Use of neogenin as brain injury diagnostic marker induced by epilepsy

Abstract

To provide a use of neogenin as a diagnostic marker for brain injury induced by epilepsy.SOLUTION: When a brain injury is induced by epilepsy, an amount of cleaved neogenin increases. Therefore, it is possible to easily and rapidly diagnose brain injury induced by epilepsy using the composition of the present invention, and the neogenin can be used as a target for developing a therapeutic agent for diseases caused by brain injury.SELECTED DRAWING: Figure 1c

Description

[Technical Field]

[0001] The present invention relates to a composition for diagnosing epilepsy-induced brain damage, a diagnostic method comprising the same, and Diagnostic kits provide information needed to diagnose epilepsy-induced brain damage Methods for screening for substances for the prevention or treatment of epilepsy-induced brain damage and This includes information on how to do this.

[0002] The present invention is disclosed in Korean Patent Application No. 10-2021- filed on April 16, 2021. This application claims priority from US Pat. No. 6,005,003, filed on Oct. 1, 2003, and all of the information disclosed in the specification and drawings of that application. The entire contents of which are incorporated herein by reference. [Background technology]

[0003] Epilepsy occurs when some nerve cells produce excessive electrical current for a short period of time. A group of chronic disorders characterized by recurrent attacks and resulting in neurobiological, psychological, cognitive, and social changes. It is a serious neurological disorder that involves aging.

[0004] Epilepsy is associated with Alzheimer's disease and stroke. It is the third most common neurological disorder, affecting approximately 0.5-2% of the world's population. In addition, new cases occur at a rate of approximately 45 per 100,000 people worldwide each year, and in Japan It is estimated that there are approximately 300,000 to 400,000 epilepsy patients in Japan.

[0005] On the other hand, hippocampal sclerosis, which is primarily characterized by neuronal death, erosis) is a lesion commonly observed in patients with temporal lobe epilepsy, and is characterized by long-term recurrent seizures. can be considered as the cause.

[0006] Hippocampal sclerosis is a disease that reduces the size of the hippocampus, and is known as a sequela of epilepsy. In addition to memory loss, psychiatric disorders such as depression and anxiety may occur, and in severe cases, death may occur. This can sometimes lead to...

[0007] Therefore, protecting neurons from excessive seizures due to epilepsy may contribute to the development of the hippocampus. There is a need to find ways to prevent loss of functionality and miswiring.

[0008] The underlying mechanism of hippocampal neuronal death is excitotoxic necrosis. oxic necrosis and gene-dependent Programmed via apoptotic signaling It has all the characteristics of programmed cell death. This provides patients with drugs that can prevent excitotoxic neuronal death while suppressing seizures. This would allow for more effective treatment of epilepsy.

[0009] On the other hand, neogenin, a receptor belonging to the IgG superfamily, has four types ( Ig-like loop domain, fibronectin III domain, Tra the nsmembrane domain and the intracellular domain, the P series domain) It has a major domain and functions as a splice enzyme in various signal pathways. There are multiple splice sites (spl) that allow for various cleavage patterns. It contains the ice site, which is involved in the differentiation of neuronal stem cells, the mediator of ion functions in the liver, It is known that it can have a wide variety of functions, including as a breast morphogen. (Cole SJ, Bradford D and Cooper HM, 200 7; Wilson NH and Key B, 2007).

[0010] Currently, the CA3 subfield of the hippocampus and the subgranular zone Neogenin is expressed in the ar zone, which is the area where neuronal stem cells and granule neurons (g It is thought that neogenin is also expressed in the nucleus neurons.

[0011] However, until now, it has not been known that neogenin is related to brain damage. stomach. Summary of the Invention [Problem to be solved by the invention]

[0012] The present inventors have developed a method for early diagnosis of epilepsy-induced brain damage and a method for predicting the same. As a result of intensive research to develop a drug that can prevent or treat brain damage caused by epilepsy, When the cleavage of neogenin is suppressed, the amount of the cleaved form of neogenin increases. The present invention was accomplished by confirming that necroptosis is reduced in the hippocampus.

[0013] Therefore, an object of the present invention is to provide a method for producing truncated neogenin. In the case of epilepsy-induced brain damage, the active ingredient is a drug that can measure the level of The present invention provides a wound diagnostic composition.

[0014] Another object of the present invention is to provide a method for treating epilepsy-induced brain damage in a population of epilepsy patients. A diagnostic composition, said composition comprising truncated neogenin The present invention is characterized by containing as an active ingredient a preparation capable of measuring the level of α- and β-glucan (α-glucan), β-glucan (α-glucan), and β-glucan (α-glucan), i.e., ... The object of the present invention is to provide a composition that

[0015] A further object of the present invention is to provide a method for treating epilepsy comprising administering the composition of the present invention as an active ingredient. The present invention provides a diagnostic kit for brain damage induced by steroids.

[0016] It is still another object of the present invention to provide a method for the treatment of cleaved nucleotides in a biological sample isolated from a subject. measuring the level of truncated neogenin. The objective of this invention is to provide a method that provides the information necessary for diagnosing induced brain damage.

[0017] It is yet another object of the present invention to provide a method for treating (a) status epilepticus (b) treating a biological sample isolated from a patient with a candidate substance; Measure the level of truncated neogenin in the sample. (c) reducing the level of cleaved neogenin compared to a group not treated with the candidate substance; said substance is a substance for the prevention or treatment of epilepsy-induced brain damage. The present invention relates to a method for screening a substance for preventing or treating epilepsy-induced brain damage, the method comprising the step of: The present invention provides a method for reducing the amount of waste generated by a fuel cell.

[0018] Yet another object of the present invention is to provide a method for treating a psoriasis-associated rheumatoid arthritis (SA) comprising: and a pharmaceutical composition for preventing or treating epilepsy-induced brain damage, comprising: That is the thing.

[0019] However, the technical problems that the present invention aims to solve are not limited to the problems mentioned above, Other problems not mentioned above will become clear to those skilled in the art from the following description. [Means for solving the problem]

[0020] To achieve the above object, the present invention provides a method for producing truncated neogenin. Epilepsy-induced seizures containing a preparation that can measure the level of eogenin as an active ingredient The present invention provides a composition for diagnosing brain injury.

[0021] In one embodiment of the present invention, the truncated neogenin is a compound that inhibits epilepsy-induced The level may be increased during the occurrence of brain injury, but is not limited to this. do not have.

[0022] In another embodiment of the invention, the epilepsy is status epilepticus. The present invention may be, but is not limited to, Streptomyces pilepticus (SE).

[0023] In yet another embodiment of the present invention, the level of the cleaved neogenin can be measured. The agent may be an antibody or aptamer specific to said truncated neogenin, It is not limited to this.

[0024] The present invention also provides a method for diagnosing epilepsy-induced brain damage in epilepsy patients. A composition for the treatment of rheumatoid arthritis, the composition comprising truncated neogenin A composition comprising a preparation capable of measuring the level of erythropoietin (neogenin) as an active ingredient. The product is provided.

[0025] The present invention also provides a method for treating epilepsy-induced seizures, comprising administering the composition of the present invention as an active ingredient. We provide a brain injury diagnostic kit.

[0026] The present invention also provides a method for detecting cleaved neogenin ( The method includes measuring the level of truncated neogenin. The present invention provides a method for providing information necessary for diagnosing brain damage induced by steroids.

[0027] The present invention also provides a method for detecting cleaved neogenin ( The method includes measuring the level of truncated neogenin. The present invention provides a method for diagnosing brain damage induced by steroids.

[0028] In one embodiment of the present invention, the subject may be a mammal, including a human. is not limited to.

[0029] In another embodiment of the present invention, the biological sample may be a hippocampal-derived sample, It is not limited to this.

[0030] In yet another embodiment of the present invention, the method further comprises measuring the cleaved neogenin. If the level of neogenin is increased compared to the level of truncated neogenin in the control group, epilepsy may result. The method may further include, but is not limited to, determining that the brain damage is induced by the It is not something that can be done.

