for group C metabolites 7 days after ONC treatment (Figures 2B, 7 and 8), group D metabolites were shown to be lower at 2 days after ONC treatment but then higher up to 7 days (see FIGS. 2B, 9 and 10).
These results indicate that the risk of developing optic neuropathy due to damage to RGC axons (2 days after ONC treatment) increases when metabolite concentrations in groups A and B in the retina are higher than normal levels (CT group). , indicating that group C and D metabolite concentrations in the retina are lower than normal levels and higher. In addition, the severity of optic neuropathy due to damage to RGC axons increased with higher concentrations of group A and D metabolites in the retina and decreased concentrations of group B and C metabolites in the retina. It shows that it increases with time.

[IMS analysis]
Next, the distribution of this metabolite group in the retinal section was analyzed by mass spectrometry using IMS. Of the metabolite groups, analysis was performed on group A metabolites (L-acetylcarnitine) and group B metabolites (PC[38:7] and PG[40:6]) as representative examples. Metabolites were also shown to be more distributed in the ONC group than in the CT group (see FIG. 11). These results support the results obtained by mass spectrometry using LC-MS.

2. Confirmation that this metabolite group is useful as a biomarker for determining optic neuropathy 2
Analysis using ONC model mice showed that the present metabolite group is useful as a biomarker for determining optic neuropathy. I did the analysis. Specifically, anterior aqueous humor was collected from each of 34 glaucoma patients (glaucoma group; see Table 2) and 37 healthy subjects (healthy group; see Table 2). 1%), remove proteins by centrifugation, and dilute twice with ultrapure water (containing 0.1% formic acid) to prepare an aqueous humor sample for LC-MS. Then, mass spectrometry using UHPLC-QTOF / MS equipped with C18 column, or mass spectrometry using LC-FT / MS equipped with HILIC column (Document "Saigusa, D. et al. PLoS One 11, e0160555, doi:10.1371/journal.pone.0160555 (2016).”), the concentration of the metabolite group (L-acetylcarnitine) was measured. Data processing was performed according to the method described in the item [Data processing] in Example 1.

As a result, the concentration of L-acetylcarnitine in the anterior aqueous humor sample in the glaucoma group was 4101±1301 (mean value±standard deviation), which was 1.3 times higher than the value in the healthy group (3102±710). A significant difference (P=0.0001352) was observed (see FIG. 12). This result is useful as a biomarker for determining the risk of developing optic neuropathy and the severity of optic neuropathy. It has been shown to be useful as a biomarker for determining the presence or absence.

3. Confirmation that this metabolite group is useful as a biomarker for determining optic neuropathy 3
Next, metabolites other than L-acetylcarnitine whose concentrations fluctuate between the glaucoma group and the healthy subject group of Example 2 were analyzed. Specifically, from each of the glaucoma group (see Table 3) consisting of a normal tension glaucoma (NTG) group and a primary open angle glaucoma (POAG) group, and a healthy subject group, an anterior aqueous humor sample, a plasma sample, and A urine sample was collected according to a standard method and subjected to mass spectrometry using UHPLC-QTOF/MS equipped with a C18 column, or mass spectrometry using LC-FT/MS equipped with a HILIC column (reference "Saigusa, D. et al. PLoS One 11, e0160555, doi:10.1371/journal.pone.0160555 (2016).”), and data processing was performed according to the method described in [Data processing] in Example 1, and metabolites was measured.

As a result, 16 metabolite groups (3-sulfoacetic acid, dimethylsulfone, ribonic acid, arabinonic acid, xylonic acid, L-hydroxyglutaric acid, hydroxycitric acid, glucaric acid, galactaric acid) were detected in the anterior aqueous humor sample. , octenoylcarnitine, betaine, L-acetylcarnitine, methylmalonylcarnitine, L-carnitine, N,N-diethylethanolamine, and L-cystine) in the glaucoma group (i.e., the NTG group and/or POAG group) was shown to increase (see Table 4 and Figures 13-25).

In addition, in the anterior aqueous humor sample, the 8 metabolite groups (dichloromethane, glutathione, L-tyrosine, L-phenylalanine, 3-mercaptolactic acid, uridine, taurine, and aminobutyric acid) were found to be associated with glaucoma compared with the healthy group. Groups (ie, NTG and/or POAG groups) were shown to decrease (see Table 5 and Figures 26-33).

