Compositions and methods for assessing edaravone responsiveness

Abstract

Compositions of the present disclosure contain edaravone and are useful to elicit a change in the expression level of a gene product in a target. The gene product is a gene product of one or more genes selected from the group consisting of KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2. The invention also relates to a method for preparing the gene product. The composition is also suitable for use as a medicament. The composition is also suitable for treating or preventing neurodegenerative diseases. The neurodegenerative disease is suitably amyotrophic lateral sclerosis (ALS). The present disclosure also provides methods of assessing the responsiveness of edaravone based on the expression level or change in expression level of a gene product.

Description

Technical Field

[0001] This application claims priority based on Japanese patent application No. 2022-106787 filed on July 1, 2022, the entire contents of which are incorporated into this specification by reference.

[0002] The present invention relates to compositions and methods for assessing edaravone responsiveness. Technical Background

[0003] Edaravone is an organic compound named 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (CAS No. 89-25-8). Edaravone is used for medical purposes as a brain protective agent or a therapeutic agent for amyotrophic lateral sclerosis (ALS), a type of neurodegenerative disease (Patent Documents 1 and 2).

[0004] ALS is an intractable disease, which starts from initial symptoms such as hand weakness, finger dyskinesia and contraction of upper limb fiber bundles, followed by symptoms such as amyotrophy and / or myasthenia, bulbar palsy and contraction of muscle fiber bundles, and finally leads to respiratory failure. It is reported that neurodegenerative diseases, such as ALS, are related to, for example, TDP-43 (43kDa TARDNA binding protein) protein. TDP-43 is such a protein, which is identified as one of the components found in the brain of patients suffering from frontotemporal lobar degeneration (FTLD) or ALS (see non-patent document 1). It is believed that the occurrence and development of neurodegenerative diseases, such as ALS, is partly due to the formation of TDP-43 protein aggregates.

[0005] [Related technologies]

[0006] [Patent document]

[0007] [Patent Document 1] Japanese Patent Publication No. H5-31523.

[0008] [Patent Document 2] U.S. Patent No. 6,933,310.

[0009] [Non-patent document]

[0010] [Non-patent document 1] Arai et al., Biochem Biophys Res Commun, 351(3): 602-11 (2006). [Summary of the Invention]

[0012] The inventors have found that exposure of a target to edaravone causes changes in the expression level of a gene product of a specific gene. In addition, the inventors have found that changes in expression levels can be useful indicators, such as biomarkers, in exploring the responsiveness to edaravone or the likelihood of having an applicable disease.

[0013] The present disclosure includes inventions related to compositions.

[0014] In one embodiment, the composition is used to cause a change in the expression level of a gene product in a target.

[0015] In one embodiment, the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0016] Additionally, the present disclosure also includes inventions related to methods of assessing responsiveness to edaravone.

[0017] In one embodiment, the assessment method includes the step of assessing whether a target is likely to be responsive to edaravone based on changes in the expression level of a gene product resulting from exposure of the target to edaravone.

[0018] In one embodiment, the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0019] Other embodiments will be apparent from the description and claims.

[0020] According to the present invention, by exposing the target to edaravone, the expression level of a specific gene product can be changed. Therefore, a beneficial effect corresponding to the gene function can be achieved, and for example, it can be used to treat or prevent a disease.

[0021] Furthermore, according to the present invention, changes in the expression of specific gene products can be used to screen for substances capable of treating or preventing diseases.

[0022] Furthermore, according to the present invention, changes in the expression of specific gene products can be used in a variety of assessments and predictions of the presence or absence of a disease, the progression of a disease, responsiveness to treatment, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] [ Figure 1 ] Figure 1 Shown are fluorescence microscopy images showing the cellular morphology of neurons expressing wild-type and mutant TDP-43 proteins when exposed to ethacrynic acid (EA). Figure 1 The images in the bottom row are Figure 1 Enlargements of the images in the upper row.

[0025] [ Figure 2 ] Figure 2 Shown are fluorescence microscopy images showing the cellular morphology of neurons expressing wild-type and mutant TDP-43 proteins when exposed to ethacrynic acid and edaravone. Figure 2 The images in the bottom row are Figure 2 Enlargement of the image in the upper row.

[0026] [ Figure 3 ] Figure 3 is a graph showing the cell survival rate of neurons expressing wild-type and mutant TDP-43 proteins according to the presence or absence of exposure to ethacrynic acid and / or edaravone.

[0027] [ Figure 4 ] Figure 4 is a schematic diagram showing the treatment profiles of the experimental groups used in the gene product expression analysis.

[0028] [ Figure 5 ] Figure 5 is a schematic diagram showing an overview of the process for gene product expression analysis.

[0029] [ Figure 6 ] Figure 6 is a graph showing the expression distribution of the experimental groups.

[0030] [ Fig. 7] Fig. 7 (a) to 7 (d) are graphs showing changes in expression of gene products depending on the presence or absence of edaravone and / or pathological conditions.

[0031] [ Fig. 8] Fig. 8 (a) to (d) are graphs showing changes in expression of other gene products depending on the presence or absence of edaravone and / or pathological conditions.

[0032] [ Fig. 9] Fig. 9 (a) to (d) are graphs showing changes in expression of other gene products depending on the presence or absence of edaravone and / or pathological conditions.

[0033] [Figure 10] Figure 10(a) and 10(b) is a graph showing changes in expression of additional gene products depending on the presence or absence of edaravone and / or pathological conditions.

[0034] [ Fig.11 ] Fig.11 is a graph showing changes in expression of additional gene products depending on the presence or absence of edaravone and / or pathological conditions.

[0035] [ Fig.12 ] Fig.12 It is a graph showing the cell survival rate of neurons expressing wild-type and mutant TDP-43 proteins and neurons not expressing TDP-43 protein according to the presence or absence of exposure to edaravone.

[0036] [ Fig.13 ] Fig.13 It is a graph showing the rate of change in neurite length in neurons differentiated from iPS cells induced from cells from ALS patients and healthy individuals, depending on the presence or absence of exposure to edaravone.

[0037] [ Fig.14 ] Fig.14 This is a graph showing the rate of change in the number of dead cells in neurons differentiated from iPS cells induced from cells from ALS patients and healthy individuals, depending on the presence or absence of exposure to edaravone.

[0038] [ Fig.15 ] Fig.15 Shown are fluorescence microscopy images showing the intracellular localization of TDP-43 in neurons differentiated from iPS cells induced from cells from ALS patients and healthy individuals when exposed to edaravone. Implementation

[0040] The present invention will be described below based on preferred embodiments of the present invention.

[0041] The present disclosure includes compositions containing edaravone. In one embodiment, the composition can be used as a drug, or it can be used as a non-drug, such as an agent.

[0042] In either case, by causing a change in the expression level of a gene product of a specific gene in the target, a beneficial effect corresponding to the type or degree of expression of the gene and its gene product can be obtained.

[0043] The details of the gene and its gene product will be described later.

[0044] In the present specification, "edaravone" includes 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one itself and its tautomers, derivatives thereof, and salts, hydrates or solvates thereof.

[0045] In one embodiment, edaravone preferably includes at least one selected from 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one and its tautomers, and salts, hydrates or solvates thereof. That is, edaravone preferably includes at least one selected from isomers represented by the following structural formula (1) and structural formula (2) and salts, hydrates or solvates thereof.

[0046] Edaravone can be produced, for example, by the production method described in European Patent Publication No. 208874.

[0047] [Chemical formula 1]

[0048]

[0049] In the present specification, unless otherwise specified, the term "gene product" is used to include transcription products, reverse transcription products and translation products from a gene, and may be one or more of these products.

[0050] The gene product may be only one or more transcription products, only one or more reverse transcription products, only one or more translation products, or a combination of one or more transcription products and one or more translation products.

[0051] Examples of transcription products include various types of RNA, such as mRNA and non-coding RNA. The transcription product is preferably mRNA, whether mature or not, more preferably mature mRNA.

[0052] An example of a reverse transcription product is cDNA.

[0053] An example of a translation product is a protein produced by transcription and translation. The translation product is preferably a protein, regardless of whether there is post-translational modification.

[0054] In one embodiment, the composition containing edaravone is preferably used as a medicament. That is, in one embodiment, the composition is a pharmaceutical composition containing edaravone as an active ingredient.

[0055] The above composition can be used as a drug, for example, to change the expression level of a specific gene product in the target to maintain brain function at a normal level or improve brain function, or to help treat or prevent diseases such as neurodegenerative diseases, muscle diseases, vascular diseases, and various inflammatory diseases.

