Method of detecting epilepsy

Abstract

To provide a novel and effective method of detecting epilepsy.SOLUTION: An epilepsy detection method is provided, comprising a step of measuring the concentration of lipid metabolite or nitric oxide metabolite in a biological sample of a subject. Or, an epilepsy marker may be provided, including a lipid metabolite or nitric oxide metabolite.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The technical field of the invention relates to methods for detecting epilepsy. [Background technology]

[0002] Epilepsy is a chronic brain dysfunction common in mammals, including humans and dogs, and is characterized by recurrent epileptic seizures. In dogs, epilepsy is estimated to occur in approximately 0.5-5.7% of the general population (Non-Patent Documents 1-3). Among epilepsies, meningoencephalitis of unknown origin (MUO) and idiopathic epilepsy (IE) are well-known causes of brain diseases accompanied by seizures in dogs.

[0003] MUO is known as an inflammatory disease of the brain and meninges of unknown origin in dogs (Non-Patent Document 4). At least some cases of MUO are caused by autoimmune inflammation (Non-Patent Documents 5-7), but the pathophysiology of most MUO cases is unknown (Non-Patent Document 4). Although a definitive diagnosis requires autopsy and histopathological approaches, clinicians are forced to make a presumptive diagnosis based on clinical diagnostic criteria (Non-Patent Document 4).

[0004] IE is known as epilepsy with no organic lesion in the brain and no identifiable cause (Non-Patent Document 8). It has been reported that 25-48% of epilepsy in dogs is IE (Non-Patent Document 1). In diagnosing IE, it is important to exclude other diseases through MRI and cerebrospinal fluid (CSF) tests (Non-Patent Document 1). With the current diagnostic criteria, there are problems with the difficulty of proactively diagnosing IE, the need for general anesthesia and sedation for MRI tests in dogs, and the high cost. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Berendt, M. & Gram, L. Epilepsy and seizure classification in 63 dogs: a reappraisal of veterinary epilepsy terminology. J. Vet. Intern. Med. 13, 14-20 (1999). [Non-Patent Document 2] Hamamoto, Y. et al. Retrospective epidemiological study of canine epilepsy in Japan using the International Veterinary Epilepsy Task Force classification 2015 (2003-2013): etiological distribution, risk factors, survival time, and lifespan. BMC Vet Res. 12, 248 (2016). [Non-Patent Document 3] Potschka, H., Fischer, A., von Ruden, EL, Hulsmeyer, V. & Baumgartner, W. Canine epilepsy as a translational model? Epilepsia 54, 571-579 (2013). [Non-Patent Document 4] Munana, KR Management of refractory epilepsy. Top Companion Anim. Med. 28, 67-71 (2013). [Non-Patent Document 5] Toda, Y. et al. Glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibody in canine necrotising meningoencephalitis. Vet. Rec. 161, 261-264 (2007). [Non-Patent Document 6] Miyake, H., Inoue, A., Tanaka, M. & Matsuki, N. Serum glial fibrillary acidic protein as a specific marker for necrotizing meningoencephalitis in Pug dogs. J. Vet. Med. Sci. 75, 1543-1545 (2013). [Non-Patent Document 7] Shibuya, M. et al. Autoantibodies against glial fibrillary acidic protein (GFAP) in cerebrospinal fluids from Pug dogs with necrotizing meningoencephalitis. J. Vet. Med. Sci. 69, 241-245 (2007). [Non-Patent Document 8] De Risio, L. et al. International veterinary epilepsy task force consensus proposal: diagnostic approach to epilepsy in dogs. BMC Vet. Res. 11, 148 (2015). Summary of the Invention [Problem to be solved by the invention]

[0006] Methods for detecting epilepsy have room for improvement.

[0007] The present invention has been made in consideration of the above circumstances, and aims to provide a novel and effective method for detecting epilepsy, a biomarker, or various methods using a biomarker. [Means for solving the problem]

[0008] The present inventors have conducted extensive research and found that specific lipid metabolites or nitric oxide metabolites are significantly increased in biological samples from MUO and IE cases. The present inventors have also found that MUO and IE can be detected by using the concentrations of lipid metabolites or nitric oxide metabolites as indicators. Based on these findings, the present invention has been completed.

[0009] According to one aspect of the present invention, there is provided the following: 1. A method for detecting epilepsy, comprising a step of measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample from a subject. 2. The detection method according to 1 above, wherein the lipid metabolite is an arachidonic acid metabolite, a linoleic acid metabolite, a dihomo-γ-linolenic acid metabolite, a lyso-platelet activating factor, a platelet activating factor, or an oleoylethanolamide. 3. The above lipid metabolite is 6-keto-prostaglandin F 1α , 20-carboxyarachidonic acid, 11,12-dihydroxyeicosatrienoic acid, 9-hydroxyoctadecadienoic acid, prostaglandin F 1α 3. The method for detecting the marker as described in 1 or 2 above, wherein the marker is a lyso-platelet activating factor or oleoylethanolamide. 4. The above nitric oxide metabolites are NO2 - Or NO3 - 4. The detection method according to any one of 1 to 3 above. 5. The detection method according to any one of 1 to 4 above, wherein the step includes a step of measuring the concentration of the lipid metabolite. 6. The detection method according to any one of 1 to 5 above, wherein the step includes a step of measuring the concentrations of the lipid metabolites and nitric oxide metabolites. 7. The above process is carried out to determine the NO2 - Or NO3 - or NO2 in blood samples. - 7. The detection method according to any one of 1 to 6 above, further comprising a step of measuring the concentration of 8. The detection method according to any one of 1 to 7 above, wherein the epilepsy is idiopathic epilepsy (IE) or meningoencephalitis of unknown origin (MUO). 9. A detection method described in any one of 1 to 8 above, further comprising a step of comparing the concentration of lipid metabolites or nitric oxide metabolites in a biological sample from the subject with the concentration of the same lipid metabolites or nitric oxide metabolites in a biological sample from a healthy subject. 10. A detection method described in any one of 1 to 9 above, which indicates that the subject is suffering from epilepsy when the concentration of lipid metabolites or nitric oxide metabolites in a biological sample of the subject is higher than the concentration of the same lipid metabolites or nitric oxide metabolites in a biological sample of a healthy subject. 11. The detection method according to any one of 1 to 10 above, wherein the biological sample is a body fluid. 12. The detection method according to any one of 1 to 11 above, wherein the biological sample is a cerebrospinal fluid sample or a blood sample. 13. A method for determining the effectiveness of treatment for epilepsy, comprising a step of measuring the concentration of lipid metabolites or nitric oxide metabolites in a biological sample from a subject. 14. The detection method according to claim 13, wherein the lipid metabolite is an arachidonic acid metabolite, a linoleic acid metabolite, a dihomo-γ-linolenic acid metabolite, a lyso-platelet activating factor, a platelet activating factor, or an oleoylethanolamide. 15. The above lipid metabolite is 6-keto-prostaglandin F 1α , 20-carboxyarachidonic acid, 11,12-dihydroxyeicosatrienoic acid, 9-hydroxyoctadecadienoic acid, prostaglandin F 1α 15. The method for determining whether the antibody is lyso-platelet activating factor, or oleoylethanolamide, according to 13 or 14 above. 16. The above nitric oxide metabolites are NO2 - Or NO3 - 16. The method for determining whether or not the above-mentioned 17. A method for determining whether or not a subject is a healthy subject, further comprising a step of comparing the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from the subject with the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject. 18. A method for determining whether a therapeutic effect is present, comprising the steps of: determining whether a concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from a subject is lower than the concentration of the lipid metabolite or nitric oxide metabolite in a biological sample from a subject before treatment or the concentration of the lipid metabolite or nitric oxide metabolite in a biological sample from a subject suffering from epilepsy; or determining whether a therapeutic effect is present if a concentration of the lipid metabolite or nitric oxide metabolite in a biological sample from a subject suffering from epilepsy is not significantly different from the concentration of the lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject. 19. The method according to any one of 13 to 18 above, wherein the biological sample is a body fluid. 20. The method according to any one of 13 to 19 above, wherein the biological sample is a cerebrospinal fluid sample or a blood sample. 21. Markers of epilepsy, including lipid metabolites or nitric oxide metabolites. 22. The marker according to claim 21, wherein the lipid metabolite is an arachidonic acid metabolite, a linoleic acid metabolite, a dihomo-γ-linolenic acid metabolite, a lyso-platelet activating factor, a platelet activating factor, or an oleoylethanolamide. 23. The above lipid metabolite is 6-keto-prostaglandin F 1α , 20-carboxyarachidonic acid, 11,12-dihydroxyeicosatrienoic acid, 9-hydroxyoctadecadienoic acid, prostaglandin F 1α 23. The marker according to claim 21 or 22, which is lyso-platelet activating factor, or oleoylethanolamide. 24. The above nitric oxide metabolites are NO2 - Or NO3 - 24. The marker according to any one of 21 to 23 above, 25. A kit for detecting epilepsy, comprising a means for quantifying lipid metabolites or nitric oxide metabolites in a biological sample from a subject. 26. A method for screening for therapeutic or alleviating agents for epilepsy, comprising the steps of administering a candidate therapeutic or alleviating agent for epilepsy to a subject and measuring the concentration of lipid metabolites or nitric oxide metabolites in a biological sample from the subject. 27. A pharmaceutical composition for use in treating epilepsy in a subject, the subject being predicted to have epilepsy by any of the methods described above in 1 to 12. 28. A method for treating epilepsy, comprising the steps of: (A) measuring a concentration of a lipid metabolite or nitric oxide metabolite in a biological sample of a subject; (B) comparing the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject with the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject; (C) determining that the subject is suffering from epilepsy if the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is higher than the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject; and (N) administering treatment for epilepsy to the subject determined to be suffering from epilepsy.