[0031] In yet another embodiment of the present invention, the control group is a normal individual or a subject with status epilepticus ( isolated from epilepsy patients without status epilepticus (SE) It may be, but is not limited to, a biological sample.

[0032] In yet another embodiment of the present invention, the level of truncated neogenin is measured by Western blotting. blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoassay Radioimmunodiffusion and immunoprecipitation analysis oprecipitation assay) It may be measured, but is not limited to this.

[0033] The present invention also provides a method for the preparation of truncated neogenin. The present invention provides a diagnostic use of a preparation capable of measuring the signal level for epilepsy-induced brain damage.

[0034] The present invention also provides a method for treating (a) status epilepticus; (SE) treating a biological sample isolated from a patient with a candidate substance; (b) isolating said sample; measuring the level of truncated neogenin; and (c) reducing the level of cleaved neogenin compared to a control untreated with the candidate substance. The substance is determined to be a substance for preventing or treating epilepsy-induced brain damage. screening for substances for the prevention or treatment of epilepsy-induced brain damage, including This provides a method for

[0035] In one embodiment of the present invention, the sample may be, but is not limited to, a sample derived from the hippocampus. It is not something that is done.

[0036] In yet another embodiment of the present invention, the measurement in step (b) is performed by Western blotting, ELISA(enzyme linked immunosorbent assay) , radioimmunoassay (RIA), radioimmunoassay (RA) immunodiffusion, and immunoprecipitation analysis The present invention may be selected from, but is not limited to, the group consisting of: It's not something like that.

[0037] The present invention also provides a method for treating a rheumatoid arthritis, comprising the steps of: A pharmaceutical composition for preventing or treating epilepsy-induced brain damage is provided.

[0038] In one embodiment of the present invention, the inhibitor is DAPT(N-[N-(3,5-Difl uorophenacetyl)-L-alanyl]-S-phenylglycin The terpolymer may be, but is not limited to, ethyl eth-butyl ester.

[0039] The present invention also provides a composition containing a neogenin cleavage inhibitor as an active ingredient. The method comprises administering a composition to an individual in need thereof to treat epilepsy-induced brain damage. A method for preventing or treating wounds is provided.

[0040] The present invention also provides a composition containing a neogenin cleavage inhibitor as an active ingredient. The present invention provides a method for preventing or treating epilepsy-induced brain damage by administering the composition of the present invention to a patient.

[0041] The present invention also provides a method for producing a drug for treating epilepsy-induced brain damage. The present invention provides a use of a neogenin cleavage inhibitor. [Effects of the Invention]

[0042] The present invention relates to the use of neogenin for diagnosing epilepsy-induced brain damage. Furthermore, the diagnostic composition of the present invention can be used to quickly and accurately diagnose and predict brain damage at an early stage. This may be a target for the development of therapeutic agents for epilepsy-induced brain damage. It has the effect of being utilized in this way.

[0043] Furthermore, DAPT, a γ-secretase inhibitor, has been shown to reduce the risk of epileptic seizures. Effectively inhibits the production of truncated neogenin and necroptosis Therefore, the pharmaceutical composition for preventing or treating brain damage induced by epilepsy is effective. It is expected that it can be used effectively as a component. [Brief explanation of the drawings]

[0044] [Figure 1] Figure 1 shows the expression of neogenin in the hippocampus on days 3 and 7 after pilocarpine-induced status epilepticus (SE). Specifically, Figure 1a shows the results of double immunofluorescence of neogenin and GFAP after pilocarpine-induced SE (scale bar = 50 μm). Figure 1b shows the results of Western blot analysis of full-length neogenin and truncated neogenin on days 3 and 7 after pilocarpine-induced SE (full-length neogenin: 240 kDa; truncated neogenin: 160 kDa). Figure 1c shows the quantification of full-length neogenin (left) and truncated neogenin (right) on days 3 and 7 after pilocarpine-induced SE. [Figure 2]Figure 2 shows the results of confirming the expression of neogenin in the hippocampus using an in vitro SE (status epilepticus) model. Specifically, Figure 2a shows the results of double immunofluorescence for neogenin and MAP2 in neurons after 24 hours of kainic acid treatment in cultured hippocampal cells (scale bar = 20 μm). Figure 2b shows the quantification of neogenin and MAP2 expression levels in neurons after 24 hours of kainic acid treatment in cultured hippocampal cells. Figure 2c shows the results of double immunofluorescence for neogenin and GFAP in astrocytes after 24 hours of kainic acid treatment in cultured hippocampal cells (scale bar = 50 μm). Figure 2d shows the quantification of neogenin and GFAP expression levels in astrocytes after 24 hours of kainic acid treatment in cultured hippocampal cells. [Figure 3] Figure 3 shows necroptosis in the hippocampus after pilocarpine-induced SE. Specifically, Figure 3a shows the results of cresyl violet staining of the CA3 region of the hippocampus and the dentate gyrus hilus on days 3 and 7 after SE (scale bar = 100 μm). Figure 3b shows the results of Western blotting for pMLKL, a molecular marker of necroptosis, on days 3 and 7 after SE. Figure 3c shows the quantification of pMLKL expression, a molecular marker of necroptosis, on days 3 and 7 after SE. [Figure 4]Figure 4 shows the relationship between inhibition of neogenin cleavage and hippocampal damage. Specifically, Figure 4a shows the results of double immunofluorescence for neogenin and MAP2 after treatment of cultured hippocampal cells with NMDA and / or DAPT1 (neogenin cleavage inhibitors) (scale bar = 20 μm). Figure 4b shows the results of MTT analysis after treatment of cultured hippocampal cells with NMDA and / or DAPT1 (*p<0.05, **p<0.01 vs. control group; #p<0.05 vs. NMDA-treated group). Figure 4c shows the results of Western blot analysis (left) and quantification (right) for MLKL and the necroptosis inhibitor pMLKL after treatment of cultured hippocampal cells with NMDA and / or DAPT1. [Figure 5] FIG. 5 shows the signaling pathway downstream of neogenin leading to necroptosis after acute insult. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present inventors have developed a method for early diagnosis of epilepsy-induced brain damage and a method for predicting the same. As a result of intensive research to develop a drug that can prevent or treat brain damage caused by epilepsy, When the cleavage of neogenin is suppressed, the amount of the cleaved form of neogenin increases. The present invention was accomplished by confirming that necroptosis is reduced in the hippocampus.

[0046] The present invention will be described in detail below.

[0047] According to one embodiment of the present invention, pilocarpine is administered to treat seizures. We confirmed that the truncated form of neogenin was increased in the hippocampus of an animal model of cancer induction. This was confirmed (see Experimental Example 1).

[0048] According to another embodiment of the present invention, cultured rat hippocampi are treated with kainic acid to induce epilepsy. Induction of seizures resulted in decreased expression of neogenin in both neurons and astrocytes. (See Experimental Example 2).

[0049] According to yet another embodiment of the present invention, an animal in which epilepsy is induced by administering pilocarpine is In the hippocampus of the model, epileptic seizures induce necroptosis, resulting in hippocampal damage. It was confirmed that (see Experimental Example 3).

[0050] According to yet another embodiment of the present invention, neurotoxicity is induced by inhibiting the cleavage of neogenin. protects neurons from epilepsy and reduces necroptosis induced by epileptic seizures It was confirmed that this was possible (see Experimental Example 4).

[0051] These results led us to conclude that if epilepsy induces brain damage, We confirmed that the amount of cleaved neogenin in cells increased compared to when it was not present. Therefore, neogenin can be used as a diagnostic marker to diagnose the onset of brain damage. I confirmed the fact.

[0052] Furthermore, the present inventors have found that when DAPT, a neogenin cleavage inhibitor, is treated, SE expression is suppressed. The expression of pMLKL, a marker of postnatal necroptosis, This suggests that brain damage is prevented by inhibiting the production of the truncated form of neogenin. It was confirmed that the severity of the wound can be prevented, improved, or treated.

[0053] Therefore, the present invention provides a method for the preparation of truncated neogenin. For diagnosing epilepsy-induced brain damage, containing a formulation capable of measuring levels as an active ingredient A composition may be provided.