[0056] In this specification, the term "target" is not particularly limited as long as the expression level of the gene product can be measured. Examples of "target" include human and non-human animals, and samples from these animals, whether there is a disease or not, and regardless of the environment, such as in vivo, ex vivo or in vitro.

[0057] The human and non-human animals are preferably mammals. That is, the target exposed to the composition is preferably a mammal or a sample derived from a mammal.

[0058] Examples of non-human animals include rodents, such as rats, mice, and guinea pigs; and non-human mammals, such as monkeys, pigs, dogs, and cats.

[0059] Examples of samples include, but are not limited to, one or more of tissues, cells, and body fluids.

[0060] Examples of tissues include brain (e.g., cerebrum and cerebellum), spinal cord, stomach, pancreas, kidney, liver, adrenal glands, skin, muscle (e.g., skeletal muscle and smooth muscle), lung, intestine (e.g., large intestine and small intestine), heart, blood vessels, etc.

[0061] Examples of cells include differentiated cells, progenitor cells or stem cells that form tissues. Taking brain-derived cells as an example, examples of brain-derived samples include neuronal cells (neurons), glial cells, neural-related differentiated cells such as astrocytes, neural stem cells, and the like.

[0062] Examples of body fluids include liquid components such as cerebrospinal fluid, blood, serum, plasma, saliva, urine, and sweat, or extracts thereof.

[0063] These samples can generally be collected from living or dead animals using commonly known methods.

[0064] As other forms of cells, a variety of cultured cells can be used, such as primary cultured cells, immortalized cell lines, or pluripotent stem cells, such as ES cells and iPS cells. Among them, iPS cells can be induced from cells from ALS patients or cells with ALS risk mutations, such as TARDBP gene heterozygous mutations. Cells differentiated from the above pluripotent stem cells, such as neurons, are also included in the targets of the present disclosure.

[0065] In one embodiment, the above-described compositions are useful for treating or preventing one or more of neurodegenerative diseases, muscle diseases, vascular disorders, and inflammatory diseases.

[0066] Examples of neurodegenerative diseases include neurodegenerative diseases accompanied by motor dysfunction and neurodegenerative diseases accompanied by cognitive dysfunction.

[0067] Examples of neurodegenerative diseases accompanied by motor dysfunction include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), arthrogryposis multiplex congenita (AMC), and the like.

[0068] Examples of neurodegenerative diseases associated with cognitive impairment include Alzheimer's disease, frontotemporal lobar degeneration (FTD), and the like.

[0069] Examples of muscle diseases include muscular dystrophy and the like.

[0070] Examples of vascular disorders include cerebral infarction, cerebral hemorrhage, and the like.

[0071] Examples of inflammatory diseases include systemic inflammatory diseases, such as multiple sclerosis and systemic sclerosis, and localized inflammatory diseases, such as stomatitis.

[0072] Among these diseases, the above composition as a pharmaceutical composition is preferably used for treating or preventing neurodegenerative diseases, more preferably for treating or preventing neurodegenerative diseases accompanied by motor dysfunction, and even more preferably for treating or preventing amyotrophic lateral sclerosis (ALS).

[0073] In the present specification, "treatment" includes inhibition of disease progression, improvement, remission, complete cure and the like.

[0074] In the present specification, "prevention" includes preventing the occurrence of a disease, preventing the recurrence of a disease, and the like.

[0075] In one embodiment, the above composition is also preferably used to treat or prevent diseases accompanied by the presence of TDP-43 mutant proteins or abnormal intracellular localization of TDP-43. Examples of such diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS) and the like. Examples of TDP-43 mutant proteins include, but are not limited to, C-terminal fragments of wild-type TDP-43 proteins (e.g., amino acid residues 208-414 from the N-terminus of wild-type TDP-43 proteins) and point mutations of wild-type TDP-43 proteins (e.g., G294A, G298S, A315T, and Q343R). Examples of wild-type TDP-43 proteins include the amino acid sequences described in Accession No.: NP_031401.1.

[0076] Regardless of the disease, by using the above composition, the expression level of a specific gene product can be changed by exposure to edaravone. Cell dysfunction or cell damage, such as cell death, can be effectively inhibited by the expression level of the gene product or the change in the expression level. Therefore, the disease can be treated or prevented, or the symptoms of the disease can be alleviated. As shown in the embodiments to be described later, the above composition is advantageous because it can treat or prevent diseases (such as ALS) that may involve TDP-43 mutant proteins. When TDP-43 mutant proteins are present, it is speculated that TDP-43 proteins aggregate in cells, making cell damage more likely to occur. Therefore, by using the above composition, cell damage can be effectively inhibited.

[0077] The edaravone-containing compositions of the present disclosure are used to induce changes in the expression levels of specific gene products.

[0078] Due to the careful study of the mechanism of action of edaravone, the inventors have found that, as shown in the examples to be described later, the expression level of the gene product of a specific gene changes depending on whether or not it is exposed to edaravone. In addition, the inventors have also found that the expression level of these gene products also changes in specific diseases. In addition, the inventors have also found that the expression changes in the presence of edaravone can help inhibit cell damage, thereby helping to predict, treat or prevent diseases caused by cell damage. That is, the inventors have found through research that the above-mentioned composition has unknown properties and new uses, thereby completing the present invention.

[0079] In this specification, the term "change" refers to an increase or decrease in the amount of a substance compared to the measured value or reference value of a comparison target, and includes, in addition to the size of the amount or ratio, the appearance of an amount that changes from an undetectable state to a detectable state, and the disappearance of an amount that changes from a detectable state to an undetectable state.

[0080] Determine as follows whether expression level changes: when determining that the measured value of the expression level of the gene product in the target is greater than or less than the measured value or the reference value of the expression level of the gene product in the target, then determine that expression level has changed.The variation of expression level can be determined according to the numerical value of measured value itself, or determines according to the size of the ratio calculated from measured value.In addition, the variation of expression can be determined according to the arithmetic mean or the size of the median calculated by a plurality of measured values ​​or ratio, or can determine that it has changed according to the significant difference that exists statistically.

[0081] The change in expression level is assessed by comparing the expression level of a measurable molecule derived from the same gene between two experimental groups as a comparison target, provided that the two experimental groups belong to or are derived from the same animal species.

[0082] Examples of molecules whose expression levels can be measured include, but are not limited to, RNA (which is a transcription product), cDNA (which is a reverse transcription product of RNA), protein (which is a translation product), and the like.

[0083] When using the ratio of measured values ​​to assess the change of expression level, when the measured value R2 of the expression level of the gene product in the experimental group for determining the target and the ratio (R2 / R1) of the measured value R2 or the measured value R1 as a reference are for example 1.01 times or higher, for example 1.10 times or higher, for example 1.30 times or higher, for example 1.50 times or higher, for example 1.70 times or higher, for example 2.0 times or higher (1 or higher log2 ratio), it can be determined that the expression level has changed. In this case, it can also be determined that the experimental group for determining the target has preferably changed so that the expression level increases.

[0084] In addition, when the R2 / R1 ratio is, for example, 0.99 times or lower, for example, 0.90 times or lower, for example, 0.80 times or lower, for example, 0.70 times or lower, for example, 0.60 times or lower, or for example, 0.50 times or lower (-1 or lower log2 ratio), it can be determined that the expression level has changed. In this case, it can also be determined that the experimental group used to determine the target has preferably changed so that the expression level is reduced.

[0085] The composition disclosed herein is suitable for causing a change in the expression level of a transcription product or a coded translation product of one or more genes selected from a group of specific genes discovered by the inventors through careful research. The transcription product and translation product whose expression level changes may be one of these, or two or more of these.

[0086] Below, the names of genes whose expression levels of gene products change due to the presence of edaravone and / or disease are illustrated, but are not limited thereto. For ease of description, unless otherwise indicated, the following genes are all expressed as human genes. However, the disclosure also includes orthologs of animal species other than humans. The base sequences or protein amino acid sequences of these genes can be searched using public databases, etc.

[0087] In this specification, the gene group (hereinafter (A) and (B)) whose expression level of gene products is changed due to the presence of edaravone and / or disease is collectively referred to as "specific genes". The description of specific genes applies to all embodiments described in this specification, unless otherwise specified.

[0088] <Specific Gene>

[0089] Examples of these genes whose expression levels of gene products are increased in the presence of disease but not in the absence of edaravone and decreased in the presence of both disease and edaravone are shown below as (A).