[0010] According to the present invention, for example, epilepsy can be detected, or a subject in whom epilepsy has been detected can be treated. [Brief description of the drawings]

[0011] [Figure 1] Lipid metabolite levels in CSF of dogs with CNS disease on simplified lipid metabolism maps. The lipid metabolite levels that were significantly altered between groups are shown on simplified maps. Dots represent individual values ​​and horizontal bars represent median values ​​for each group. All values ​​are expressed as ratios to internal standard (IS). The abbreviations next to the arrows refer to the mediators of those pathways. AA, arachidonic acid; COX, cyclooxygenase; CYP, cytochrome P450; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxy-10E,12Z-octadecadienoic acid; HpODE, hydroperoxy-10E,12Z-octadecadienoic acid; IE, idiopathic epilepsy; LA, linoleic acid; LOX, lipoxygenase; MUO, meningoencephalitis of unknown origin; PAF, platelet activating factor; PG, prostaglandin; * and **, statistical significance of p<0.05 and 0.01. [Diagram 2] Correlation between CSF lyso-PAF levels and cell counts. Pearson correlation analysis was performed with XLSTAT. Lyso-PAF levels were positively correlated with cell counts in the CSF of MUO dogs (R=0.926, p<0.001). The solid line represents the fitted regression line, and the dotted line represents the variate normal ellipse (p=0.950). [Diagram 3] Plasma lipid metabolite levels in dogs with CNS disease on simplified lipid metabolism maps. Significantly altered lipid metabolite levels between groups are shown on simplified maps. Dots represent individual values, and horizontal bars represent median values ​​for each group. All values ​​are expressed as ratios to internal standard (IS). Abbreviations next to the arrows refer to mediators of those pathways. AA, arachidonic acid; COX, cyclooxygenase; CYP, cytochrome P450; DGLA, dihomo-gamma-linolenic acid; DHET, dihydroxyeicosatrienoic acid; EET, epoxy 5Z,8Z,14Z eicosatrienoic acid; HETE, hydroxyeicosatrienoic acid; IE, idiopathic epilepsy; OEA, oleylethanolamide; MUO, meningoencephalitis of unknown origin; PG, prostaglandin; * and **, statistical significance of p<0.05 and 0.01. [Figure 4] Levels of NOx in CSF and plasma of dogs with central nervous system disease. IE, idiopathic epilepsy; MUO, meningoencephalitis of unknown origin; NS, not significant; * and **, statistical significance of p<0.05 and 0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, an embodiment of the present invention will be described in detail. Note that, in order to avoid repetition and complexity, description of similar contents will be omitted as appropriate.

[0013] In one embodiment of the present invention, the term "lipid metabolite" includes a lipid degradation product generated by the degradation by enzyme-dependent or enzyme-independent oxidation in a living body. Examples of lipid metabolites include arachidonic acid metabolites, linoleic acid metabolites, dihomo-γ-linolenic acid metabolites, eicosapentaenoic acid metabolites, or docosahexaenoic acid metabolites. Examples of lipid metabolites include arachidonic acid metabolites (e.g., cyclooxygenase metabolites of arachidonic acid (e.g., 6-keto-prostaglandin F 1α , prostaglandin I2, 11β-13,14-dihydro-15-keto-PGF 2α, 13,14-dihydro-15-keto-PGJ2, tetranor-PGDM, 20-hydroxy-PGE2, 15-keto-PGE2, 13,14-dihydro-15-keto-tetranor-PGE2, tetranor-PGEM, 15-keto-PGF 2α , 13,14-dihydro-15-keto-tetranor-PGF 1α , tetranor-PGFM, PGK2, 13,14-dihydro-15-keto-tetranor-PGF 1β , 13,14-dihydro-15-keto-PGE2, 6-keto-PGF 1α , TXB2), cytochrome p450 metabolites of arachidonic acid (e.g., 20-carboxyarachidonic acid, 20-HETE, 11,12 DHET, 11,12 EET, 17-HETE), lipoxygenase metabolites of arachidonic acid (e.g., 5-LOX metabolites of arachidonic acid such as 5-HpETE), enzyme-independent oxidative metabolites of arachidonic acid (e.g., 8-iso-15(R)-PGF 2α )), linoleic acid metabolites (e.g., 9-HODE or 9-HpODE), dihomo-γ-linolenic acid metabolites (e.g., PGF 1α ), lyso-platelet activating factor, platelet activating factor, or oleoylethanolamide, preferably characterized in that its concentration in a biological sample from a subject suffering from epilepsy is significantly higher than its concentration in a biological sample from a healthy subject.