[0054] As used herein, the term "neogenin" refers to the NEO 1 gene. It is a protein encoded by the ATPase inhibitor ATPase, which is highly expressed in the hippocampus. GenBank: AAC51287.1, GenBank: AAB17263.1 It is a protein known to have a unique sequence, and there are many other proteins that correspond to it in other organisms besides humans. The neogenin may include, without limitation, all neogenins derived from individuals of the same kind.

[0055] Unless otherwise specified in the present invention, neogenin is the full-length uncleaved form (fu ll-length) neogenin.

[0056] As used herein, the term "truncated neogenin" refers to enin) is a splice enzyme of various signaling pathways. It may be one of several truncated forms, preferably one of the four truncated sites in neogenin. It targets the P domain of the GABA receptor and induces the proteolytic enzyme γ-secretase (GAMMA). It may also be in a form in which the P domain is cleaved by a-secretase.

[0057] The "P domain" is the intracellular domain of neogenin. It consists of P1, P2, and P3 domains, and there are also corresponding proteins in non-humans. Conserved domains in neogenins derived from various individuals, such as the P domain, The domain may be a domain having the sequence of SEQ ID NO: 1 or 2.

[0058] According to one embodiment of the present invention, the truncated neogenin comprises the P1 domain portion of the P domain. The molecular weight of the full-length neogenin was 240 kDa, but the P1 domain was The molecular weight of neogenin from which the cleaved amino acid moiety was found to be 160 kDa (see FIG. 5).

[0059] In the present invention, the truncated neogenin is a compound that inhibits epilepsy-induced brain damage. The level may increase when generated.

[0060] As used herein, the term "epilepsy" refers to an abnormal overactivity of the brain. Hypersynchronization causes some nerve cells to generate excessive electrical currents in a short period of time, resulting in repeated seizures (seizures). A group of chronic disorders that cause neurobiological, psychological, cognitive, and social changes. Epilepsy is a serious neurological disorder that causes abnormal hyperexcitability of nerve cells. Excitotoxicity due to excitability is induced The hippocampus is one of the brain regions where pathological damage caused by epilepsy is common. hippocampal sclerosis (hippocampal sclerosis), collagenization within (gliosis), abnormal neurogenesis and dentate gyrus granule cell abnormal cell architecture (cytoarchitectural abnormal ity of dentate granule cells) and abnormal synaptic circuit patterns aberrant synpatic circuits may occur, As the above-mentioned pathological phenomena in the hippocampus progress, chronic epileptic seizures (CHRO) nic epileptic seizures occur, resulting in cognitive and memory impairment as well as This can lead to the development of formulation-refractory, intractable epilepsy that does not respond to drug treatment.

[0061] In the present invention, the epilepsy is status epilepticus. icus;SE).

[0062] The above-mentioned "status epilepticus (SE)" means Epileptic seizures lasting more than 30 minutes or repeated seizures without regaining consciousness It means a state in which

[0063] In the present invention, the epilepsy may occur in mammals including humans, For example, the mammal is a human, a dog, a cat, a mouse, a rabbit, a horse, a sheep, or a hamster. , hedgehog, ferret, or guinea pig may be.

[0064] As used herein, the term "necroptosis" refers to the process by which protoplasts It means programmed necrosis, which appears as a result of cell death. Unlike apoptosis, necroptosis is caused by the catabolism of caspases. This is cell death that occurs without caspase activation and occurs primarily in pathological conditions.

[0065] In the present invention, the preparation capable of measuring the level of the cleaved neogenin is The antibody or aptamer may be specific to the selected neogenin.

[0066] As used herein, the term "antibody" refers to a specific antibody directed against an antigenic site. For purposes of the present invention, an antibody refers to a protein molecule specifically directed against a marker protein. This refers to antibodies that bind to the antibody, and includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. In addition, a part of a whole antibody may also be included in the antibody of the present invention, as long as it has antigen-antibody binding ability. Any kind of immunoglobulin that specifically binds to the truncated neogenin of the present invention is included. For example, intact antibodies having two full-length light chains and two full-length heavy chains are included. Not only the antibodies but also functional fragments of antibody molecules, i.e., Fab, which have antigen-binding function, The antibodies of the present invention include F(ab'), F(ab')2, and Fv. Any antibody capable of specifically binding to neogenin, such as a humanized antibody or a chimeric antibody, may be used. Individual and recombinant antibodies are also included.

[0067] As used herein, the term "aptamer" refers to a molecule that binds to a target molecule in a sample. A substance that can specifically bind to the analyte to be analyzed and has a stable tertiary structure by itself. By meaning double-stranded nucleic acid (DNA, RNA, or modified nucleic acid), we specifically target The presence of the protein can be confirmed. Aptamers can be produced by a general method for producing aptamers. The oligonucleotides are selective for the target protein to be confirmed and have high binding strength. After determining and synthesizing the nucleotide sequence, the 5' and 3' ends of the oligonucleotides are -SH, -COOH, -OH or NH 2, but is not limited to this.

[0068] The composition of the present invention, which comprises an antibody specific to neogenin, detects known proteins. The method may further include the preparations necessary for sensing known proteins using the composition. The method may be used without limitation to detect neogenin in a subject (in the present invention, a biological sample obtained from a subject). The level of

[0069] As used herein, the term "diagnosis" refers to the diagnosis of a subject for a particular disease or disorder. to determine the susceptibility of a subject to a particular disease Determining whether or not you currently have a disease or illness, or whether or not you have contracted a particular disease or illness Determining a subject's prognosis, or therametics etrics) (e.g., monitoring the state of an object to provide information on therapeutic efficacy) (including monitoring).

[0070] The present invention also provides a method for diagnosing epilepsy-induced brain damage in epilepsy patients. A composition for the treatment of rheumatoid arthritis, the composition comprising truncated neogenin A composition comprising a preparation capable of measuring the level of erythropoietin (neogenin) as an active ingredient. The composition can be provided.

[0071] In the present invention, the patient group is a group of patients with status epilepticus. The patient population may also be a group of patients suffering from SE.

[0072] The present invention also includes a composition for diagnosing epilepsy-induced brain damage according to the present invention. A kit for diagnosing brain damage caused by epilepsy can be provided.

[0073] The kit of the present invention includes not only an antibody that recognizes a target protein as a marker, but also and one or more other component compositions, solutions, or devices suitable for the analytical method. That's fine.

[0074] The present invention also provides a method for detecting cleaved neogenin ( The method includes measuring the level of truncated neogenin. This may provide a method for providing the information necessary to diagnose brain damage induced by steroids.

[0075] The present invention also provides a method for detecting cleaved neogenin ( The method includes measuring the level of truncated neogenin. The present invention provides a method for diagnosing brain damage induced by steroids.

[0076] The method for diagnosing epilepsy-induced brain damage according to the present invention is This allows for rapid diagnosis before brain damage worsens, and allows for immediate action such as administering therapeutic drugs. It is possible to respond.

[0077] Therefore, the method for diagnosing epilepsy-induced brain damage according to the present invention is These compounds may also be effectively used to treat brain damage induced by steroids.

[0078] In the present invention, the subject may be a mammal, including a human, and may be a person suffering from epilepsy. The individual may be one who has or is suspected of having an induced brain injury.

[0079] In the present invention, the biological sample may be a sample derived from the hippocampus.

[0080] In the present invention, the measured level of cleaved neogenin is Increased levels of genin compared with normal levels of genin are associated with epilepsy-induced brain damage. The method may further include the step of determining.

[0081] In the present invention, the control group is a group of normal individuals or individuals with status epilepticus. It refers to biological samples isolated from epilepsy patients without epilepsy (SE). do.

[0082] In the present invention, the level of the cleaved neogenin is measured by the protein synthesis method known in the art. There are no particular limitations on the method of measurement as long as it is a method for measuring expression of proteins. blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoassay Radioimmunodiffusion and immunoprecipitation analysis oprecipitation assay) Good too.