[0090] In one embodiment, the above composition is used to reduce the expression level of the gene product in the target with disease for any single gene, any combination of two or more genes, or a combination of all genes from genome (A). In one embodiment, the above disease is preferably a neurodegenerative disease.

[0091] [Genome(A)]

[0092] KAZALD1 (Kazal-type serine peptidase inhibitor domain 1)

[0093] SBK1 (SH3 binding domain kinase 1)

[0094] ·SCN2A (Sodium voltage-gated channel alpha subunit 2)

[0095] UBE2L6 (ubiquitin-conjugating enzyme E2 L6)

[0096] ALPL (alkaline phosphatase)

[0097] NTM (neurotrimin)

[0098] PTTG1 (sister chromatid separation regulator; Securin)

[0099] ITGB4 (integrin subunit β4)

[0100] HAUS4 (HAUS augmin-like complex subunit 4)

[0101] ·DCTD (dCMP deaminase)

[0102] MT2A (Metallothionein 2A)

[0103] ASF1B (anti-silencing function 1B histone chaperone)

[0104] FCSK (fucokinase)

[0105] MAST1 (microtubule-associated serine / threonine kinase 1)

[0106] Examples of genes whose expression levels of gene products are decreased in the presence of disease but not edaravone, and increased in the presence of both disease and edaravone are shown below as (B).

[0107] In one embodiment, the above composition is used to increase the expression level of a gene product in a disease-affected target of the genome (B).

[0108] In one embodiment, the above-mentioned disease is preferably a neurodegenerative disease.

[0109] [Genome (B)]

[0110] FAIM2 (Fas apoptosis inhibitory molecule 2)

[0111] KAZALD1 is a gene that encodes a class of proteins belonging to the insulin growth factor (IGF) binding protein superfamily. Changes in the expression of KAZALD1 can promote cell proliferation or changes in IGF function or expression levels. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant proteins (which is a pathological state of neurodegenerative diseases) and further decreased in the presence of edaravone.

[0112] Changes in the expression of IGF may be associated with changes in the risk of neurodegenerative diseases (such as ALS) through some mechanisms. Therefore, it is believed that reducing the expression level of KAZALD1 is useful in reducing the risk of occurrence or development of neurodegenerative diseases by regulating IGF activity, and improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0113] SBK1 is a gene encoding a class of serine / threonine protein kinases. Changes in the expression of SBK1 can promote cancer progression or cell proliferation. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant protein (which is a pathological state of neurodegenerative diseases), and further decreased in the presence of edaravone.

[0114] It is believed that reducing the expression level of SBK1 can be used to inhibit the occurrence of cell damage by inhibiting the formation of TDP-43 protein aggregates through phosphorylation, or to improve the possibility of treating or predicting the prognosis of neurodegenerative diseases (such as ALS).

[0115] SCN2A is a gene encoding a class of proteins belonging to the voltage-gated sodium channel family in excitatory neurons. The expression changes of SCN2A can promote the occurrence of epilepsy or developmental disorders. In addition, during neuronal cell damage, the function of SCN2A becomes overactive, which may aggravate cell damage. It has been found that the expression level of the gene product of this gene increases when there is a TDP-43 mutant protein (which is a pathological state of neurodegenerative diseases), and further decreases when there is edaravone.

[0116] By reducing the expression level of SCN2A, the occurrence of cell damage can be suppressed. In addition, it is considered to be important because with the reduction of the expression level of SCN2A, the possibility of treating or predicting the prognosis of neurodegenerative diseases (such as ALS) can be improved. In particular, it is considered to be advantageous to reduce the expression level of SCN2A because the function of SCN2A can be suppressed during the occurrence of neuronal cell damage, so the progress of cell damage can be effectively suppressed.

[0117] UBE2L6 is a gene encoding a class of proteins belonging to the ubiquitin conjugating enzyme family. It is believed that changes in the expression of UBE2L6 promote protein degradation and inhibit autophagy in pathological states (such as cancer). It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant proteins (which are pathological states of neurodegenerative diseases) and further decreased in the presence of edaravone.

[0118] When autophagy is inhibited, the degradation of abnormal proteins (such as TDP-43 protein aggregates) may be hindered, which may lead to the progression of pathological conditions. Therefore, by reducing the expression level of UBE2L6, autophagy can be promoted and the degradation of abnormal proteins can be promoted. Therefore, it is believed that it is useful in inhibiting the occurrence of cell damage or improving the possibility of treating or predicting the prognosis of neurodegenerative diseases (such as ALS).

[0119] ALPL is a gene encoding a class of proteins belonging to the alkaline phosphatase family. ALPL is highly expressed in the axons or dendrites of neurons. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant proteins (which is a pathological state of neurodegenerative diseases), and further reduced in the presence of edaravone.

[0120] Increased ALPL expression levels can lead to the occurrence or progression of neurodegenerative diseases (such as ALS) through some mechanisms in neuronal cells. Therefore, it is believed that reducing the expression level of ALPL is important in inhibiting the occurrence of cell damage or improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0121] NTM is a gene encoding a class of proteins belonging to the IgLON family of cell adhesion molecules. Changes in the expression of NTM can promote the elongation of neurites and axons, or cell-to-cell adhesion. It has been found that the expression level of the gene product of this gene increases in the presence of TDP-43 mutant proteins (which are pathological states of neurodegenerative diseases), and further decreases in the presence of edaravone. In autoimmune neurological diseases involving anti-IgLON family antibodies, phosphorylated tau proteins can accumulate in the thalamus and brainstem tegmentum, possibly leading to symptoms similar to bulbar palsy type motor neuron disease. Therefore, it is believed that reducing the expression level of NTM is important in reducing the accumulation of abnormal proteins or side effects associated with accumulation, thereby increasing the possibility of treatment or prognosis prediction for a variety of neurodegenerative diseases (such as bulbar palsy type ALS).

[0122] PTTG1 is a gene encoding securin, a protein that promotes cell mitosis. Changes in the expression of PTTG1 can promote the regulation of cell division or tumor formation. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant protein (which is a pathological state of neurodegenerative diseases) and further decreased in the presence of edaravone.

[0123] Increased PTTG1 expression levels can promote the occurrence or progression of neurodegenerative diseases (such as ALS) through some mechanisms related to cell division. Therefore, it is believed that reducing the expression level of PTTG1 is important in inhibiting the occurrence of cell damage or improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0124] ITGB4 is a gene encoding a protein belonging to the integrin family. Changes in the expression of ITGB4 can promote cell proliferation, adhesion and migration, or tissue formation and repair. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant protein (which is a pathological state of neurodegenerative diseases), and further decreased in the presence of edaravone.

[0125] Increased ITGB4 expression levels can promote the occurrence or progression of neurodegenerative diseases (such as ALS) through some mechanisms related to cell proliferation or repair. Therefore, it is believed that reducing the expression level of ITGB4 is important in inhibiting the occurrence of cell damage or improving the possibility of treating or predicting the prognosis of neurodegenerative diseases (such as ALS).

[0126] HAUS4 is a gene that encodes a class of proteins that promote microtubule formation during cell mitosis. Changes in the expression of HAUS4 can promote the regulation of cell division or the elongation of axons and dendrites. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant proteins (which is a pathological state of neurodegenerative diseases) and further reduced in the presence of edaravone.

[0127] In ALS disease animal models, it has been confirmed that HAUS4 expression levels increase and can promote the occurrence or progression of neurodegenerative diseases (such as ALS). Therefore, it is believed that reducing the expression level of HAUS4 is important in promoting the formation or functional maintenance of normal neuronal cells, inhibiting the occurrence of cell damage, or improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0128] DCTD is a gene encoding a protein (dCMP deaminase) that promotes the deamination of dCMP to dUMP. Changes in the expression of DCTD can promote the regulation of nucleic acid biosynthesis. It has been found that the expression level of the gene product of this gene increases when there is a TDP-43 mutant protein (which is a pathological state of neurodegenerative diseases), and further decreases when there is edaravone.

[0129] The upregulation of DCTD can lead to an imbalance of substrates or products in nucleic acid biosynthesis, which may make DNA mutations more likely to occur during DNA replication. Therefore, it is believed that reducing the expression level of DCTD is important in the formation or function maintenance of normal neuronal cells, inhibiting the occurrence of cell damage, or improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0130] MT2A is a gene encoding a class of proteins belonging to the metallothionein family. Changes in the expression of MT2A can promote heavy metal metabolism, such as intracellular heavy metal concentrations or cell apoptosis. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant proteins (which is a pathological state of neurodegenerative diseases), and further reduced in the presence of edaravone.