[0014] In one embodiment of the present invention, "6-keto-prostaglandin F 1α " is also known as 6-keto-PGF 1α This compound may also be written as 9α,11α,15S-trihydroxy-6-oxo-prost-13E-en-1-oic acid. This compound may have the structure of the compound with CAS Registry Number 58962-34-8.

[0015] In one embodiment of the present invention, "20-carboxyarachidonic acid" may be alternatively referred to as 20-carboxy-AA. This compound may be alternatively referred to as 5Z,8Z,11Z,14Z-eicosatetraenedioic acid. This compound may have the structure of the compound having CAS Registry Number 79551-84-1.

[0016] In one embodiment of the present invention, "11,12-dihydroxyeicosatrienoic acid" may be alternatively referred to as 11,12-DHET. This compound may be alternatively referred to as (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid. This compound may have the structure of the compound having CAS Registry Number 192461-95-3.

[0017] In one embodiment of the present invention, "9-hydroxyoctadecadienoic acid" may be alternatively referred to as 9-HODE. This compound may be alternatively referred to as (±)-9-hydroxy-10E,12Z-octadecadienoic acid. This compound may have the structure of the compound with CAS Registry Number 98524-19-7.

[0018] In one embodiment of the present invention, "prostaglandin F 1α " is also known by the alias PGF 1α This compound may also be written as 9α,11α,15S-trihydroxy-prost-13E-en-1-oic acid. This compound may have the structure of the compound with CAS Registry Number 745-62-0.

[0019] In one embodiment of the present invention, "lyso-platelet activating factor" may be alternatively referred to as lyso-PAF. This compound may be alternatively referred to as 1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine or 1-O-octadecyl-sn-glyceryl-3-phosphorylcholine. This compound may have the structure of the compound having CAS Registry Number 52691-62-0 or 74430-89-0. Lyso-platelet activating factor may be lyso-PAF C-16 or lyso-PAF C-18.

[0020] In one embodiment of the present invention, "oleoylethanolamide" may be alternatively referred to as OEA. This compound may be alternatively referred to as N-(cis-9-octadecenoyl)ethanolamine. This compound may have the structure of the compound with CAS Registry Number 111-58-0.

[0021] In one embodiment of the present invention, the "nitric oxide metabolite" is, for example, a nitrite ion (NO - ) and nitrate ion (NO3 - ). The amount of nitric oxide metabolites may be measured, for example, by diazotization reaction absorbance measurement, chemiluminescence method, electrode method, electron spin resonance method, or Griess method. These methods include improved methods. The diazotization reaction absorbance measurement may include, for example, a step of reacting a nitric oxide metabolite with an amine, or a step of measuring the absorbance of a diazo compound. The diazotization reaction absorbance measurement may be performed, for example, by adding an aromatic amine to a diazo compound generated by reaction of a nitric oxide metabolite with an aromatic primary amine in an acidic solution, and measuring the absorbance of the red color of the diazo compound generated by coupling.

[0022] In one embodiment of the present invention, "epilepsy" typically includes chronic brain diseases that cause repeated seizures due to excessive firing activity of cerebral nerve cells. In terms of seizure symptomatology, epileptic seizures include those in which partial symptoms are observed due to local excitation of the cerebrum (focal seizures) and those in which symptoms are observed throughout the body accompanied by loss of consciousness due to excitation that has spread to the entire cerebrum (generalized seizures). Typically, epilepsy classifications defined by etiology include "symptomatic epilepsy" in which organic lesions that cause seizures are observed in the brain, and "idiopathic epilepsy (IE)" in which no organic lesions are present in the brain and the cause of seizures is not found to be other than genetic predisposition (mainly corresponding to idiopathic epilepsy in humans).

[0023] In one embodiment of the present invention, "meningoencephalitis of unknown origin (MUO)" is typically a type of symptomatic epilepsy. Symptoms may include epileptic seizures, circling, difficulty standing, or reduced level of consciousness. MUO includes, for example, granulomatous meningoencephalomyelitis (GME), necrotizing meningoencephalitis (NME), or necrotizing leukoencephalitis (NLE).

[0024] In one embodiment of the present invention, the term "biological sample" refers to a sample separated from a living body, and includes bodily fluids such as blood (including plasma), urine, saliva, nasal mucus, sweat, tears, feces, cerebrospinal fluid, etc. A cerebrospinal fluid sample or a blood sample is particularly preferred for increasing detection accuracy.

[0025] In one embodiment of the present invention, a "subject" includes a human or non-human mammal (e.g., dog, cat, monkey, mouse, rat, rabbit, horse, cow, pig, or sheep). The mammal may be, for example, a mammal classified in the family Hominidae (e.g., Homo), Canidae (e.g., Canis), Felidae (e.g., Cat), Muridae (e.g., Murinae or Rattus), Leporidae (e.g., Leporinae), Equidae (e.g., Horse), Bovidae (e.g., Bovidae or Ovis), or Suidae (e.g., Sus). Subjects include, for example, subjects suffering from epilepsy, subjects suspected of having epilepsy, subjects diagnosed with epilepsy, or subjects in which symptoms of epilepsy have been observed. Subjects include patients.

[0026] According to one embodiment of the present invention, a novel method for detecting epilepsy is provided. This method for detecting epilepsy includes, for example, a step of measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample of a subject. According to this method for detecting epilepsy, as demonstrated in the examples described below, epilepsy can be detected.

[0027] In one embodiment of the present invention, the detection method includes a step of (A) measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample of a subject. The concentration of a lipid metabolite or a nitric oxide metabolite can be measured by a known method, for example, by mass spectrometry or immunoassays such as ELISA and immunochromatography. Examples of mass spectrometry include liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MSMS), high performance liquid chromatography-mass spectrometry (HPLC-MS), high performance liquid chromatography-tandem mass spectrometry (HPLC-MSMS), or gas chromatography-mass spectrometry (GC-MS). Immunoassays include, for example, analytical methods using detectably labeled anti-lipid metabolite antibodies, or detectably labeled antibodies against anti-lipid metabolite antibodies (secondary antibodies). According to the labeling method of the antibody, it is classified into enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA), etc., and any of these can be used in the present invention. From the viewpoint of accurately measuring the concentration of lipid metabolites having similar structures, measurement by mass spectrometry (particularly, LC-MSMS or HPLC-MSMS) is preferable. The concentration of lipid metabolites may be measured, for example, by the method described in Takenouchi S. et al. J. Vet. Med. Sci. 84, 689-693 (2022). The concentration of nitric oxide metabolites may be measured by the method described in the above embodiment related to nitric oxide metabolites. The detection method may be rephrased as a test method.