[0083] The term "measurement" as used herein means measuring a desired substance (a marker protein in the present invention). To measure and confirm the presence (expression) of a desired substance or the level of a desired substance The meaning of the present invention includes measuring and confirming changes in the expression level of the target gene. Measuring the expression level of a protein means measuring the presence or absence of expression (i.e., expression (measure the presence or absence of) or measure the qualitative or quantitative change level of said protein The measurement may be carried out by any method, including, without limitation, qualitative (analytical) and quantitative methods. The types of qualitative and quantitative methods for measuring protein levels are well known in the art. These include the experimental methods well known in the art and described herein. Protein level comparison methods are well known in the art. The detection of the protein can be performed by detecting the presence or absence of cleaved neogenin or by detecting the amount of expression of the protein. This means checking for an increase (upregulation) or decrease (downregulation).

[0084] In the present invention, the biological sample is used to diagnose epilepsy-induced brain damage. Anything collected from a subject for this purpose may be used without limitation, for example, tissue, cells, etc. The biological tissue may include cells, blood, serum, plasma, saliva, and urine, and is preferably a biological tissue. a sample or cell sample, such as a cell, tissue, organ, or fine needle aspirate from diseased tissue; Consists of core needle biopsy specimens and vacuum aspiration biopsy specimens The biological sample may be used for detection or diagnosis. It may be pretreated before use, for example by homogenization, filtration, This may include distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. To increase the detection sensitivity of protein markers, for example, The samples were analyzed by anion exchange chromatography, affinity chromatography, and size exclusion chromatography. Size exclusion chromatography ), liquid chromatography, sequential extraction ) or gel electrophoresis.

[0085] The present invention also provides a method for treating epilepsy-induced brain damage, comprising the steps of: It is possible.

[0086] (a) Cleaved neogenin in a biological sample isolated from a subject measuring the level of ATP-dependent neogenin;

[0087] (b) The level of cleaved neogenin was compared with the level of cleaved neogenin in the control group. determining that the brain damage is epilepsy-induced if the level of the brain damage increases with the level of the epilepsy-induced brain damage; and

[0088] (c) Treating epilepsy-induced brain damage.

[0089] In the present invention, the treatment in the step (c) may be a drug treatment, a surgical treatment, a vagus nerve stimulation treatment, or the like. Methods such as surgery and the ketogenic diet may also be used.

[0090] In the present invention, the drug is an anticonvulsant, such as Tegretol. l), Dilantin, Mysolin, Orwheel (Orfil), phenobarbital, Rivotril ivotril, Zarontin, Sabril, Lami Lamictal, Zonisamide, Topamax opamax, Trileptal, and Neurontin The anticonvulsant may be one or more anticonvulsants selected from the group consisting of It is not limited.

[0091] In the present invention, the drug contains the neogenin cleavage inhibitor of the present invention as an active ingredient. The pharmaceutical composition may be, but is not limited to, a pharmaceutical composition containing the compound.

[0092] In the present invention, the surgical treatment includes temporal lobectomy, epileptogenic lesion removal, localized brain ablation, and the like. One or more of the following procedures are performed: cortical resection, epileptic tract blockade, and hemispherectomy. It may be present, but is not limited to this.

[0093] The present invention also provides a method for treating (a) status epilepticus; (SE) treating a biological sample isolated from a patient with a candidate substance; (b) isolating said sample; measuring the level of truncated neogenin; and (c) reducing the level of cleaved neogenin compared to a control untreated with the candidate substance. The substance is determined to be a substance for preventing or treating epilepsy-induced brain damage. screening for substances for the prevention or treatment of epilepsy-induced brain damage, including The present invention may provide a method for performing the above-mentioned processing.

[0094] In the present invention, the sample may be a sample derived from the hippocampus.

[0095] In the present invention, the measurement in the step (b) is carried out by Western blot, ELISA (enzyme-linked immunosorbent assay), or the like. yme linked immunosorbent assay), radioimmunoassay (R IA, radioimmunoassay, radial immunodiffusion diffusion, immunoprecipitation analysis ssay).

[0096] The present invention also provides a method for treating a rheumatoid arthritis, comprising the steps of: A pharmaceutical composition for preventing or treating epilepsy-induced brain damage may be provided.

[0097] In the present invention, the inhibitor is a gamma-secretase inhibitor. ase inhibitor), for example, (5S)-(tert-butoxycarbonyl) (4R)-hydroxy-(2R)-benzylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanonyl )-L-Leucyl-L-phenylalaninamide ((5S)-(tert-butoxyc arbonylamino)-6-phenyl-(4R)-hydroxy-(2R) -benzylhexanoyl)-L-leu-L-phe-amide,L-685 ,458),(S,S)-2-[2-(3,5-difluorophenyl)-acetylamino ]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[ e][1,4]diazepin-3-yl)-propionamide ((S,S)-2-[2-( 3,5-difluorophenyl)-acetylamino]-N-(1-me thyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo [e][1,4]diazepin-3-yl)-propionamide,comp ound E), and DAPT (N-[N-(3,5-Difluorophenace tyl)-L-alanyl]-S-phenylglycinet-butyl es ter), and preferably DAPT (N-[N-(3,5- Difluorophenacetyl))-L-alanyl]-S-phenylg lycinet-butyl ester), but is not limited thereto, Any substance that can inhibit the cleavage of genin may be included.

[0098] In the present invention, a "pharmaceutical composition" refers to a pharmaceutical composition produced for the purpose of preventing or treating a disease. Each of these may be formulated into various forms by conventional methods and used. Oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups It may be formulated into various dosage forms, and is used in the form of an external preparation, suppository, and sterile injection solution. That's fine.

[0099] The pharmaceutical composition according to the present invention can be prepared by adding suitable carriers, excipients, etc., which are commonly used in the manufacture of pharmaceutical compositions. The excipient may further include, for example, a diluent, a binder, a disintegrant, The group consisting of lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives It may be one or more selected from:

[0100] The pharmaceutical compositions of the present invention can be formulated into powders, granules, sustained-release granules, or the like by conventional methods. , enteric-coated granules, liquids, eye drops, elucic preparations, emulsions, suspensions, alcohol preparations, lozenges , perfumes, lemonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, Soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts , dry extract, fluid extract, injection, capsule, perfusion solution, plaster, lotion, Pastes, sprays, inhalants, patches, sterile injections, or topical preparations such as aerosols The topical preparation may be formulated and used in the form of a cream, gel, patch, spray, or the like. ointments, plasters, lotions, liniments, pastes or cataplasms, etc. The dosage form may be:

[0101] Carriers, excipients and diluents that may be included in pharmaceutical compositions according to the present invention include lactose, lactic acid bacteria ... Dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol ethanol, erythritol, maltitol, starch, acacia gum, alginate, gelatin Calcium phosphate, calcium silicate, cellulose, methyl cellulose, Amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil .

[0102] When formulating a drug, the fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. that are usually used are The formulation is prepared using a diluent or excipient such as an antiperspirant.

[0103] As an additive for the tablets, powders, granules, capsules, pills and lozenges according to the present invention, Corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose , D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate Sodium, Calcium Sulfate, Sodium Chloride, Sodium Bicarbonate, Purified Lanolin, Microcrystalline Cellulose, dextrin, sodium alginate, methylcellulose, carboxymethyl Sodium cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl Starch, hydroxypropyl methylcellulose (HPMC), HPMC1928, HP MC2208, HPMC2906, HPMC2910, propylene glycol, casein , calcium lactate, excipients such as Primozel, gelatin, gum arabic, ethanol, Tempura flour, cellulose acetate phthalate, carboxymethyl cellulose, carboxymethyl cellulose Calcium caseinate, glucose, purified water, sodium caseinate, glycerin, stearic acid, Sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose Microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl cellulose lacquer, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropyl Cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl Binders such as pyrrolidone may be used, hydroxypropyl methylcellulose, thiazolinone, Corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl Turch, sodium carboxymethylcellulose, sodium alginate, carboxymethyl Chilled cellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid , 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate , formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum ( Guar gum), baking soda, polyvinylpyrrolidone, calcium phosphate, gelling starch, Rubia gum, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white Disintegration of sugar, magnesium aluminum silicate, D-sorbitol liquid, hard anhydrous silicic acid, etc. Agent, calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil (Hydrogenated vegetable oil), talc, lycopodium, kaoli Ingredients include: petrolatum, sodium stearate, cocoa butter, sodium salicylate, salicylic acid Magnesium, polyethylene glycol PEG4000, PEG6000, liquid paraffin , hydrogenated soybean oil (Lubri wax), aluminum stearate, stearate Lead, sodium lauryl sulfate, magnesium oxide, macrogol, Synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, Raffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, acetic acid Lubricants such as sodium, sodium oleate, dl-leucine, and hard anhydrous silica are used. may be used.