[0131] It is believed that the upregulation of MT2A is partly caused by oxidative stress. Therefore, it is believed that reducing the expression level of MT2A is important in promoting the maintenance of normal neuronal cell function by regulating the response to oxidative stress, inhibiting the occurrence of cell damage, or improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0132] ASF1B is a gene that encodes a class of proteins belonging to the histone chaperone H3 / H4 family. Changes in the expression of ASF1B can promote the regulation of cell division, proliferation, and aging. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant proteins (which is a pathological state of neurodegenerative diseases), and further decreased in the presence of edaravone.

[0133] Increased ASF1B expression levels may promote the occurrence or progression of neurodegenerative diseases (such as ALS) through a mechanism related to cell division or proliferation. Therefore, it is believed that reducing the expression level of ASF1B is important in inhibiting the occurrence of cell damage or improving the possibility of treating or predicting the prognosis of neurodegenerative diseases (such as ALS).

[0134] FCSK is a gene that encodes a class of proteins that belong to the phosphotransferase family and use alcohol as a receptor. Changes in the expression of FCSK can promote the metabolism of fructose and mannose. It has been found that the expression level of the gene product of this gene is increased in the presence of TDP-43 mutant protein, which is a pathological state of neurodegenerative diseases, and further decreased in the presence of edaravone.

[0135] Increased FCSK expression levels can promote the occurrence or progression of neurodegenerative diseases (such as ALS) through a certain mechanism related to intracellular glucose metabolism. Therefore, it is believed that reducing the expression level of FCSK is important in inhibiting the occurrence of cell damage or improving the possibility of treatment or prognosis prediction of neurodegenerative diseases (such as ALS).

[0136] MAST1 is a gene encoding a class of serine / threonine protein kinases. The expression changes of MAST1 are mainly related to brain development and can promote the expression of postsynaptic or synaptic terminals of neuromuscular junctions. It has been found that the expression level of the gene product of this gene increases in the presence of TDP-43 mutant proteins (which is a pathological state of neurodegenerative diseases) and further decreases in the presence of edaravone.

[0137] It is believed that reducing the expression level of MAST1 is important in inhibiting the occurrence of cell damage by inhibiting the formation of TDP-43 protein aggregates through phosphorylation, or in improving the possibility of treating or predicting the prognosis of neurodegenerative diseases such as ALS.

[0138] FAIM2 is a gene that encodes a class of anti-apoptotic proteins that protect cells from Fas-induced apoptosis. This gene is also known as protein lifesaver 2-like (LOC102551901), as a rat ortholog. Changes in the expression of FAIM2 can promote the size of the cerebellum or the thickness of the internal granular layer, as well as the development of Purkinje cells. It has been found that the expression level of the gene product of this gene is reduced in the presence of TDP-43 mutant proteins, which is a pathological state of neurodegenerative diseases, and further increased in the presence of edaravone.

[0139] Since apoptosis can be induced during neuronal damage, it is believed that the expression level of FAIM2 is reduced. Therefore, it is believed that increasing the expression level of FAIM2 is important in improving the possibility of treating or predicting the prognosis of neurodegenerative diseases (such as ALS) by maintaining the survival of neuronal cells.

[0140] In addition, FAIM2 is related to the regulation of autophagy. Therefore, increasing the expression level of FAIM2 can inhibit the formation of mutant SOD1 (superoxide dismutase 1) protein aggregates, which is a pathological state of neurodegenerative diseases (such as ALS), or promote the degradation of abnormal protein (such as TDP-43 and SOD1) aggregates. Therefore, it is considered to be important in improving the possibility of treatment or prognosis prediction for neurodegenerative diseases (such as ALS).

[0141] The exposure method of the above composition is not particularly limited. Examples of the exposure method include administration to a living body, contact with a sample, and the like.

[0142] In the case of administration to humans or non-human animals, examples include oral administration and parenteral administration. In this case, the composition containing edaravone is preferably a pharmaceutical composition containing edaravone.

[0143] Examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal injection, injection into the digestive tract, inhalation, etc. These administrations may be single bolus administration or multiple bolus administrations, or may be continuous administration, such as infusion.

[0144] The contact with the sample can be performed, for example, using a liquid in which the above-mentioned composition is dissolved or dispersed.

[0145] The above composition may be in a solid state or a liquid state at 1 atmosphere and 20° C., depending on the intended use.

[0146] When the composition is in a solid state, examples of the dosage form include tablets, capsules, powders, fine granules, granules, suppositories and the like.

[0147] When the composition is in a liquid state, examples of the dosage form include liquid, syrup, suspension, injection, infusion and the like.

[0148] The liquid may be a solution containing a solvent or a dispersion containing a dispersant.

[0149] The above composition may further contain additives as required. These additives are preferably pharmaceutically acceptable.

[0150] In one embodiment, when an additive is included in the composition, the additive may constitute the remainder of the composition alone as a single type of additive or as a combination of two or more types of additives.

[0151] As additives, those used in the art can be used without particular limitation. As additives, for example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, colorants, diluents, vehicles, solvents or solubilizers, isotonic agents, pH regulators, stabilizers, propellants, adhesives, etc. can be used. These additives can be used independently of each other, or two or more additives can be used in combination.

[0152] As a carrier or diluent, water, water containing electrolytes (e.g., saline), monohydric alcohols having 1 to 3 carbon atoms (e.g., methanol, ethanol, propanol), or aqueous liquids for cell culture can be used. These aqueous liquids can be used independently, or two or more aqueous liquids can be used in combination as solvents or dispersants.

[0153] The content of edaravone in the composition can be appropriately changed according to the applicable target. However, taking the case of administration to a living mammal as an example, regardless of the administration method, the content of edaravone can be set to an amount preferably administered per day within the range of 0.01 μg / kg body weight or more and 10 mg / kg body weight or less.

[0154] In one embodiment, taking the case of administration to adult humans as an example, regardless of the administration form, the content of edaravone in the composition can be set to an amount for daily administration within the range of preferably 10 mg or more and 150 mg or less, more preferably 20 mg or more and 120 mg or less, and even more preferably 90 mg or more and 120 mg or less.

[0155] The above content can also be set, for example, as a therapeutically effective amount.

[0156] When exposed to a sample (e.g., tissue or cells collected from an animal or an in vitro sample), taking liquid form as an example, the content of edaravone in the composition is preferably greater than 0.1 μmol / L and less than 1000 μmol / L, more preferably greater than 0.5 μmol / L and less than 500 μmol / L, even more preferably greater than 0.75 μmol / L and less than 300 μmol / L, still more preferably greater than 10 μmol / L and less than 300 μmol / L, and most preferably greater than 50 μmol / L and less than 250 μmol / L.

[0157] Regardless of the form, when the content of edaravone is within the above range, it can effectively inhibit the occurrence of adverse reactions (such as cell damage) or the development of the disease toward the target.

[0158] The above is a description of an embodiment of a composition containing edaravone. In the following, other embodiments of the present invention are described. In the following description, the contents different from the above-mentioned embodiment are mainly described, and for the contents not specifically described, the matters described in this description are appropriately applied as long as the effects of the present invention can be achieved.

[0159] In one embodiment, the expression level of the gene product of the above-mentioned specific gene or the variation of the expression level can be used as an index to assess whether the target is likely to suffer from a disease. That is, the present embodiment relates to a method for assessing the possibility of suffering from a disease. By adopting this assessment method, it is possible to promote the early detection of the disease or the assessment of the appropriate treatment demand, thereby possibly promoting the improvement of the patient's quality of life.

[0160] In one embodiment, the disease that can be assessed using the present method is preferably a neurodegenerative disease, more preferably ALS, and even more preferably ALS associated with the presence of a TDP-43 mutant protein or abnormal intracellular localization of TDP-43.

[0161] In this specification, when describing the embodiment of the method, unless otherwise specified, the method may include only one step, or may include performing multiple steps simultaneously or in any order. In addition, in the case of an embodiment that may include multiple steps, unless otherwise specified, one or more other steps may be further inserted between two steps, two steps may be performed continuously so that no other steps are inserted therebetween, or two or more steps may be performed simultaneously. In addition, as long as the effect of the present invention can be achieved, the steps according to the embodiments described in this specification may be appropriately combined.