[0028] In one embodiment of the present invention, the detection method may further include a step of determining the presence or absence of epilepsy for the subject from whom the biological sample was collected based on the concentration of the lipid metabolite or nitric oxide metabolite measured in the above step (A). This step may include a step (B) of comparing the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject with the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject. Here, if the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject, it may be indicated that the subject suffers from epilepsy. That is, the detection method of one embodiment of the present invention may further include a step (C) of determining that the subject suffers from epilepsy if the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject. The term "significantly different" in step (C) means that the concentration of a lipid metabolite or nitric oxide metabolite is either higher or lower than that of a healthy subject, and for example means that the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from a subject is higher, preferably significantly higher, than the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject (for example, the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from a subject is statistically significantly higher than the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject, or is about 1.1 times or more, about 1.25 times or more, about 1.5 times or more, about 1.75 times or more, about 2.0 times or more, about 3 times or more, about 5 times or more, about 10 times or more, about 50 times or more, or about 100 times or more). The concentration of lipid metabolites or nitric oxide metabolites in a biological sample from a healthy subject can be the average value calculated by measuring the concentrations of lipid metabolites or nitric oxide metabolites in biological samples taken from multiple healthy subjects in advance. The term "suffering epilepsy" is also used to mean cases where there is a possibility of suffering from epilepsy. The term "same" can be used when a metabolite of the same type or name as that used in a test subject is used in a comparison subject (e.g., a healthy subject).

[0029] According to the detection method of one embodiment of the present invention, epilepsy can be detected in a subject. Therefore, this detection method can be used to assist in the diagnosis of epilepsy, and the determination of whether a subject has epilepsy or not can ultimately be made by a doctor or veterinarian, possibly in combination with other findings. In one embodiment of the present invention, the detection method includes a method for assisting detection or a method for detection.

[0030] According to the detection method of one embodiment of the present invention, epilepsy can be quantitatively detected based on a biological sample collected from a subject. That is, the detection method of one embodiment of the present invention is advantageous in that epilepsy can be detected simply and accurately while reducing the burden on the patient.

[0031] In another aspect, one embodiment of the present invention provides a method for diagnosing epilepsy. According to this method, it is possible to diagnose whether or not a subject suffers from epilepsy by using lipid metabolites or nitric oxide metabolites in a biological sample as an indicator. The method for diagnosis of one embodiment of the present invention includes a step (A') of measuring the concentration of lipid metabolites or nitric oxide metabolites in a biological sample of a subject, similar to the detection method of one embodiment of the present invention. The method for diagnosis of one embodiment of the present invention may further include a step (B') of comparing the concentration of lipid metabolites or nitric oxide metabolites in the biological sample of the subject with the concentration of the same lipid metabolites or nitric oxide metabolites in a biological sample of a healthy subject, and may further include a step (C') of determining that the subject suffers from epilepsy when the concentration of lipid metabolites or nitric oxide metabolites in the biological sample of the subject is significantly different from the concentration of the same lipid metabolites or nitric oxide metabolites in the biological sample of the healthy subject. The steps (A'), (B') and (C') correspond to the steps (A), (B) and (C), respectively, and can be carried out according to the description of the embodiment of the detection method of the present invention.

[0032] In another aspect, one embodiment of the present invention provides a method for determining the therapeutic effect on epilepsy. According to the method for determination of one embodiment of the present invention, the therapeutic effect on epilepsy can be determined using lipid metabolites or nitric oxide metabolites in a biological sample as an indicator. In one embodiment of the present invention, the method for determination includes a method for assisting the determination or a method for the determination.

[0033] The determination method of one embodiment of the present invention includes, similarly to the embodiment of the detection method of the present invention, a step (D) of measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample of a subject. The measurement of the concentration of a lipid metabolite or a nitric oxide metabolite can be performed in the same manner as the embodiment of the detection method of the present invention.

[0034] The method of one embodiment of the present invention may further include a step of determining the therapeutic effect against epilepsy for the treated subject based on the concentration of the lipid metabolite or nitric oxide metabolite measured in the above step (D). This step may include a step (E) of comparing the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject treated for epilepsy with the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject. Here, the therapeutic effect may be indicated when the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject treated for epilepsy is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the subject before treatment or the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the epileptic subject, or when the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject is not significantly different. That is, the determination method of the present invention may further include a step of (F) determining that there is a therapeutic effect when the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample of a subject receiving treatment for epilepsy is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of the subject before treatment or the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a subject suffering from epilepsy (preferably, when it is significantly different in a direction approaching the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject), or when it is not significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject.The term "significantly different" in step (F) means that the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is either higher or lower than that in the subject before treatment or in the subject with epilepsy, and for example means that the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is lower, preferably significantly lower, than the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the subject before treatment or in the subject with epilepsy (for example, the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is statistically significantly lower than the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the subject before treatment or in the subject with epilepsy, or is about 0.9-fold or less, about 0.75-fold or less, about 0.5-fold or less, about 0.25-fold or less, about 0.1-fold or less, about 0.05-fold or less, or about 0.01-fold or less). Furthermore, "not significantly different" in step (F) means that the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is equivalent to the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject (for example, the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is not statistically significantly different from the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the healthy subject, or is more than about 0.8 times and less than about 1.2 times, or more than about 0.9 times and less than about 1.1 times). The concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject before treatment can be a value obtained by measuring the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject before treatment. The concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the epileptic subject can be an average value calculated by measuring the lipid metabolite or nitric oxide metabolite concentration in advance by collecting biological samples from multiple epileptic subjects. In addition, the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample from the healthy subject can be an average value calculated by measuring the concentrations of the lipid metabolite or nitric oxide metabolite in biological samples collected in advance from a plurality of healthy subjects. In addition, the subject to be treated in the method of one embodiment of the present invention is preferably a subject suffering from epilepsy.In addition, in one embodiment of the present invention, a "subject suffering from epilepsy" includes a subject whose results of other testing methods indicate epilepsy, and preferably a subject who has been diagnosed by a physician or veterinarian as having epilepsy.

[0035] The treatment for epilepsy, the therapeutic effect of which can be judged by the judgment method of one embodiment of the present invention, includes, for example, drug therapy. In one embodiment of the present invention, drug therapy includes treatment with a therapeutic drug for epilepsy. In one embodiment of the present invention, the therapeutic drug for epilepsy includes an antiepileptic drug. In one embodiment of the present invention, examples of the antiepileptic drug include Na channel inhibitors (e.g., carbamazepine, phenytoin, lamotrigine, zonisamide, etc.), Ca channel inhibitors (e.g., ethosuximide, gabapentin, etc.), glutamate receptor inhibitors (e.g., topiramate, perampanel, etc.), synaptic vesicle release inhibitors (e.g., levetiracetam, etc.), GABA transaminase inhibitors (e.g., valproic acid, etc.) or GABA analogues (e.g., gabapentin, pregabalin, etc.) (these compounds may be salts thereof).

[0036] According to the determination method of one embodiment of the present invention, the therapeutic effect can be determined in a subject who has received treatment for epilepsy, and therefore the effectiveness of the treatment for epilepsy administered to the subject can be verified. If no therapeutic effect is observed, the treatment can be immediately discontinued and a different treatment plan can be made. Therefore, the determination method of one embodiment of the present invention is advantageous in that it can prevent unnecessary medication, which can contribute to reducing medical costs and the burden on patients. The determination method of one embodiment of the present invention is useful for preventing and managing epilepsy.

[0037] In another aspect, an embodiment of the present invention provides an epilepsy marker comprising a lipid metabolite or a nitric oxide metabolite, or the use of a lipid metabolite or a nitric oxide metabolite as an epilepsy marker. In one embodiment of the present invention, the "epilepsy marker" comprises a substance whose presence or amount is an indicator of the presence or absence of epilepsy and the severity of the symptoms, and can be used as a marker for detecting, identifying, evaluating, etc. epilepsy. That is, according to one embodiment of the present invention, a lipid metabolite or a nitric oxide metabolite can be used as a disease identification marker for epilepsy, and a lipid metabolite or a nitric oxide metabolite can be used to evaluate the severity of epilepsy.