[0104] The additives of the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monosodium glutamate, and the like. Sucrose stearates, polyoxyethylene sorbitol fatty acid esters (twin Esters), Polyoxyethylene monoalkyl ethers, Lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, hydroxide Sodium, Propylene Glycol, Polyvinylpyrrolidone, Ethylcellulose, Carboxymethyl Sodium cellulose and the like may also be used.

[0105] The syrup of the present invention may contain a solution of sucrose, other sugars or sweeteners. Fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as needed. may be used.

[0106] The emulsion of the present invention may contain purified water, and may contain, as needed, emulsifiers, preservatives, stabilizers, Fragrances and the like may also be used.

[0107] Suspensions according to the present invention include those containing acacia, tragacanth, methylcellulose, carboxymethylcellulose, cellulose, sodium carboxymethylcellulose, microcrystalline cellulose, alginate Sodium acetate, hydroxypropyl methylcellulose (HPMC), HPMC1828, Suspending agents such as HPMC2906 and HPMC2910 may be used, and if necessary, Surfactants, preservatives, stabilizers, coloring agents, fragrances may also be used.

[0108] The injections according to the present invention contain distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, Injection, Dextrose Injection, Dextrose + Sodium Chloride Injection, Peasy ( PEG), lactated Ringer's injection, ethanol, propylene glycol, fixed oil Canola oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, myristate Solvents such as isopropyl, benzene benzoate, sodium benzoate, sodium salicylate , sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene Glycol, twines, nidic acid amide, hexamine, dimethylacetamide, etc. solubilizers, weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia), Niacin and ammonium acetate), organic compounds, proteins, albumin, peptones, gums buffering agents such as sodium chloride, isotonicity agents such as sodium sulfite (NaHSO3) and dihydrogen peroxide Carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2S O3), nitrogen gas (N2), stabilizers such as ethylenediaminetetraacetic acid, sodium bisulfite 0.1% sodium formaldehyde sulfoxylate, thiourea, ethylenediaminetetraacetic acid Sulfating agents such as disodium aminetetraacetate and sodium acetone bisulfite, Dimethyl alcohol, chlorobutanol, procaine hydrochloride, glucose, calcium gluconate Pain-relieving agents such as CMC sodium, sodium alginate, Twin 80, monosteryl A suspending agent such as aluminum phosphate may also be included.

[0109] The suppository of the present invention contains cocoa butter, lanolin, witepsol, polyethylene glycol cellulose, glycerol, gelatin, methylcellulose, carboxymethylcellulose, stearic acid and oleic acid mixture, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa Butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate Twin or Span, Imhausen, Monolen (Monosteer Propylene glycol phosphate), glycerin, Adeps solidus us), Butyrum Tego-G, Cebesperma 16 bes Pharma 16), Hexalide Base 95, Cotomar, Hydrocoat SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Hydropostol (Id ropostal), Massa estrarium (A, AS , B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospos Tal-En, Paramount-B, Sposhiro (OSI, OSIX, A, B, C, D, H, L ), Suppository base IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299) , Spostal (N, Es), Wecoby (W, R, S, M, Fs), Tezestar Triglise A base such as Lido base (TG-95, MA, 57) may also be used.

[0110] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. Such a solid formulation may contain at least one or more excipients, such as starch, calcium carbonate, or the like, in addition to the extract. calcium carbonate, sucrose It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0111] Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, and syrups. In addition to the commonly used simple diluents water and liquid paraffin, various excipients, e.g. Wetting agents, sweeteners, flavoring agents, preservatives, etc. may also be included. This includes solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. The agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. may be used.

[0112] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. "Practically effective amount" means a dose sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level depends on the type and severity of the patient's disease, the activity of the drug, and the sensitivity to, administration time, administration route and excretion rate, treatment period, and concurrently used drugs may be determined by factors including and other factors well known in the medical field. .

[0113] The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents. The conventional therapeutic agent may be administered sequentially or simultaneously, either singly or in combination. Taking all of the above factors into consideration, the maximum dose can be achieved with the minimum amount without side effects. It is important to administer an amount that provides an effect, and this is well known in the art to which the present invention pertains. This may be readily determined by one of ordinary skill in the art.

[0114] The pharmaceutical compositions of the present invention may be administered to an individual by a variety of routes. As can be expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, spinal injection Peripheral space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, oral or nasal spray, dermal administration, transdermal administration, etc. This may also be done.

[0115] The pharmaceutical composition of the present invention may be selected based on the disease to be treated, the route of administration, the age, sex, weight and disease of the patient. It is determined by the type of drug that is the active ingredient, along with various related factors such as the severity of the condition. .

[0116] The present invention also provides a composition containing a neogenin cleavage inhibitor as an active ingredient. The method comprises administering a composition to an individual in need thereof to treat epilepsy-induced brain damage. A method for preventing or treating wounds is provided.

[0117] The present invention also provides a composition containing a neogenin cleavage inhibitor as an active ingredient. The present invention provides a method for preventing or treating epilepsy-induced brain damage by administering the composition of the present invention to a patient.

[0118] The present invention also provides a method for producing a drug for treating epilepsy-induced brain damage. The present invention provides a use of a neogenin cleavage inhibitor.

[0119] In the present invention, an "individual" is an individual in need of treatment for epilepsy-induced brain damage. It refers to a subject, more specifically a human or non-human primate, mouse, By "animals" is meant mammals such as animals, rats, dogs, cats, horses, and cows.

[0120] In the present invention, "administration" refers to providing a specific composition of the present invention to an individual by any suitable method. It means to do.

[0121] As used herein, the term "prevention" refers to the prevention of the occurrence of epilepsy-induced brain damage. By preemptively administering a pharmaceutical composition according to the present invention before the onset of epilepsy, brain damage induced by epilepsy can be prevented. This refers to any action that inhibits or delays the injury caused by an epileptic seizure. It inhibits necroptosis, a type of cell death that occurs in neurons. This includes protecting neurons by

[0122] As used herein, the term "treatment" refers to the treatment of a patient with a disease or condition resulting from the administration of a pharmaceutical composition according to the present invention. Anything that reverses or favorably modifies the symptoms of epilepsy-induced brain damage means the act of

[0123] The present invention also provides a method for treating a rheumatoid arthritis, comprising the steps of: A food composition for preventing or ameliorating epilepsy-induced brain damage can be provided.

[0124] When the neogenin cleavage inhibitor of the present invention is used as a food additive, The inhibitors may be added neat or with other foods or food ingredients, and are typically The amount of the active ingredient to be mixed may vary depending on the purpose of use (prevention, health or Generally, when producing a food or beverage, the amount of the present invention may be determined appropriately depending on the intended purpose (therapeutic treatment). The neogenin cleavage inhibitor is used in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, it is not permitted to add any additives for health and hygiene purposes or for health regulation purposes. In the case of long-term intake, the amount may be less than the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount greater than the above range.

[0125] There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, Sausages, bread, chocolate, candies, snacks, sweets, pizza, ramen Other noodles, gum, dairy products including ice cream, various soups, drinking water, There are teas, energy drinks, alcoholic beverages and vitamin complexes, and they are health supplements in the usual sense. Includes all functional foods.