[0162] Specifically, the method includes a step of evaluating whether the target is likely to suffer from a disease based on the expression level or the change in the expression level of the gene product of one or more genes in the specific genes in the target.

[0163] The applicable target of the present method is preferably a mammal or a sample from a mammal. More specifically, the applicable target of the present method is preferably the brain of a mammal or a sample from the brain.

[0164] In addition, as an applicable target of the present method, a sample collected from a living or dead mammal can be preferably used, and the mammal is more preferably a human or a non-human animal.

[0165] In one embodiment, the method can also be performed as a method to assist in detecting a disease from a target, and the disease is preferably a neurodegenerative disease.

[0166] In one embodiment, the method can also be performed under in vitro conditions.

[0167] In one embodiment, the method includes step A1, which measures the expression level of the gene product of a specific gene in the target. That is, in this step A1, the expression level of the transcription product or the encoded translation product of one or more specific genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2 is determined. In the present method, one or more types of gene products of a specific gene can be used as an indicator of the possibility of having a disease (e.g., a neurodegenerative disease) or as a biomarker for evaluation.

[0168] Measurement of expression levels can be performed in vitro or in vivo.

[0169] When the expression level of a gene product is quantified with the transcription product as the target, for example, RNA can be extracted from the sample as the measurement target using a conventional method, and if necessary, after obtaining cDNA as a reverse transcription product, the measurement can be performed using methods known in the art, such as Northern blotting, RT-PCR, nucleic acid array, and RNA-seq. In addition, if necessary, the measured value can be corrected according to the expression level of housekeeping genes such as GAPDH and β-actin.

[0170] When the translation product (i.e., protein) is used as a target to quantify the expression level of the gene product, immunological methods known in the art, such as ELISA, flow cytometry, Western blotting or immunohistochemical staining, can be used, for example, using a sample or its solution as a measurement target for measurement. If necessary, the measured value can be corrected according to the protein expression level of the above-mentioned housekeeping gene.

[0171] As other methods of quantitative translation product, mass spectrometry, high performance liquid chromatography, gas chromatography, NMR analysis or a combination of these methods can be used. Examples of other methods of measuring the target of the translation product include measuring the activity of the translation product and considering the measured activity as the expression level, or administering a ligand that specifically binds to the translation product in vivo.

[0172] In one embodiment, in addition to step A1, the method further comprises step B1 of evaluating whether the target is likely to suffer from a disease based on the measured expression level or change in the expression level of the gene product.

[0173] In order to evaluate the possibility of the disease in step B1, for example, a method including at least one of the following steps B11, B12 and B13 may be adopted.

[0174] In one embodiment, the method may include, for example, performing at least one of steps B12 and B13 after step A1.

[0175] In one embodiment, the method may include, for example, performing step B11 before, after or simultaneously with step A1, and then performing steps B12 and B13 in this order.

[0176] Step B11 is a step in which a normal target, i.e. a healthy animal not affected by the disease or a sample from the animal is used as a control group to measure the expression level of the gene product in the sample. The expression level of the gene product in the control group can also be used as a reference value.

[0177] Step B12 is a step of comparing the expression level of the gene product in the normal target (control group) with the expression level of the gene product in the evaluation target.

[0178] Step B13 is a step of comparing the normal target (control group) with the evaluation target, and when a change in the expression level of the gene product of one or more genes among specific genes is confirmed, evaluating the evaluation target as having a high possibility of having a disease.

[0179] From the perspective of improving the accuracy of the evaluation, the evaluation of the change in expression level is preferably performed based on the presence or absence of a statistically significant difference.

[0180] Comparison of expression levels between experimental groups can be performed by comparing transcription products from the same gene or translation products from the same gene under the same or similar experimental conditions.

[0181] Specifically, as shown in the examples described later, due to the presence of a disease (e.g., a neurodegenerative disease), the expression level of the gene product of one or more genes belonging to the genome (A) in the above-mentioned specific gene can be increased compared to a normal target. Therefore, when the expression level of the gene product of one or more genes belonging to the genome (A) is increased compared to the expression level in a normal target, it can be assessed that the assessment target or the animal from which the target is obtained has a high probability of suffering from a disease (e.g., a neurodegenerative disease).

[0182] On the other hand, when the expression level of the gene product of one or more genes belonging to the genome (A) among the above-mentioned specific genes is decreased or remains unchanged compared to the expression level in the normal target, the evaluation target or the animal from which the target is obtained can be evaluated as having a low possibility of suffering from a disease (e.g., a neurodegenerative disease).

[0183] From the perspective of improving the accuracy of the assessment of the possibility of disease and making it easier to exclude the possibility of diseases other than the disease being assessed, it is preferred to use two or more of the specific genes as targets and compare the expression levels of the gene products for assessment, and it is more preferred to use all of the above-mentioned specific genes as targets and compare the expression levels of the gene products for assessment.

[0184] In addition, the expression level of the gene product of the gene belonging to the genome (B) in the above-mentioned specific gene may be reduced compared to the normal target due to the presence of the disease. Therefore, when the expression level of the gene product of the gene belonging to the genome (B) is reduced compared to the expression level in the normal target, it can be evaluated that the evaluation target or the animal from which the target is obtained has a high possibility of suffering from a disease (e.g., a neurodegenerative disease).

[0185] On the other hand, when the expression level of the gene product of genome (B) increases or remains unchanged compared to the expression level in the normal target, the evaluation target or the animal from which the target is obtained can be assessed to have a low possibility of suffering from a disease (e.g., a neurodegenerative disease).

[0186] From the perspective of further improving the accuracy of the evaluation, it is preferred to use all genes belonging to genomes (A) and (B) as targets and perform the evaluation by comparing the expression levels of the gene products.

[0187] Alternatively or additionally, the possibility of the presence or absence of a disease can also be assessed based on a cutoff value for the expression level of a gene product by setting the cutoff value as a reference value or threshold. For example, when the expression level of the gene product of one or more genes in the above-mentioned specific genes increases compared to the cutoff value (genome (A)) or decreases compared to the cutoff value (genome (B)), it can be assessed that the assessment target or the animal from which the target is derived has a high possibility of suffering from a disease (e.g., a neurodegenerative disease).

[0188] "Cut-off value" can be set to such expression level, for example, when the presence or severity of the disease is assessed based on the cut-off value, it satisfies at least one of sensitivity and specificity at a predetermined level. In addition, the expression level of the gene product in a healthy animal or healthy human body can also be used as the cut-off value. The cut-off value can be appropriately set according to the type of disease or the severity of the assessment. In addition, in order to assess the severity of the disease, multiple cut-off values ​​can be set to stratify the possibility of being ill.

[0189] In the following description, the contents different from the above-mentioned embodiment are mainly described, and the matters described in this specification will be appropriately applied as long as the effects of the present invention can be achieved for the contents not specifically described.

[0190] In one embodiment, it is possible to evaluate whether a target is likely to be responsive to edaravone by using the expression level or the change in expression level of the gene product of the above-mentioned specific gene as an indicator. That is, the present embodiment relates to a method for evaluating responsiveness to edaravone. The method allows for earlier and simpler evaluation and prediction of the effect of edaravone on a disease.

[0191] The applicable target of the present method is preferably a target suffering from or suspected of suffering from a neurodegenerative disease. The applicable target is more preferably a patient suffering from a neurodegenerative disease or a mammal as a disease model, or a sample from such a mammal. In this case, the mammal is more preferably a human or non-human animal. More specifically, the applicable target of the present evaluation method is preferably a sample collected from a patient, or the brain of a mammal or a sample from the brain.

[0192] Furthermore, the disease that is a target for the present method is preferably a neurodegenerative disease, more preferably ALS, and even more preferably ALS accompanied by the presence of a TDP-43 mutant protein or abnormal TDP-43 localization in cells.

[0193] Specifically, the method preferably uses a target that has or is suspected of having a disease and has been exposed to edaravone. The method includes a step of evaluating whether the target is likely to be responsive to edaravone based on changes in the expression levels of the gene products of one or more genes in the specific genes in the target.

[0194] In one embodiment, the present method can also be used as a method to assist in predicting whether a target (eg, a target suffering from or suspected of suffering from a neurodegenerative disease) will effectively respond to edaravone treatment.

[0195] In one embodiment, the method can also be performed under in vitro conditions.