[0038] Since the determination method according to one embodiment of the present invention can be used to determine the effectiveness of a therapeutic drug or alleviator for epilepsy, one embodiment of the present invention can also provide a screening method for therapeutic drugs or alleviators for epilepsy. That is, according to another aspect, one embodiment of the present invention provides a screening method for therapeutic drugs or alleviators for epilepsy, comprising the steps of (G) administering a candidate therapeutic drug or alleviator for epilepsy to a subject, and (H) measuring the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from the subject. This screening method can further include a step of determining whether or not the candidate therapeutic drug or alleviator for epilepsy has a therapeutic effect based on the concentration of the lipid metabolite or nitric oxide metabolite measured in the step (H). This step may include a step of (I) comparing the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample from the subject after administration of the candidate therapeutic drug or alleviator for epilepsy with the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject. Here, it may be shown that the candidate therapeutic agent or alleviator has a therapeutic effect if the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample of a subject after administration of the candidate therapeutic agent or alleviator is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a subject before administration of the candidate therapeutic agent or alleviator or the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a subject suffering from epilepsy, or if it is not significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject. That is, the screening method of one embodiment of the present invention may further include a step of (J) determining that a therapeutic effect is present when the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample of a subject after administration of a candidate therapeutic drug or alleviator for epilepsy is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a subject before administration of the candidate therapeutic drug or alleviator for epilepsy or the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a subject suffering from epilepsy (preferably, when the concentration is significantly different in a direction approaching the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject), or when the concentration is not significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject, thereby making it possible to select a candidate having a therapeutic effect.The term "significantly different" in step (J) means that the concentration of the lipid metabolite or nitric oxide metabolite is either higher or lower than that of the subject or the subject suffering from epilepsy before administration of the candidate therapeutic or alleviator, and for example means that the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is lower, preferably significantly lower, than the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the subject or the subject suffering from epilepsy before administration of the candidate therapeutic or alleviator (for example, the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is statistically significantly lower than the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of the subject or the subject suffering from epilepsy before administration of the candidate therapeutic or alleviator, or is about 0.9-fold or less, about 0.75-fold or less, about 0.5-fold or less, about 0.25-fold or less, about 0.1-fold or less, about 0.05-fold or less, or about 0.01-fold or less). Furthermore, "not significantly different" in step (J) means that the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is equivalent to the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of a healthy subject (for example, the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of the subject is not statistically significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in the biological sample of a healthy subject, or is more than about 0.8 times and less than about 1.2 times, or more than about 0.9 times and less than about 1.1 times). The screening method of one embodiment of the present invention can be carried out according to the description of the embodiment of the detection method and determination method of the present invention. In the screening method of one embodiment of the present invention, the subject to which a candidate therapeutic drug or alleviator for epilepsy is administered is preferably a subject suffering from epilepsy. When carrying out the screening method of one embodiment of the present invention, a mammal other than a human can be used as the subject. The therapeutic drug for epilepsy that is the subject of the screening method of one embodiment of the present invention is the same as that described in the determination method of the present invention.In one embodiment of the present invention, the epilepsy alleviating agent includes foods (e.g., food compositions), quasi-drugs (e.g., medicated cosmetics), feeds (e.g., pet foods), cosmetics (e.g., cosmetic compositions), and skin care compositions that have the function of alleviating epilepsy symptoms, and the above foods include supplements, foods for specified health uses, foods with functional claims, and nutritional foods. In addition, the term "alleviation" is used to mean the same as improvement.

[0039] In another aspect, one embodiment of the present invention provides a method for identifying an epilepsy marker in a lipid metabolite or nitric oxide metabolite in a biological sample, comprising the steps of (K) measuring the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from a subject suffering from epilepsy and the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject, and (L) comparing the two measured concentrations. The identification method of one embodiment of the present invention may further include a step of determining the lipid metabolite or nitric oxide metabolite as an epilepsy marker based on the result of the comparison of concentrations performed in the above step (L). In this step, if the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample from the subject suffering from epilepsy is significantly different from the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample from the healthy subject, the lipid metabolite or nitric oxide metabolite is shown to be an epilepsy marker. That is, the identification method of one embodiment of the present invention may further include a step of (M) determining that a lipid metabolite or nitric oxide metabolite is an epilepsy marker when the concentration of the lipid metabolite or nitric oxide metabolite in a biological sample from a subject suffering from epilepsy is significantly different from the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject. The term "significantly different" in step (M) means that the concentration of a lipid metabolite or nitric oxide metabolite is either higher or lower than that of a healthy subject, and for example, means that the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample of a subject is higher than that of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject, preferably significantly higher (for example, the concentration of a lipid metabolite or nitric oxide metabolite in a biological sample of a subject is statistically significantly higher than that of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject, or is about 1.1 times or more, about 1.25 times or more, about 1.5 times or more, about 1.75 times or more, about 2.0 times or more, about 3 times or more, about 5 times or more, about 10 times or more, about 50 times or more, or about 100 times or more). The identification method of one embodiment of the present invention can be carried out according to the description of the embodiment of the detection method and determination method of the present invention.

[0040] A treatment for epilepsy can be administered to a subject in which epilepsy is detected by the detection method of one embodiment of the present invention or a subject diagnosed with epilepsy by the diagnosis method of one embodiment of the present invention. Thus, in another aspect, one embodiment of the present invention provides a method for treating epilepsy, comprising: (A) measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample of the subject; (B) comparing the concentration of the lipid metabolite or the nitric oxide metabolite in the biological sample of the subject with the concentration of the lipid metabolite or the nitric oxide metabolite in the biological sample of the subject with the concentration of the same lipid metabolite or the nitric oxide metabolite in the biological sample of the healthy subject; (C) determining that the subject suffers from epilepsy when the concentration of the lipid metabolite or the nitric oxide metabolite in the biological sample of the subject is significantly different from the concentration of the same lipid metabolite or the nitric oxide metabolite in the biological sample of the healthy subject; and (N) administering treatment for epilepsy to the subject determined to suffer from epilepsy. The steps of detecting and determining epilepsy (i.e., steps (A), (B) and (C)) in the treatment method of one embodiment of the present invention can be carried out according to the description of the embodiment of the detection method of the present invention and the embodiment of the diagnosis method of the present invention. Treatment for epilepsy can be carried out according to the description of the embodiment of the determination method of the present invention.

[0041] In another aspect, one embodiment of the present invention provides a kit for detecting epilepsy, comprising a means for detecting (e.g., a means for measuring concentration (e.g., a means for quantifying)) a ​​lipid metabolite or a nitric oxide metabolite in a biological sample of a subject. The kit of one embodiment of the present invention is typically a kit for detecting epilepsy according to the above-mentioned embodiment of the detection method of the present invention. The means for detecting lipid metabolites includes, for example, a substance that specifically binds to a lipid metabolite, including, for example, an antibody against a lipid metabolite. The means for detecting lipid metabolites also includes a mass spectrometer used in the above-mentioned mass spectrometry. The means for detecting nitric oxide metabolites includes, for example, a reagent (e.g., amines) used in the above-mentioned diazotization reaction absorbance measurement method, chemiluminescence method, electrode method, electron spin resonance method, or Griess method. The kit of one embodiment of the present invention may include a buffer solution or an instruction manual.