[0126] The health drink composition according to the present invention can be prepared in the same manner as a conventional drink, by adding various flavorings or natural carbohydrates. The natural carbohydrates mentioned above may include glucose and fructose. monosaccharides such as maltose and sucrose; disaccharides such as dextrose; Polysaccharides such as cellulose and cyclodextrins, and xylitol, sorbitol, Sweeteners include thaumatin, stevia extract, and sugar alcohols such as erythritol. Natural sweeteners such as cereals and synthetic sweeteners such as saccharin and aspartame are used. The proportion of the natural carbohydrates is generally about 0.05g per 100mL of the composition of the present invention. 1 to 0.20 g, or about 0.04 to 0.10 g.

[0127] In addition to the above, the compositions of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, , pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters Adjustments, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. In addition, the composition of the present invention may contain natural fruit juice, fruit juice, It may also contain fruit pulp for the production of juice and vegetable drinks. The proportions of such additives are not critical, but Generally, the amount is selected from the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the invention. It is target.

[0128] In the present invention, the food composition may be a functional health food composition, It is not limited.

[0129] In this specification, the term "healthy functional food" refers to a food for specified health uses (FOSHU). It is the same term as "special health use (FoSHU)" and is used for not only providing nutrition but also for It means food with high medical and therapeutic effects that is processed to efficiently function in the body. The food product is formulated into tablets, capsules, powders, granules, etc. to provide a useful effect in preventing or improving obesity. It may be prepared in various forms such as granules, liquid, pills, etc.

[0130] The health functional food of the present invention can be produced by a method commonly used in the art. During production, raw materials and ingredients that are commonly added in the industry may be added. Unlike pharmaceuticals, it is made from food ingredients and is said to be free of side effects that can occur when taking pharmaceuticals for a long period of time. It has the advantage of being highly portable.

[0131] The terms used in the present invention are currently as broad as possible while taking into consideration the functions in the present invention. The general terms used have been chosen without regard to the intention or intent of those skilled in the art. This may change depending on precedents, the emergence of new technologies, etc. In addition, in certain cases, the applicant may choose In some cases, the meaning of the term is specified in detail in the description of the invention. Therefore, the terms used in the present invention are not merely names of terms, but also have meanings that the terms have. It should be defined based on taste and the overall context of the present invention.

[0132] Throughout the specification of the present invention, when a part "comprises" a certain element, this is Unless otherwise stated, the term "component" refers to a device that includes other elements and does not exclude other elements. The terms "about," "degree," and "degrees" used throughout the present specification mean that "Substantially" and the like are intended to be examples of the manufacturing and material tolerances inherent in the stated meaning. The numerical values ​​are used in the sense of numerical values ​​or their approximate values, and are used in the sense of accurate and absolute values ​​to facilitate understanding of the present invention. To prevent unscrupulous infringers from exploiting disclosures that refer to quantitative figures. Used for.

[0133] Throughout the present specification, the term "combinations of these" contained in Markush-style expressions is used. The term "se" is selected from the group of components listed in the Markush form of expression. It means a mixture or combination of one or more of the above elements, selected from the group consisting of the above elements. "includes one or more of the following" means "includes one or more of the following"

[0134] (Mode for carrying out the invention)

[0135] Preferred examples are presented below to aid in understanding the present invention. The following examples are provided so that the present invention may be more readily understood. The contents of the disclosure are not limited.

[0136] [Example] Example 1. Experimental animals Six-week-old C57BL / 6N male mice (Koatech, Gyeonggi-do, Republic of Korea) and Spr We used 18-day-old embryonic Guinea-Dawley rats (Samtako, Gyeonggi-do, Republic of Korea) as the target animals. The ambient temperature is 22±1℃ and the lighting time is 12 hours (lights on from 8:00 to 20:00). The animals were kept with access to food and water. Animal experiments were conducted in accordance with the ethical committee of the Catholic University of Japan ( CUMS-2020-0103-01) and the Animal Care Committee of the Animal Research Institute of Dankook University (DK U-16-024) and has been approved by the National Institute of Health for the Care and Use of Laboratory Animals. The study was carried out in accordance with the guidelines (NIH Publication No. 80-23, revised 1996).

[0137] Example 2. Preparation of an epilepsy-induced mouse model The pilocarpine-induced epilepsy model has been previously reported. As shown (Cho KO et al., 2015; Jang HJ et al., 20 19;Jeong KH et al.,2011;Kim JE and Cho K Specifically, mice were administered scopolamine methyl nitrate (sc opolamine methyl nitrate;catalog no.S225 0; Sigma-Aldrich, St. Louis, MO) 2 mg / kg and tert-butyl terbutaline hemisulfate salt; catalog no. P6503; Sigma-Aldrich, California)2 mg / kg each was intraperitoneally administered, and 30 minutes later, pilocarpine hydrochloride (pilocarpine hydrochloride) was administered. pine hydrochloride; catalog no. P6503; Missouri Pilocarcinoma (Sigma-Aldrich, St. Louis, CA) was administered at 280 mg / kg. After pin administration, the severity and duration of behavioral seizures were monitored for approximately 3 hours. The Racine Scale (Racine RJ, 1972) was used to determine the cial clonus, stage 2: head nodding, stage 3: forelim b clonus, stage 4: rearing, stage 5: rearing and falling g). Mice with persistent generalized convulsive seizures (stages 3 to 5) were classified as SE (Status Encephalopathy). This was considered to be indicative of status epilepticus and was discarded for further study. Three hours after the onset of SE, 10 mg of diazepam was administered. The seizure was terminated by intraperitoneal administration of 100 mg / kg. After the seizure was terminated, the temperature was maintained at 100°C to promote recovery. Place the animals in an incubator at 30°C for 2 days or until normal body temperature is reached. They were then allowed to recover by providing wet chow until they could tolerate it. The animals were reared in the rearing environment described in Example 1.

[0138] Example 3. Primary cell culture Hong N, Kim MH, Min CK, Kim H The method was modified from that described in J and Lee JH, 2017. 16. Maternal Sprague-Dawley rats were anesthetized with 5% urethane and 18-day-old fetuses were explanted from the uterus. Rat fetuses were obtained. The hippocampus was separated from the brain of the rat fetus using forceps under a stereomicroscope. Separate and place in HEPES-buffered Hanks' salt solution (20 mM HEPES, 137mM NaCl, 1.3mM CaCl2, 0.4mM Mg SO4,0.5mM MgCl2,5.0mM KCl,0.4mM KH2PO4,0 0.6mM Na2HPO4, 3.0mM NaHCO3 and 5.6mM Glucose The cells were placed in a 5 ml pipette and flame-narrowed pipette. Dissociate by trituration with a stool pipette, pellet by centrifugation, and then add 2% B-27 supplement ement, 0.25% Glutamax I, and penicillin, streptomycin, and amphotericin B at 100 U / ml and 1 0.00μg / ml and 0.025μg / ml, without L-glutamine, pH 7. Dissociated cells were resuspended in 0.2 mg / ml of matrimonial medium. Matrigel (BD Biosciences, Bedford, Massachusetts, USA) A 25 mm circular cover glass precoated with CI (science) was used. Plate at a density of 110,000 cells in 10% CO2 and 90% air. The cells were grown at 37°C in a humidified atmosphere. Fresh medium was added by exchanging 75% of the medium. Medium was fed on days 3, 7, and 10. Cells used in experiments were mitotic for at least 12 days. The cells were cultured without mitotic inhibitors.