[0196] Hereinafter, in the present embodiment, the experimental group to which the target who has a disease or is suspected of having a disease and has been exposed to edaravone belongs is also referred to as a "disease-exposed group".

[0197] In one embodiment, the method includes step A2, which measures the expression level of the gene product of a specific gene in a target (exposed disease group) suffering from or suspected of having a disease and having been exposed to edaravone. That is, in this step, the expression level of the transcription product of one or more specific genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, FAIM2 or the translation product encoded thereof is measured in a target suffering from a disease or suspected of having a disease and having been exposed to edaravone.

[0198] The measurement of the expression level of the transcription product or the translation product can be performed using the same measurement method as in the above-mentioned embodiment.

[0199] Whether the target used in the present method has a disease or is suspected of having a disease can be evaluated using, for example, the methods in the above embodiments. In addition or alternatively, when a neurodegenerative disease is evaluated as a form of disease, the target can also be evaluated for having or suspected of having a neurodegenerative disease based on the presence of a TDP-43 mutant protein or based on the abnormal intracellular localization of TDP-43. For example, the presence of a TDP-43 mutant protein or the abnormal intracellular localization of TDP-43 can be determined by applying the above-mentioned immunological method to a sample, such as a tissue or a cell.

[0200] Furthermore, for example, animals (including humans) that have been evaluated as having or suspected of having a neurodegenerative disease by performing an examination on their organisms, such as electromyography, or samples from such animals can be used.

[0201] Exposure of the target to edaravone can be performed, for example, by in vivo administration, or after collecting a sample from an animal that has not been exposed to edaravone, the sample can be contacted with edaravone under in vitro conditions.

[0202] For example, in the case where edaravone has been administered in vivo, expression levels can be measured on a sample collected from an organism, such as a human patient or an animal, after administration. In this case, the sample is considered to be a target that has been exposed to edaravone.

[0203] In either case, exposure to edaravone may be with edaravone itself, or may be with a composition according to the above-described embodiments.

[0204] The exposure amount of the target to edaravone can be appropriately changed according to the purpose. For example, in the case of in vivo administration, the exposure amount of edaravone can be set preferably in the range of 0.01 μg / kg body weight or more and 10 mg / kg body weight or less per day.

[0205] In one embodiment, taking the case of administration to adult humans as an example, regardless of the administration form, the exposure amount of edaravone can be set to an amount that can be administered daily within the range of preferably 10 mg or more and 150 mg or less, more preferably 20 mg or more and 120 mg or less, and even more preferably 90 mg or more and 120 mg or less.

[0206] For in vivo administration, for example, the above-mentioned oral and / or parenteral administration methods can be employed.

[0207] When exposing a sample collected from an animal, such as a tissue or cell or an in vitro sample, the exposure concentration of edaravone is preferably 0.1 μmol / L or more and 1000 μmol / L or less, more preferably 0.5 μmol / L or more and 500 μmol / L or less, even more preferably 0.75 μmol / L or more and 300 μmol / L or less, still more preferably 10 μmol / L or more and 300 μmol / L or less, and most preferably 50 μmol / L or more and 250 μmol / L or less.

[0208] The exposure time of the sample to edaravone can be appropriately changed according to the purpose. For example, as long as the concentration is as described above, the exposure time may be preferably 1 hour to 120 hours, more preferably 12 hours to 60 hours.

[0209] In the present method, from the perspective of further improving the accuracy of the assessment, the target may be further exposed to a stress inducer while, before, or after being exposed to edaravone.

[0210] Examples of stress inducers include compounds that cause stress in the exposed target, making it more susceptible to cell damage. Examples of such compounds include one or more of endoplasmic reticulum stress agents, osmotic stress agents, oxidative stress agents, etc. These are preferably used in an in vitro environment.

[0211] Examples of endoplasmic reticulum stress agents include thapsigargin, tunicamycin, dithiothreitol, and the like.

[0212] Examples of osmotic stress agents include sorbitol, sodium chloride, and the like.

[0213] Examples of oxidative stress agents include ethacrynic acid, arsenites (eg, sodium arsenite), quinone compounds (eg, tert-butylhydroquinone), and the like.

[0214] When a stress inducer is included, the content of the stress inducer may be appropriately changed depending on the type and combination of the compounds, but may be each independently set to, for example, 0.1 μmol / L or more and 1000 μmol / L or less, for example, 1 μmol / L or more and 100 μmol / L or less.

[0215] The exposure time to the stress inducer may be in the same range as the exposure time to edaravone.

[0216] In one embodiment, in addition to step A2, the method further comprises step B2 of evaluating whether the target is likely to be responsive to edaravone based on the changes in the measured expression levels of the gene products.

[0217] To assess the likelihood of response to edaravone in step B2, for example, a method comprising at least one of the following steps B21, B22 and B23 may be employed.

[0218] In one embodiment, the method may include performing at least one of steps B22 and B23, for example, after step A2.

[0219] In one embodiment, the method may include, for example, performing step B21 before or after step A2, or performing step B21 simultaneously with step A2, and then performing steps B22 and B23 in that order.

[0220] Step B21 is a step of measuring the expression level of the gene product in the unexposed disease group using the target having or suspected of having the disease and not exposed to edaravone as the unexposed disease group. From the perspective of improving the accuracy of the evaluation, it is preferred that the target in the unexposed disease group is the same as the target in the exposed disease group except for the presence or absence of exposure to edaravone.

[0221] Step B22 is a step of comparing the expression level of the gene product in the target belonging to the non-exposed disease group with the expression level of the gene product in the evaluation target (exposed disease group).

[0222] Step B23 is a step of comparing the disease-unexposed group with the disease-exposed group, and when a change in the expression level of the gene product of one or more genes in the specific genes is confirmed, evaluating a high possibility that the target is responsive to edaravone.

[0223] The evaluation of expression level changes can be performed by setting an arbitrary threshold, or can be performed based on statistically significant differences. From the perspective of improving the accuracy of the evaluation, the evaluation of expression level changes is preferably performed based on the presence or absence of statistically significant differences.

[0224] Specifically, among the above-mentioned specific genes, the expression level of the gene product of one or more genes belonging to the genome (A) can be increased compared to a normal target in the presence of a disease (e.g., a neurodegenerative disease), and can be further reduced by exposure to edaravone compared to a case where only the disease (e.g., a neurodegenerative disease) is present. Therefore, for one or more genes in the genome (A), when the expression level of the gene product in the exposed disease group is reduced compared to the expression level of the gene product in the unexposed disease group, it can be assessed that the target suffering from or suspected of suffering from a disease (e.g., a neurodegenerative disease) has a high possibility of being responsive to edaravone.

[0225] On the other hand, for the gene set (A), when the expression level of the gene product in the exposed disease group increases or remains unchanged compared to the expression level of the gene product in the unexposed disease group, it can be assessed that the target suffering from or suspected of suffering from a disease (e.g., a neurodegenerative disease) has a low possibility of being responsive to edaravone.

[0226] From the perspective of improving the accuracy of the responsiveness assessment to edaravone, it is preferred to target multiple genes among the specific genes and compare the expression levels of the gene products for assessment, and it is more preferred to target all of the above-mentioned specific genes and compare the expression levels of the gene products for assessment.

[0227] In addition, among the above-mentioned specific genes, the expression level of the gene product of the gene belonging to the genome (B) can be reduced compared to the normal target in the presence of a disease (e.g., a neurodegenerative disease), and can be further increased by exposure to edaravone compared to the case where only the disease (e.g., a neurodegenerative disease) exists. Therefore, for the genes in the genome (B), when the expression level of the gene product in the exposed disease group is increased compared to the expression level of the gene product in the unexposed disease group, it can be evaluated that there is a high possibility that the target suffering from or suspected of suffering from the disease (e.g., a neurodegenerative disease) is responsive to edaravone.

[0228] On the other hand, for the gene set (B), when the expression level of the gene product in the exposed disease group is decreased or remains unchanged compared to the expression level of the gene product in the unexposed disease group, it can be assessed that the target having or suspected of having a disease (e.g., a neurodegenerative disease) has a low possibility of being responsive to edaravone.

[0229] From the perspective of improving the accuracy of the evaluation, it is preferred to target all genes belonging to genomes (A) and (B) and compare the expression levels of the gene products for evaluation.

[0230] From the perspective of more accurately evaluating the possibility of responsiveness to edaravone, it is also preferred to further use a normal target, i.e., a healthy animal not affected by the disease or a sample from the animal as a control group, and compare the expression level of the gene product in the evaluation target. That is, in this embodiment, it is preferred to use two or three experimental groups of the exposed disease group, the unexposed disease group and the normal group for comparison.