[0042] In a kit of one embodiment of the present invention, when the means for detecting a lipid metabolite is an antibody, the kit may contain reagents (and optionally equipment) necessary to measure the concentration of the lipid metabolite by immunoassay using the antibody.

[0043] The kit according to one embodiment of the present invention may be a kit for measuring the concentration of a lipid metabolite by a sandwich method or a competitive method, which may include a microtiter plate, an anti-lipid metabolite antibody for capture, an anti-lipid metabolite antibody labeled with alkaline phosphatase or peroxidase, or a substrate for alkaline phosphatase or a substrate for peroxidase.

[0044] The kit according to one embodiment of the present invention may also be a kit for measuring the concentration of a lipid metabolite by a sandwich method using a secondary antibody. The kit may include a microtiter plate, an anti-lipid metabolite antibody for capture, an anti-lipid metabolite antibody as a primary antibody, and an antibody against the anti-lipid metabolite antibody labeled with alkaline phosphatase or peroxidase as a secondary antibody, or a substrate for alkaline phosphatase or a substrate for peroxidase.

[0045] The above kit can be used, for example, as follows. First, a capture antibody is fixed to a microtiter plate, and a target biological sample is appropriately diluted and added thereto, followed by incubation, removal of the sample, and washing. Next, a primary antibody is added, incubation and washing are performed, and an enzyme-labeled secondary antibody is added and incubated, followed by addition of a substrate to develop color. The concentration of lipid metabolites can be determined by measuring the color development using a microtiter plate reader or the like. When a competitive method is used, a capture antibody is fixed to a microplate, and a biological sample and an enzyme-labeled antigen whose concentration is known in advance are simultaneously reacted in the same microplate. After the reaction, the concentration of lipid metabolites can be determined by detecting the enzyme activity remaining in the microplate.

[0046] In the kit of one embodiment of the present invention, the labeled antibody is not limited to an enzyme-labeled antibody, but may be an antibody labeled with a radioactive substance (25I, 131I, 35S, 3H, etc.), a fluorescent substance (fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescent material, etc.), a luminescent substance (luciferase, luciferin, aequorin, etc.), nanoparticles (gold colloid, quantum dots), etc. Also, a biotinylated antibody may be used as the labeled antibody, and labeled avidin or streptavidin may be added to the kit.

[0047] Further, an example of the kit according to the embodiment of the present invention is a kit for measuring the concentration of lipid metabolites by immunochromatography. This kit can be configured such that an antibody storage section in which a first anti-lipid metabolite antibody labeled with gold colloid or the like is stored is connected to a determination section in which a second anti-lipid metabolite antibody (preferably an antibody that recognizes another epitope of the lipid metabolite) is immobilized in a line on a cellulose membrane or the like, the two sections being connected by a thin groove.

[0048] The above kit can be used, for example, as follows. First, when a biological sample is added to the antibody storage section or the biological sample receiving section adjacent to the antibody storage section, the labeled antibody and the lipid metabolite bind to each other in the antibody storage section to form a lipid metabolite-labeled antibody complex, and the complex moves to the determination section through the groove by capillary action. Next, when the complex is captured by the immobilized second anti-lipid metabolite antibody, a red line appears in the determination section due to the plasmon effect of the gold colloid, and the presence of the lipid metabolite can be detected. In this case, the kit can be provided in the form of a stick, such as a test stick, and the test stick may further include absorbent paper for absorbing the biological sample, a desiccant, etc.

[0049] In the kit according to one embodiment of the present invention, when the detection means for detecting lipid metabolites is a mass spectrometer, an internal standard device may be included in addition to the mass spectrometer. By using an internal standard, the extraction efficiency and ionization efficiency for each analysis can be corrected during measurement by a mass spectrometer. An example of an internal standard used in mass spectrometry is a deuterated lipid metabolite.

[0050] In addition to the above, the kit of one embodiment of the present invention can be carried out according to the description of the embodiment of the detection method and determination method of the present invention.

[0051] In another aspect, one embodiment of the present invention provides a pharmaceutical composition or palliative for use in treating or alleviating epilepsy in a subject, the subject being predicted to have epilepsy by the method according to the embodiment of the detection method described above (e.g., a method for detecting epilepsy comprising a step of measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample of the subject). This pharmaceutical composition or palliative can treat or alleviate a subject suffering from epilepsy. This pharmaceutical composition or palliative may contain any antiepileptic drug, for example, the above-mentioned Na channel inhibitor, Ca channel inhibitor, glutamate receptor inhibitor, synaptic vesicle release inhibitor, GABA transaminase inhibitor, or GABA analog drug. The pharmaceutical composition or palliative may include an anti-epileptic drug, such as, for example, carbamazepine, phenytoin, lamotrigine, zonisamide, ethosuximide, gabapentin, topiramate, perampanel, levetiracetam, valproic acid, gabapentin, pregabalin, docosahexaenoic acid, eicosapentaenoic acid, or salts thereof.

[0052] In another aspect, one embodiment of the present invention provides a method for detecting a lipid metabolite or a nitric oxide metabolite in a biological sample from a subject, the method comprising a step of measuring a concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample from a patient (e.g., a subject suspected of epilepsy or a subject having epilepsy). This method makes it possible to evaluate whether or not a subject has developed epilepsy. This method may comprise a step of obtaining (e.g., collecting) a biological sample from the subject. In carrying out this method, any of the above-mentioned embodiments of the detection method and determination method of the present invention may be employed.

[0053] In one embodiment of the present invention, when using the above-mentioned methods (e.g., epilepsy detection method, diagnosis method, treatment effect evaluation method, treatment method), markers, kits, or pharmaceutical compositions, for example, detection of lipid metabolites or nitric oxide metabolites in a biological sample is performed. 1α、 MUO may be detected by measuring the concentrations of 20-carboxy-AA, 9-HODE, or lyso-PAF. - , NO3 - MUO or IE may be detected by measuring the concentration of PGF in plasma. 1α MUO may be detected by measuring the concentration of 20-carboxy-AA, OEA, or 11,12-DHET in plasma. IE may be detected by measuring the concentration of NO2 in plasma. - MUO may be detected by measuring the levels of the lipid metabolites or nitric oxide metabolites in the test subject. When the levels of the lipid metabolites or nitric oxide metabolites in the test subject are significantly higher than those in healthy subjects, the test subject may be evaluated as suffering from MUO or IE.

[0054] In one embodiment of the present invention, "measuring the concentration" includes detecting that a sample contains a lipid metabolite or nitric oxide metabolite at a certain concentration or more, measuring the concentration qualitatively or quantitatively, or measuring the amount. In one embodiment of the present invention, "significantly" may be evaluated using the Kruskal-Wallis test, Steel-Dwass multiple comparison test, or Pearson's correlation analysis, and may be in a state of p<0.05 or p<0.01. Alternatively, it may be in a state of substantial difference.

[0055] All publications and publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "or."