In plasma samples, 14 metabolite groups (pyroglutamine, LPC (18:2) sn-1, LPE (18:2), LPC (18:2) sn-2, androsterone sulfate, testosterone Sulfuric acid, phenolsulfuric acid, lactic acid, D-glucose, hydroperoxylinoleic acid, PC (34:2), thiomorpholine-3-carboxylic acid, LPC (18:2), and L-carnitine) compared to the healthy group, A greater increase was shown in the glaucoma group (ie, the NTG group and/or the POAG group) (see Table 6 and Figures 34-47).

In addition, in plasma samples, 29 metabolite groups (LPC (18:0), methyl palmitate, isopropyl myristate, LPE (22:6), LPC (17:0) sn-1, LPC (14 : 0), LPC (17:0) sn-2, LPC (15:0), oleic acid, p-cresol sulfate, palmitic acid, linoleic acid, stearic acid, indoxyl sulfate, hydroxybutyric acid, docosahexaenoic acid, myristic acid , hydroxybenzoic acid, N-phenylacetylglutamine, arachidonic acid, aconitic acid, pyridoxal, LPC(16:0), PC(36:2), PC(38:4), PC(40:6), proline betaine, and ceramide (d18:1/16:0)) were shown to decrease in the glaucoma group (that is, the NTG group and/or the POAG group) compared to the healthy group (Table 7 and Figures 48-74 reference).

In addition, in urine samples, eight metabolite groups (methyladenosine, succinyladenosine, dodecanoylcarnitine, O-(7-carboxyheptanoyl)carnitine, O-(8-carboxyoctanoyl)carnitine), O- (11-carboxyundecanoyl)carnitine, maleic acid, and fumaric acid) were shown to be increased in the glaucoma group (i.e., the NTG group and/or the POAG group) compared to the healthy group (Table 8). and Figures 75-81).

In addition, in urine samples, one type of this metabolite group (citric acid) was shown to decrease in the glaucoma group (that is, the NTG group and / or the POAG group) compared to the healthy group (Table 9 and FIG. 82).

The above results indicate that the metabolite groups of [Group E] to [Group J] are useful as biomarkers for determining the presence or absence of onset of optic neuropathy (e.g., glaucoma such as NTG and POAG). there is

According to the determination method or biomarker for determination of the present invention, the risk of onset of optic neuropathy, the presence or absence of onset, and the severity of optic neuropathy can be determined with high accuracy. It is expected that appropriate treatment will be available and the number of blind people due to optic nerve damage will be reduced. In addition, the screening method of the present invention contributes to the development of prophylactic (protective) or therapeutic agents (drugs) for optic neuropathy.

Claims (6)

A method for assisting diagnosis of the presence or absence of glaucoma, comprising the following steps (a-1) and (b-1) .
(a-1) A step of measuring the concentration of a fatty acid in a biological sample collected from a subject , wherein the fatty acid is one or more selected from the group consisting of oleic acid, stearic acid, and myristic acid. is a fatty acid of step (a-1);
(b-1) A step of comparing the fatty acid concentration measured in step (a-1) with the fatty acid concentration in a control subject who does not develop glaucoma , wherein the fatty acid concentration measured in step (a-1) step (b-1) , when the concentration is lower than the concentration of fatty acid in the control subject , indicating that the subject is likely to have developed glaucoma ; 2. The method of claim 1, wherein the biological sample is a blood sample, urine sample, anterior aqueous humor sample, or retinal sample. A biomarker for determining the presence or absence of onset of glaucoma , comprising a fatty acid, wherein the fatty acid is one or more fatty acids selected from the group consisting of oleic acid, stearic acid, and myristic acid. and
the concentration of said fatty acid in a biological sample taken from a person suffering from glaucoma is lower than the concentration of said fatty acid in a control person who does not develop glaucoma ;
The biomarker for determination . The biomarker for determination according to claim 3 , wherein the biological sample is a blood sample, a urine sample, an anterior aqueous humor sample, or a retinal sample. A screening method for a therapeutic agent for glaucoma , comprising the following steps (A) to (C).
(A) A step of measuring the concentration of a fatty acid in a biological sample collected from a glaucomatous non-human animal to which a test substance is administered , wherein the fatty acid is selected from the group consisting of oleic acid, stearic acid, and myristic acid. step (A), wherein one or more fatty acids are
(B) comparing the concentration of the fatty acid measured in step (A) with the concentration of the fatty acid in a glaucomatous non-human animal to which the test substance is not administered;
(C) When the fatty acid concentration measured in step (A) is higher than the fatty acid concentration in non-human animals with glaucoma to which the test substance is not administered, the test substance is considered to be effective in treating glaucoma . evaluating; 6. The screening method according to claim 5 , wherein the biological sample is a blood sample, urine sample, anterior aqueous humor sample, or retinal sample.

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