[0139] Hippocampal astrocytes (Min JO et al., 2015) The dissociated cells were cultured using a modified method. Plate at a density of 25 flasks per day, humidified with 10% CO2 and 90% air. The cells were grown in a humidified atmosphere at 37°C. Fresh medium was supplied every 3–4 days until the cells reached maturity. n / EDTA (catalog no. T4174; St. Louis, MO, Sigma a-Aldrich), diluted 1:9, plated, and stored for approximately 1 hour before use. The cells were grown to 80-90% confluence. , ~95% pure astrocytes, GFAP (glial fibrillary acid Immunopositivity for antibodies against idic protein ), but not markers of neurons or oligodendrocytes. showed no immunoreactivity to

[0140] Example 4. MTT assay Rat hippocampal neurons were cultured in a 96-well plate at 1.4 × 10 4 cells density The cells were seeded with 100 μM NMDA (N- methyl-D-aspartate; catalog no. M3262; Missouri Sigma-Aldrich, St. Louis, MO) and / or 1 μM DAPT (N-[N -(3,5)-Difluorophenacetyl)-L-alanyl]-Sp henylglycine t-butyl ester;catalog no.D5 942; Sigma-Aldrich, St. Louis, MO) treated neurons MTT (3-[4,5-dimethylthiazol-2-yl]methylthiazol-2-yl) was incubated at a final concentration of 0.5 mg / ml for 4 h. azol-2-yl-2,5-dipheyltetrazolium bromide Catalog no. M5655; Sigma-Aldrich, St. Louis, Missouri The solution was then removed and the mixture was placed in a humidified bath. Dimethyl sulfoxide was added to produce an insoluble purple The formazan product was dissolved. The absorbance was measured on a microplate Measurement was performed at a wavelength of 570 nm using a reader. Formazan absorbance in the cells indicates 100% viability. .

[0141] Example 5. Immunofluorescence and Histological Evaluation d histologic assessment) Mice were anesthetized on day 3 or 7 after SE and treated with 0.1 M phosphine at pH 7.4. The hatch buffer contains saline and 4% paraformaldehyde. aldehyde) was transcardially perfused. The brains were removed and fixed in paraformaldehyde for 24 hours, then placed in 30% sucrose for 3 days. The tissue was then cryoprotected with sucrose solution. Tek (Sakura Finetechnical, Tokyo, Japan) Then, serial coronal sections (30 μm thick) were cut into Sectioning was performed in a cryostat and further processed for staining.

[0142] To perform double immunofluorescence, The tissue sections were incubated with 10% normal goat serum in 0.01M PBS for 1 hour. After incubation with Neogenin (1:200; Litho, Colorado) overnight at 4°C, Luton, Novus Biologics) and GFAP (1:400; California (Chemicon International, Inc., Temecula) antibodies and incubations. The next day, the tissue sections were incubated with Alexa Fluor 488-c conjugated anti-rabbit IgG (1:300; California, Carlsbad, Invitrogen) and Cy3-conjugated anti -mouse IgG (1:500; Jackson, West Grove, Pennsylvania) Finally, the cells were incubated with a confocal microscope (ImmunoResearch). Confocal microscope (LSM 700; Jena, Germany) The tissue sections were mounted in a microscope (Carl Zeiss, Co., Ltd.) and observed.

[0143] For histological evaluation, tissues were placed on slides and analyzed at 100%, 95%, 90%, 80%, and 100% resolution. After rehydration in 10% and 70% ethanol and tap water for 3 minutes each, 0.1% cresyl violet staining solution for 1 minute Solution (catalog no. C5042; Sigma-Aldrich, St. Louis, MO) Excessive staining was prevented by incubation in 95% ethanol and 0.1% ice vinegar. After removal with glacial acetic acid, the slides were then stored in 100% ethanol, 50% ethanol / xylene, and 100% xylene solutions. The tissue sections were then dehydrated and mounted in DPX mountant (catalog no. 06522; Mounted upright (Sigma-Aldrich, St. Louis, MO). Visualization was performed using a microscope (BX51; Olympus Corp., Tokyo, Japan). did.

[0144] Example 6. Immunocytochemistry Hippocampal neurons were fixed in methanol at -20°C for 10 min and then incubated for 5 min. After permeabilizing the cells with 0.3% Triton X-100, they were incubated in 10% PBS for 1.5 hours. Blocking was performed with ovine serum albumin. , in a solution supplemented with 0.2% bovine serum albumin. anti-MAP2 (catalog no. M9942; St. Louis, MO, USA) igma-Aldrich) or anti-GFAP (Ballinn, Massachusetts) Millipore) and rabbit anti-neogen (Dallas, Texas) Santa Cruz Biotechnology, Inc.) antibody (1:100 After washing with PBS, the cells were incubated overnight at 4°C with Ale xa Fluor 488-conjugated anti-rabbit IgG( Invitrogen) and Alexa Fluor 555-conjugated Immunoprecipitation with anti-mouse IgG (Invitrogen) for 1.5 hours at room temperature After washing with PBS, the cell nuclei were counterstained with DAPI. Next, the coverslip was placed in Vector Stain. ld mounting medium (California, Burlingame, Vecto The slides were mounted on Alexa Fluor 4000 (Alexa Fluor 4000) and Alexa Fluor 4000 (Alexa Fluor 4000) slides. Fluor 488 (excitation, 488 nm; emission, >519 nm) and Alexa Flu or 555 (excitation, 555 nm; emission, >565 nm) labeled neurons. Observation was performed using a spot microscope (LSM 700; Carl Zeiss, Co., Ltd.).

[0145] Example 7. Western Blot Chilled brain tissue ProEX TM CETi lysis buffer The lysates were lysed using a lysate preparation (TransLab Biosciences, Daejeon, Republic of Korea). The supernatant was collected for protein extraction by centrifugation. The same amount of protein (30-40 μg) was added to 8% SDS-polyacrylamide gel. The gel was separated and the membrane was then coated with polyvinylidene difluoride (PVDF). The membrane was then transferred to a 0.1% fluoride membrane. 3% bovine in Tris-buffered saline containing Tween-20 After blocking with serum albumin, anti- eogenin (1:2,000; Novus Biologicals), anti- MLKL(mixed lineage kinase domain-like ps eudokinase) (1:1,000; Danvers, Massachusetts, Cell Signaling Technology), and anti-pMLKL (phosp horylated MLKL)(1:1,000;Cell Signaling T The membrane was then incubated with the antibody (antibody) at 4°C overnight. Horseradish peroxidase-conjugated s Common anti-rabbit or anti-mouse antibody (1:2, 000; Novus Biologicals) for 1 hour. . enhanced chemiluminescence detection ki The bands were detected using a ELISA kit (Elpis Biotech, Inc., Daejeon, Republic of Korea). After that, the loading control group was nal anti-β(1:2,000;Santa Cruz Biotechnol ogy, Inc.) or anti-α tubulin antibody (1:2, 000; Santa Cruz Biotechnology, Inc.) The lot was reprobed. Relative quantification of band immunoreactivity was performed using Image Densitometric analysis was performed using a Quant LAS 4000 (Fujifilm, Tokyo, Japan). This was carried out by

[0146] Example 8. Statistical Analysis All data are expressed as mean ± SEM. All statistical analyses were performed using Prism 9 ( GraphPad Software, Inc., La Jolla, CA For in vivo Western blot analysis, the ROUT test (Q = 1%) was performed. ) based on the Kolmogorov-Smirnov or The Shapiro-Wilk normality test was performed. (truncated form of neogenin) In the case of Tasset, the Kruskal-Wallis test was followed by Dunn's post A hoc test was performed. For datasets that passed the normality test, the same SDs ( One-W according to the assumption of standard deviations ay (MLKL and pMLKL) or Brown-Forsythe (full-length neogenin ) After performing ANOVA, Benjamini, Krieger, and Yekutiel A post hoc two-stage step-up method was performed. For tro experiments, statistical analysis was performed using Student's t-test for single comparisons or Multiple statistical comparisons were performed using one-way methods with Bonferroni post-test. y ANOVA was used. A p-value < 0.05 indicates statistical significance. It was considered to be there.

[0147] [Experimental Example] Experimental Example 1. Neogenin expression in the hippocampus after pilocarpine-induced SE After acute pilocarpine-induced seizures, neogenin expression was confirmed in the hippocampus. As a result of the fluorescence, as shown in Figure 1a, neurons and GFAP- Neogenin immunoreactivity was observed in both astrocytic and astrocyte-positive cells. This effect persisted for up to 7 days after pilocarpine administration. Western blot results (Fig. 1b) As shown in Fig. 1c and 1d, the full-length nerve was formed on the third day after pilocarpine administration. The levels of ogenin were shown to be decreased when compared with the levels in the sham group. On the other hand, recovery was confirmed after 3 and 7 days after pilocarpine-induced SE. The levels of the cleaved form of neogenin were significantly elevated in the eyes, but this was not observed in the sham group. The results showed that acute seizures induced by pilocarpine were not observed in the hippocampus. It is shown that it induces the cleavage of neogenin.