[0056] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may be adopted. Furthermore, the present invention may be adopted in combination with each of the configurations or features described in the above embodiments, or independently. EXAMPLES

[0057] The present invention will be further explained below with reference to examples, but is not limited to these.

[0058] In this example, the concentrations of lipid metabolites and nitric oxide metabolites in cerebrospinal fluid (CSF) and plasma of dogs with meningoencephalitis of unknown origin (MUO) and idiopathic epilepsy (IE) were measured using a liquid chromatography tandem mass spectrometer and a NOx analyzer, respectively. The results and experimental method are shown below.

[0059] result Sample information A total of 32 dogs were included in this study: 11 with IE, 12 with MUO, and 9 clinically healthy dogs.

[0060] Dog breeds included beagles (n=9), chihuahuas (n=9), toy poodles (n=6), Maltese poodles (n=2), papillons (n=2), pugs (n=2), Yorkshire terriers (n=1), and mixed breeds (n=1). There were 11 males (7 neutered) and 21 females (14 neutered). Median age was 5.0 years (range: 1-14 years) and median weight was 4.2 kg (range: 1.9-11.3 kg). Of the 23 symptomatic dogs, 13 had generalized seizures, 3 had focal seizures, and 7 had no seizures but had other neurological abnormalities such as paralysis, lameness, head tilt, or blindness. Median time since last seizure was 12.5 days (range: 1-26 days). Seven of the 11 dogs diagnosed with IE and four of the 12 dogs diagnosed with MUO were already prescribed antiepileptic drugs (AEDs), and one dog with IE and seven dogs with MUO were prescribed prednisolone.

[0061] Lipid production profile in CSF Based on our previous study using urine samples (Takenouchi S. et al. J. Vet. Med. Sci. 84, 689-693 (2022)), 196 lipid metabolites were measured in CSF and plasma using LC-MS / MS. Some samples were not collected for this study. Substantial plasma samples were not stored from 13 dogs, and CSF samples were not stored from 4 dogs. Of the 196 lipids, 127 were not detected in CSF, whereas 69 were within the standard reference range.

[0062] Of these, four types of lipids in the CSF were significantly different between the groups. Figure 1 shows these lipid metabolites along with a schematic diagram of lipid metabolism. 6-Keto-prostaglandin F, a metabolite of PGI2, 1α (6-keto-PGF 1αCSF levels of 20-carboxyarachidonic acid (20-carboxy-AA), 9-hydroxyoctadecadienoic acid (9-HODE), a metabolite of linoleic acid, and lyso-platelet activating factor (lyso-PAF) were significantly higher in the MUO group than in the control group. Lyso-PAF levels were also significantly higher in the MUO group than in the IE group (p<0.05) and were positively correlated with CSF cell counts (R=0.926, p<0.001, Figure 2). The lyso-PAF measured in this study was lyso-PAF C-16.

[0063] Lipid production profile in plasma Of the 114 lipid metabolites detected in plasma, only four were significantly different between groups (Figure 3). Plasma concentrations of 20-carboxy-AA were higher in IE dogs than in healthy control dogs, but not in MUO dogs. Similarly, plasma concentrations of oleoylethanolamide (OEA) and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET) were higher in IE dogs than in healthy control dogs. Furthermore, concentrations of 11,12-DHET were higher in IE dogs than in MUO dogs. PGF, a metabolite of dihomo-γ-linolenic acid, was also higher in IE dogs than in MUO dogs. 1α Plasma concentrations of were statistically higher in dogs with MUO than in IE and healthy control dogs (p<0.05).

[0064] NOx production in CSF or plasma NOx levels were assessed in CSF and plasma samples (Figure 4). In dogs with MUO and IE, NO2 - , NO3 - and their total CSF levels were higher than those of healthy control dogs. - was higher in MUO dogs than in IE and healthy control dogs, but NO3 - There were no significant differences between groups in levels or the sum of NO2- and NO3-.

[0065] Experimental Method Animals and sample collection This study was a retrospective, single-center study conducted at the University of Tokyo Animal Medical Center from October 2018 to April 2020. All patients were client-owned dogs. CSF and plasma samples from clinical cases were initially collected for diagnosis by veterinarians. After use for the initial purpose, samples were stored when a significant amount remained and approved for use in research. Clinically healthy dogs were kept at the Veterinary Surgery Laboratory of the University of Tokyo as a control group. Written informed consent was obtained from all owners for use in research after clinical use. All methods followed the relevant guidelines and regulations (University of Tokyo Animal Experimentation Committee; P18-115, 21-020), and this study was conducted in accordance with the ARRIVE guidelines.

[0066] All patients were clinically diagnosed with IE or MUO at our hospital. The diagnostic procedure for IE at Tier II confidence level was performed according to De Risio, L. et al. BMC Vet. Res. 11, 148 (2015). Neurological examination, cranial MRI, and CSF analysis were normal, and reactive seizures were excluded by medical history, physical examination, and blood tests. MUO was diagnosed according to Granger, N. et al. Vet. J. 184, 290-297 (2010). Clinical signs and MRI appearances, such as breed predisposition, were observed, but other causes of responsive seizures and structural epilepsy were negative.

[0067] Plasma and CSF samples were obtained as clinically indicated. Blood was collected from the vein into heparinized tubes and centrifuged at 500×g for 5 min. The supernatant was stored at −80°C as plasma samples. CSF samples were collected by puncture of the cerebellum-medullary cisterna oblongata with a 23-gauge or 25-gauge needle under isoflurane anesthesia. Routine analysis was performed, and the supernatant was collected after centrifugation and stored at −80°C as CSF samples.

[0068] Comprehensive analysis of lipids by LC-MS / MS CSF samples were centrifuged at 20,000×g for 10 min at 4°C, and 50 μl of the supernatant was mixed with 750 μl of 1% formic acid and 10 μl of internal standard solution. This mixture was applied to a solid-phase extraction cartridge (OASIS μElution plate (Waters, MA, USA)) pretreated with 200 μl of methanol and distilled water. Plasma samples were centrifuged in the same manner as CSF, and 100 μl of the supernatant was mixed with 100 μl of organic solvent (methanol:acetonitrile = 1:1, v / v, containing 5N hydrochloric acid) and 10 μl of internal standard solution for deproteinization. After centrifugation, 100 μl of the supernatant was mixed with 700 μl of 1% formic acid. The mixture was applied to an Oasis PRiME HLB cartridge (Waters, MA, USA).

[0069] After washing with 200 μl of distilled water and 200 μl of hexane, the lipid fraction of the CSF or plasma samples was eluted with 50 μl of methanol. The eluted sample solution was filtered through an Ultrafree-MC Centrifugal Filter (Merck KGaA, Darmstadt, Germany). The collected analytes were used for measurement.

[0070] Five microliters of the analyte was injected into a high-performance liquid chromatograph (Nexera 2, Shimadzu, Kyoto, Japan) equipped with a mass spectrometer (LCMS-8060, Shimadzu). 196 lipid mediators were measured and analyzed using Method Package for Lipid Mediators Version 3 with LabSolutions software (Shimadzu). Each lipid was identified by its retention time and selected reaction monitor ion transition. The level of each lipid mediator in the sample was evaluated by comparing the peak area ratio calculated by the following formula: peak area of ​​each metabolite / peak area of ​​internal standard.