[0231] The expression level of the gene product of the above-mentioned specific gene can be increased or decreased compared to the normal target in the presence of the disease, and can be further decreased or increased by exposure to edaravone. Based on this useful expression profile, the present embodiment can assess the possibility of the therapeutic effect of edaravone administration or help effectively predict the therapeutic responsiveness.

[0232] Specifically, step B2 in this embodiment preferably includes at least one of the following steps B24, B25, and B26.

[0233] In one embodiment, the method may include performing at least one of steps B25 and B26, for example, after step A2.

[0234] In one embodiment, the method may include, for example, performing step B24 before or after step A2, or performing step B24 simultaneously with step A2, and then performing steps B25 and B26 in this order.

[0235] Step B24 is a step of measuring the expression level of the gene product in the normal target (control group). The normal target is preferably not exposed to edaravone.

[0236] Step B25 is a step of comparing the expression level of the gene product in the target (disease-exposed group) that has or is suspected of having a disease and has been exposed to edaravone with the expression level of the gene product in the normal target (control group).

[0237] Step B26 is a step of evaluating the possibility that the target suffering from or suspected of suffering from the disease is responsive to edaravone and that edaravone is highly effective for the target when it is confirmed that the expression level of the gene product in the target in the exposure disease group is not changed compared to the expression level of the gene product in the normal target for one or more specific genes. When evaluating in this way, for example, the possibility that the exposure amount or exposure frequency of edaravone in the evaluation target is appropriate can also be evaluated.

[0238] On the other hand, for one or more specific genes, when it is confirmed that the expression level of the gene product in the target in the exposure disease group has changed compared to the expression level of the gene product in the normal target, it can be evaluated that there is a possibility that the evaluation target is not responsive to edaravone, or it can be evaluated that there is a possibility that the response to edaravone is low and the effect of edaravone is low, or it can be evaluated that the response to edaravone is high and the effect of edaravone is very high. When evaluating in this way, depending on the increase or decrease in the expression level or the degree of increase or decrease, it can also be evaluated that, for example, there is a possibility that the exposure amount or exposure frequency of edaravone in the evaluation target is excessive or insufficient.

[0239] In addition, taking genome (A) as an example, when the expression level of the gene product in the exposed disease group is lower than the expression level of the gene product in the unexposed disease group and higher than the expression level of the gene product in the normal target (control group), it can also be evaluated that there is a possibility that the evaluated target has low responsiveness to edaravone, or there is a possibility that the exposure amount or exposure frequency of edaravone is insufficient.

[0240] Based on such an assessment, for example, a further step of determining whether appropriate treatment of the target is needed can be performed to allow monitoring of the effectiveness of edaravone treatment or to aid in treatment optimization. Examples of such a need include increasing or decreasing the amount or frequency of exposure to edaravone, or implementing measures other than exposure to edaravone.

[0241] In any case, the evaluation of the change in expression level can be performed by setting an arbitrary threshold value, or can be performed based on statistically significant differences. From the perspective of improving the accuracy of the evaluation, the evaluation of the change in expression level is preferably performed based on the presence or absence of statistically significant differences.

[0242] As another embodiment of the method for evaluating edaravone responsiveness, for example, step B2 in the present embodiment preferably includes steps B21 and B24, and the following steps B27 and B28.

[0243] In one embodiment, the method may include, for example, performing steps A1, B21, and B24 simultaneously or in any order, and then performing steps B27 and B28 in that order.

[0244] Step B27 is a step of comparing the expression level of the gene product of the specific gene in the healthy individuals (control group) with the expression level of the gene product of the specific gene in the unexposed disease group.

[0245] Step B28 is a step of assessing the possibility that the target in the unexposed disease group or the patient or animal from which the target was sampled responds to edaravone treatment when a change in expression level between the two groups is determined.

[0246] That is, the present embodiment is an example of a method for discriminating responsiveness to edaravone using the expression level of a gene product of a specific gene that is increased or decreased due to a disease compared to a healthy individual as a biomarker.

[0247] In one embodiment, the present method can also be used as a method to help predict whether a target having or suspected of having a disease will effectively respond to edaravone treatment.

[0248] When the control group is compared with the unexposed disease group, for one or more genes in the genome (A), when the expression level of the gene product in the unexposed disease group is higher than the expression level of the gene product in the normal group, it can be assessed that the target having or suspected of having a disease (e.g., a neurodegenerative disease) has a high possibility of being responsive to edaravone.

[0249] On the other hand, when the control group is compared with the unexposed disease group, for genome (A), when the expression level of the gene product in the unexposed disease group is lower than or equal to the expression level of the gene product in the normal group, it can be assessed that the target having or suspected of having a disease (e.g., a neurodegenerative disease) has a low possibility of being responsive to edaravone.

[0250] Similarly, for genes belonging to gene group (B), when the control group is compared with the unexposed disease group, when the expression level of the gene product in the unexposed disease group is lower than the expression level of the gene product in the normal group, it can be assessed that the target having or suspected of having a disease (e.g., a neurodegenerative disease) has a high possibility of being responsive to edaravone.

[0251] On the other hand, when the control group is compared with the unexposed disease group, for the genome (B), when the expression level of the gene product in the unexposed disease group is higher than or equal to the expression level of the gene product in the normal group, it can be assessed that the target having or suspected of having a disease (e.g., a neurodegenerative disease) has a low possibility of being responsive to edaravone.

[0252] In the following description, the contents different from the above-mentioned embodiment are mainly described, and for the contents not specifically described, the matters described in this specification are appropriately applied as long as the effects of the present invention are achieved.

[0253] In one embodiment, by using the expression level of the gene product of the above-mentioned specific gene or the change of the expression level as an index, any candidate substance can be selected as a substance capable of treating or preventing neurodegenerative diseases. That is, the present embodiment relates to a method for screening a substance capable of treating or preventing neurodegenerative diseases. Utilizing this method, it is possible to use a method different from the previous one to determine whether an unknown substance is a substance capable of treating or preventing neurodegenerative diseases. Therefore, the substance can be screened as a drug candidate at an earlier stage, which contributes to the early development of a drug.

[0254] Specifically, the method for screening substances capable of treating or preventing neurodegenerative diseases includes: step A3, which exposes the target to a test substance; and step B3, which selects the test substance as a substance capable of treating or preventing neurodegenerative diseases based on changes in the expression level of the gene product of a specific gene in the target exposed to the test substance.

[0255] In the present method, edaravone is preferably excluded from the test substance. That is, it is preferred that the target used for substance evaluation in the present method is exposed to the test substance but not to edaravone.

[0256] In the present method, the target exposed to the test substance is preferably a mammal suffering from or suspected of suffering from a neurodegenerative disease, or a sample from a mammal suffering from or suspected of suffering from a neurodegenerative disease, more preferably, the mammal is a human. More specifically, the applicable target of the present method is preferably the brain of a mammal or a sample from the brain.

[0257] Furthermore, the neurodegenerative disease as a target for evaluation is preferably ALS, and more preferably ALS accompanied by the presence of a TDP-43 mutant protein or abnormal TDP-43 intracellular localization.

[0258] The test substance is not particularly limited, and examples thereof include organic low molecular weight compounds, nucleic acids, proteins, peptides, antibodies, natural compounds and the like.

[0259] In step A3, the method of exposing the target to the test substance is not particularly limited, and may be performed in vitro or in vivo, for example, using a method similar to that in the above-described embodiment.

[0260] For example, when the target is exposed to the test substance in vitro, it can be performed by contacting the target with a liquid in which the test substance is dissolved or dispersed. Examples of the liquid include saline, buffer or culture medium containing the test substance.

[0261] For example, when the target is exposed to the test substance in vivo, the test substance itself or a liquid in which the test substance is dissolved or dispersed can be administered to the mammal. The administration method may be oral or parenteral administration.

[0262] In step B3, the expression level of the gene product of the specific gene is measured, and based on the change in the measured expression level, the test substance is determined to be a substance capable of treating or preventing a neurodegenerative disease.

[0263] In order to determine that the test substance is the expected substance in step B3, a method including at least one of the following steps B31, B32 and B33 may be used.

[0264] In one embodiment, the method may include performing at least one of steps B32 and B33, for example, after step A3.