[0148] Experimental Example 2. Neogenin expression in hippocampal cells cultured in an in vitro seizure model It is commonly used to create an SE model to generate an in vitro seizure model. (BeN-Ari Y, 1985; Nadler JV, 1979) Cultured rat hippocampal cells were treated with 150 μM of 150 μM niacinic acid for 24 hours. After 24 hours of treatment with 0 μM kainic acid or vehicle (control group), Figures 2a and 2b show As shown, neogenin and MAP2 expression in neurons, and astrocytes, was confirmed by confocal microscopy. Neogenin and GFAP expression in glial cells was visualized. Furthermore, kainic acid treatment induces neogenin expression in both neurons and astrocytes. Kainic acid significantly reduced the expression of MAP2 (P<0.05 vs. control group). The expression of GFAP was significantly decreased (P<0.01 vs. control group), but increased. (P<0.05 vs. control group), which indicates the induction of reactive astrocytes. Therefore, neogenin was detected in both neurons and astrocytes in an in vitro seizure model. It was confirmed that the expression of

[0149] Experimental Example 3. Hippocampal damage and necroptosis after pilocarpine-induced SE tosis) To confirm the SE-induced hippocampal damage, cresyl violet staining (cre As a result, as shown in Figure 3a, The hippocampus of the control group (sham group) was divided into the CA3 pyramidal cell layer. It is completed by the 1 layer and the hilus of the dentate gyrus. However, after pilocarpine-induced SE, the cells showed normal morphology. CA3 pyramidal cell layer and dentate gyrus hilus Pyknotic cells (pyknots) in the hilus of dentate gyrus On the 7th day, pyramidal cells were observed. In both the dentate gyrus and dentate hilar cells, The clear nuclear pyknotic morphology (pykno) corresponds to the increase in cleaved neogenin shown in Figure 1c. tic morphology) was observed. To assess the type of necroptosis, we performed a cytotoxic assay using pMLKL, an important molecular marker for necroptosis. Western blotting was performed. As a result, as shown in Figure 3b and Figure 3c, the sham group and In contrast, pMLKL expression in the hippocampus increased 3 days after pilocarpine-induced SE. The results showed that the level of the SE-induced vasopressin increased on the first day of treatment, but decreased to the control level on the seventh day after the acute attack. This means that hippocampal necroptosis is induced within three days after birth.

[0150] Experimental Example 4. Effect of inhibition of neogenin cleavage on neuronal survival Excitotoxicity similarly affects neogenin expression To confirm this, cultured hippocampal neurons were treated with 100 μM NMDA. As shown in Figure 4a, NMDA stimulated the MAP2-positive rat hippocampus. It was shown to significantly decrease the expression of neogenin in neurons (P<0.05 In particular, as shown in Figure 4b, this decrease in neogenin expression was Treatment of cells with 1 μM of DAPT, an inhibitor of ogenin cleavage, for 24 hours improved the It was confirmed that the effect was neuroprotective (P<0.05 vs. control group). To confirm whether or not there is a difference, MTT analysis was performed. As a result, as shown in Figure 4b, NMDA induces the death of hippocampal neurons, whereas 1 μM DAPT treatment inhibits cell viability. The results showed that the hippocampal neurons were significantly improved in the presence of GABA (P<0.01 vs. control group). Inhibition of oxogenin cleavage protects against NMDA-induced neurotoxicity This means that

[0151] Furthermore, to confirm whether DAPT1 can suppress necroptosis, we As a result of comparing the expression level of pMLKL by blotting, as shown in Figure 4c, DAPT1 treatment It was confirmed that NMDA-induced necroptosis was reduced by the treatment.

[0152] As mentioned above, when damage is induced in the hippocampus by an acute epileptic seizure, the full-length The levels of neogenin are decreased, whereas the levels of the truncated form of neogenin are increased. Furthermore, when neogenin cleavage inhibitors were treated, necroptosis was suppressed. It has been shown that the expression of pMLKL, a marker for neogenin cleavage, is reduced. The inhibitor can be used as an active ingredient in a substance for preventing, improving or treating brain damage caused by epilepsy. It was confirmed that this is the case.

[0153] The foregoing description of the present invention is given by way of example only and is intended to be illustrative and not restrictive of the principles of the present invention. A person with sufficient knowledge may create the invention in other specific forms without changing the technical idea or essential features of the invention. It will be understood that the above-described embodiments can be easily modified in various ways. It should be understood in all respects that this is illustrative and not restrictive. [Industrial Applicability]

[0154] The present invention relates to the use of neogenin for diagnosing epilepsy-induced brain damage. Furthermore, the diagnostic composition of the present invention can be used to quickly and accurately diagnose and predict brain damage at an early stage. This may be a target for the development of therapeutic agents for epilepsy-induced brain damage. In addition, DAPT, a gamma-secretase inhibitor, can be used to treat epileptic seizures. Induced production of cleaved neogenin and necroptosis It effectively inhibits epilepsy-induced brain damage, making it a useful pharmaceutical composition for preventing or treating epilepsy-induced brain damage. The compound can be usefully used as an active ingredient in compositions and has industrial applicability.

Claims

1. A composition for assessing brain damage induced by epilepsy, comprising as an active ingredient a preparation capable of measuring the level of truncated neogenin.

2. The composition for assessment according to claim 1, wherein the level of the truncated neogenin increases during the occurrence of epilepsy-induced brain damage.

3. The composition for determination according to claim 1, wherein the epilepsy is status epilepticus (SE).

4. The composition for determination according to claim 1, wherein the preparation capable of measuring the level of the cleaved neogenin is an antibody or aptamer specific to the cleaved neogenin.

5. A composition for assessing epilepsy-induced brain damage in a group of epilepsy patients, comprising: The composition for determining brain damage induced by epilepsy, characterized in that it contains, as an active ingredient, a preparation capable of measuring the level of truncated neogenin.

6. A kit for assessing brain damage induced by epilepsy, comprising the composition according to any one of claims 1 to 5 as an active ingredient.

7. A method for providing information necessary for determining epilepsy-induced brain damage, comprising measuring the level of truncated neogenin in a biological sample isolated from a subject.

8. 8. The method of claim 7, wherein the subject is a mammal, including a human.

9. The method according to claim 7, wherein the biological sample is a sample derived from the hippocampus.

10. The method of claim 7, further comprising determining that the brain damage is induced by epilepsy if the measured level of truncated neogenin is increased compared to the level of truncated neogenin in a control group.

11. 11. The method of claim 10, wherein the control group is a biological sample isolated from a normal individual or an epilepsy patient without status epilepticus (SE).

12. The method of claim 7, wherein the level of cleaved neogenin is measured by one or more methods selected from the group consisting of Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.

13. (a) treating a biological sample isolated from a patient with status epilepticus (SE) with a candidate substance; (b) measuring the level of truncated neogenin in the sample; and (c) A method for screening for a substance for preventing or treating brain damage induced by epilepsy, comprising a step of determining that the substance is a substance for preventing or treating brain damage induced by epilepsy if the substance reduces the level of cleaved neogenin compared to a group not treated with the candidate substance.

14. The screening method according to claim 13, wherein the sample is a hippocampal-derived sample.

15. 14. The method of claim 13, wherein the measurement in step (b) is selected from the group consisting of Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.

16. A pharmaceutical composition for preventing or treating brain damage induced by epilepsy, comprising a neogenin cleavage inhibitor as an active ingredient.

17. The pharmaceutical composition according to claim 16, wherein the inhibitor is DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester).

18. Use of a neogenin cleavage inhibitor for the manufacture of a drug for treating brain damage induced by epilepsy.

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