[0071] NOx measurement Nitrite ion (NO2 - ) and nitrate ion (NO3 -For the measurement of NOx, 30 μl of CSF or plasma samples were used with an ENO-20 NOx analyzer (Eicom, Kyoto, Japan) according to the manufacturer's instructions. Sodium nitrite and sodium nitrate (Wako, Osaka, Japan) were used as standard solutions.

[0072] statistical analysis All statistical analyses were performed using XLSTAT Life Science (version 2021.2.2.1141, Addinsoft, Paris, France), an add-in for MS Excel (Microsoft Corporation, Redmond, Washington, US). Kruskal-Wallis test, Steel-Dwass multiple comparison test, and Pearson correlation analysis were used for comparisons between groups. The significance level was set at p-values ​​< 0.05 for all statistical comparisons.

[0073] We confirmed the elevation of several specific lipid and nitric oxide metabolites in the CSF and plasma of dogs with MUO or IE. This indicates that lipid and nitric oxide metabolites in the CSF and plasma may be useful biomarkers for the diagnosis of CNS diseases, and that lipid profiles may be related to the pathophysiological mechanisms of these diseases. In particular, the method of detecting specific lipid and nitric oxide metabolites in plasma is an innovative method for diagnosing CNS diseases without the use of anesthesia or sedation. Because canine epileptic seizure disorders are excellent translational models for humans, these results are mutually beneficial for human medicine.

[0074] The embodiment has been described above. However, this embodiment is merely an example, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A method for detecting epilepsy, comprising a step of measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample from a subject.

2. The detection method according to claim 1, wherein the lipid metabolite is an arachidonic acid metabolite, a linoleic acid metabolite, a dihomo-γ-linolenic acid metabolite, a lyso-platelet activating factor, a platelet activating factor, or an oleoylethanolamide.

3. The lipid metabolite is 6-keto-prostaglandin F 1α , 20-carboxyarachidonic acid, 11,12-dihydroxyeicosatrienoic acid, 9-hydroxyoctadecadienoic acid, prostaglandin F 1α 3. The method according to claim 1, wherein the antibody is selected from the group consisting of lyso-platelet activating factor, ... and oleoylethanolamide.

4. The nitric oxide metabolite is NO 2 - Or NO 3 - The detection method according to any one of claims 1 to 3,

5. The detection method according to any one of claims 1 to 4, wherein the step includes a step of measuring a concentration of the lipid metabolite.

6. The detection method according to any one of claims 1 to 5, wherein the step includes a step of measuring the concentrations of the lipid metabolites and the nitric oxide metabolites.

7. The process comprises: 2 - Or NO 3 - or NO concentration in blood samples 2 - The method according to any one of claims 1 to 6, further comprising a step of measuring the concentration of

8. The detection method according to any one of claims 1 to 7, wherein the epilepsy is idiopathic epilepsy (IE) or meningoencephalitis of unknown origin (MUO).

9. The detection method according to any one of claims 1 to 8, further comprising a step of comparing the concentration of a lipid metabolite or nitric oxide metabolite in the subject's biological sample with the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample from a healthy subject.

10. The detection method according to any one of claims 1 to 9, wherein the subject is indicated to be suffering from epilepsy when the concentration of lipid metabolites or nitric oxide metabolites in the subject's biological sample is higher than the concentration of the same lipid metabolites or nitric oxide metabolites in a biological sample from a healthy subject.

11. The detection method according to any one of claims 1 to 10, wherein the biological sample is a body fluid.

12. The detection method according to any one of claims 1 to 11, wherein the biological sample is a cerebrospinal fluid sample or a blood sample.

13. A method for determining the effectiveness of a treatment for epilepsy, comprising a step of measuring the concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample from a subject.

14. The detection method according to claim 13, wherein the lipid metabolite is an arachidonic acid metabolite, a linoleic acid metabolite, a dihomo-γ-linolenic acid metabolite, a lyso-platelet activating factor, a platelet activating factor, or an oleoylethanolamide.

15. The lipid metabolite is 6-keto-prostaglandin F 1α , 20-carboxyarachidonic acid, 11,12-dihydroxyeicosatrienoic acid, 9-hydroxyoctadecadienoic acid, prostaglandin F 1α , lyso-platelet activating factor, or oleoylethanolamide.

16. The nitric oxide metabolite is NO 2 - Or NO 3 - The method according to any one of claims 13 to 15,

17. The method of any one of claims 13 to 16, further comprising a step of comparing the concentration of a lipid metabolite or nitric oxide metabolite in the biological sample of the subject with the concentration of the same lipid metabolite or nitric oxide metabolite in a biological sample of a healthy subject.

18. The method of any one of claims 13 to 17, wherein a therapeutic effect is indicated when the concentration of the lipid metabolite or nitric oxide metabolite in the subject's biological sample is lower than the concentration of the lipid metabolite or nitric oxide metabolite in the subject's biological sample before treatment or the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of a subject suffering from epilepsy, or is not significantly different from the concentration of the lipid metabolite or nitric oxide metabolite in the biological sample of a healthy subject.

19. The method according to any one of claims 13 to 18, wherein the biological sample is a body fluid.

20. The method according to any one of claims 13 to 19, wherein the biological sample is a cerebrospinal fluid sample or a blood sample.

21. Markers of epilepsy, including lipid metabolites or nitric oxide metabolites.

22. 22. The marker of claim 21, wherein the lipid metabolite is an arachidonic acid metabolite, a linoleic acid metabolite, a dihomo-gamma-linolenic acid metabolite, a lyso-platelet activating factor, a platelet activating factor, or an oleoylethanolamide.

23. The lipid metabolite is 6-keto-prostaglandin F 1α , 20-carboxyarachidonic acid, 11,12-dihydroxyeicosatrienoic acid, 9-hydroxyoctadecadienoic acid, prostaglandin F 1α 23. The marker of claim 21 or 22, which is lyso-platelet activating factor or oleoylethanolamide.

24. The nitric oxide metabolite is NO 2 - Or NO 3 - The marker according to any one of claims 21 to 23,

25. A kit for detecting epilepsy, comprising a means for quantifying lipid metabolites or nitric oxide metabolites in a biological sample from a subject.

26. A method for screening for therapeutic or alleviating agents for epilepsy, comprising the steps of administering a candidate therapeutic or alleviating agent for epilepsy to a subject and measuring the concentration of lipid metabolites or nitric oxide metabolites in a biological sample from the subject.

27. A pharmaceutical composition for use in treating epilepsy in a subject, the subject being predicted to have epilepsy by the method of any one of claims 1 to 12.

28. A method for treating epilepsy, comprising the steps of: (A) measuring a concentration of a lipid metabolite or a nitric oxide metabolite in a biological sample of a subject; (B) comparing the concentration of the lipid metabolite or the nitric oxide metabolite in the biological sample of the subject with the concentration of the same lipid metabolite or the nitric oxide metabolite in a biological sample of a healthy subject; (C) determining that the subject is suffering from epilepsy if the concentration of the lipid metabolite or the nitric oxide metabolite in the biological sample of the subject is higher than the concentration of the same lipid metabolite or the nitric oxide metabolite in the biological sample of the healthy subject; and (N) administering treatment for epilepsy to the subject determined to be suffering from epilepsy.

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