[0265] In one embodiment, the method may include, for example, performing step B31 before or after step A3, or performing step B31 simultaneously with step A3, and then performing steps B32 and B33 in this order.

[0266] Step B31 is a step of using targets who have not been exposed to the test substance and edaravone and who suffer from or are suspected of suffering from a neurodegenerative disease as a non-exposed group, and measuring the expression level of the gene product of the specific gene in the non-exposed group.

[0267] Step B32 is a step of comparing the expression level of the gene product in the non-exposed group with the expression level of the gene product in the target (test substance exposed group) that has been contacted with the test substance.

[0268] Step B33 is a step of comparing the unexposed group with the exposed group, and for one or more specific genes, when a change in the expression level of the gene product is confirmed, determining the test substance to be a substance capable of treating or preventing a neurodegenerative disease and selecting the substance.

[0269] The determination of the change in expression level can be performed by setting an arbitrary threshold, or can be performed based on statistically significant differences. From the perspective of improving the accuracy of determination, the determination of the change in expression level is preferably performed based on the presence or absence of statistically significant differences.

[0270] The expression level of the gene product of one or more genes belonging to the genome (A) in the above-mentioned specific gene may increase due to the presence of neurodegenerative diseases. Then, the test substance with similar efficacy to edaravone can reduce the expression level of the gene product in the target. Therefore, when the expression level of the gene product of one or more genes in the specific gene in the test substance exposure group is reduced compared with the expression level in the unexposed group, it can be determined that the test substance is a substance that can treat or prevent neurodegenerative diseases.

[0271] On the other hand, when the expression level of the gene products of one or more genes belonging to the genome (A) in a specific gene in the test substance exposure group increases or remains unchanged compared to the expression level in the unexposed group, it can be determined that the test substance is not a substance capable of treating or preventing neurodegenerative diseases, or the possibility of this is low.

[0272] The expression level of the gene product of the gene belonging to the genome (B) in the above-mentioned specific gene may be reduced due to the presence of neurodegenerative diseases. Then, the test substance with similar efficacy to edaravone can increase the expression level of the gene product in the target. Therefore, when the expression level of the gene product of the gene in the test substance exposure group is increased compared with the expression level in the unexposed group, it can be determined that the test substance is a substance that can treat or prevent neurodegenerative diseases.

[0273] On the other hand, when the expression level of the gene product of the gene belonging to the genome (B) in the test substance exposure group is decreased or remains unchanged compared with the expression level in the unexposed group, it can be determined that the test substance is not a substance capable of treating or preventing neurodegenerative diseases, or this possibility is low.

[0274] From the viewpoint of improving the accuracy and reproducibility of test substance determination and more efficient screening, determination is preferably performed by comparing the expression levels of gene products of a plurality of genes among specific genes, and more preferably by comparing all of the above specific genes.

[0275] From the perspective of more accurately determining the contribution of the test substance to the treatment or prevention of the disease, step B3 preferably includes step B35, which further uses the target that has not been exposed to the test substance but has been exposed to edaravone as the edaravone exposure group, and compares the expression level of the gene product in the test substance exposure group. That is, preferably using three experimental groups including the test substance exposure group, the edaravone exposure group and the unexposed group to compare the expression level of each target. That is, in this embodiment, the unexposed group can be used as a negative control, and the edaravone exposure group can be used as a positive control.

[0276] The targets used in these three experimental groups are preferably mammals suffering from or suspected of suffering from neurodegenerative diseases, or samples from mammals suffering from or suspected of suffering from neurodegenerative diseases. More specifically, the applicable targets of the present determination method are preferably mammalian brains or samples from mammalian brains.

[0277] In this case, it is preferable to prepare groups having the same concentrations of the test substance to be exposed and edaravone in terms of improving the accuracy and repeatability of the evaluation.

[0278] In one embodiment of the present method, as step B34, a step of measuring the expression level of the gene product of a specific gene in the edaravone exposure group is performed. When step B34 is performed, it can be performed before step B35.

[0279] In one embodiment, the method may include performing steps A3, B31 and B34 simultaneously or in any order, and then performing (a) steps B32 and B33, and / or (b) step B35 in this order. When performing step groups (a) and (b), at least step B32 may be performed before steps B33 and B35, and steps B33 and B35 may be performed in any order or simultaneously.

[0280] Step B35 is a step of comparing the expression level of the gene product in the test substance exposure group with the expression level of the gene product in the target that has not been exposed to the test substance but has been exposed to edaravone.

[0281] Due to the existence of neurodegenerative diseases, the expression level of the gene product of a specific gene can be increased or decreased. Then, a test substance with similar efficacy to Edaravone can increase or decrease the expression level of the gene product of a specific gene. Therefore, for one or more specific genes, when confirming that the expression level of the gene product in the target exposed to the test substance is not changed compared with the expression level of the gene product in the target exposed to Edaravone, the test substance can be selected as a substance that can treat or prevent neurodegenerative diseases. In addition, it can also be determined that the test substance becomes a high possibility of a material with the same efficacy as Edaravone in treating or preventing neurodegenerative diseases.

[0282] On the other hand, for one or more specific genes, when it is confirmed that the expression level of the gene product in the test substance exposure group has changed compared to the expression level of the gene product in the edaravone exposure group, it can be determined that the test substance has a low possibility of becoming a substance capable of treating or preventing neurodegenerative diseases. In addition, it can be determined that the test substance does not have the same efficacy as edaravone in treating or preventing neurodegenerative diseases, or has a low possibility of efficacy.

[0283] In addition, for example, for one or more genes belonging to genome (A) in a specific gene, when the expression level of the gene product in the test substance exposure group is reduced compared with the expression level of the gene product in the unexposed group, and increased compared with the expression level of the gene product in the edaravone exposure group, it can be evaluated that the efficacy of the test substance may be less than the efficacy of edaravone. For genes belonging to genome (B) in a specific gene, when the expression level of the gene product in the test substance exposure group is increased compared with the expression level of the gene product in the unexposed group, and reduced compared with the expression level of the gene product in the edaravone exposure group, it can be evaluated that the efficacy of the test substance may be less than the efficacy of edaravone. According to such evaluation, measures can be taken to enhance efficacy, such as changing the test substance used or adjusting the exposure amount or exposure frequency of the test substance.

[0284] In any case, from the perspective of improving the accuracy of determination, determination of changes in expression levels is preferably performed based on the presence or absence of a statistically significant difference.

[0285] In one embodiment, in order to determine whether the test substance is the expected substance in step B3, for example, determination can also be made by comparing the results of the expression levels in the test substance exposure group with the results of the expression levels in the normal target.

[0286] Specifically, in one embodiment, step B3 preferably includes at least one of the following steps B36, B37 and B38.

[0287] In one embodiment, the method may include performing at least one of steps B37 and B38, for example, after step A3.

[0288] In one embodiment, the method may include, for example, performing step B36 before or after step A3, or performing step B36 simultaneously with step A3, and then performing steps B37 and B38 in this order.

[0289] In one embodiment, the method may adopt a combination of two or all of the above steps B31, B32 and B33, or a combination of the above steps B34 and B35.

[0290] Step B36 is a step of measuring the expression level of the gene product of the specific gene in the control group using a normal target that has not been exposed to the test substance and edaravone as a control group.

[0291] Step B37 is a step of comparing the expression level of the gene product in the normal target (control group) with the expression level of the gene product in the target (test substance exposure group) that has been in contact with the test substance and suffers from or is suspected of suffering from a neurodegenerative disease.

[0292] Step B38 is a step of comparing the normal target (control group) with the exposure group, and for one or more specific genes, when it is confirmed that there is no change in the expression level of the gene product, determining that the test substance is a substance capable of treating or preventing neurodegenerative diseases and selecting the substance.

[0293] The determination of expression level changes can be performed by setting an arbitrary threshold, or can be performed based on statistically significant differences. From the perspective of improving the accuracy of determination, the determination of expression level changes is preferably performed based on the presence or absence of statistically significant differences.

[0294] The expression level of the gene products of one or more of the above-mentioned specific genes may increase or decrease due to the presence of neurodegenerative diseases. Therefore, when the test substance is likely to be a substance capable of treating or preventing neurodegenerative diseases, the expression level of the gene products of one or more of these specific genes in the test substance exposure group may decrease or increase.

[0295] Therefore, when it is confirmed that, for one or more specific genes, the gene product in the test substance exposure group has no change in expression level compared to that in the normal target, it can be determined that the test substance is a substance capable of treating or preventing a neurodegenerative disease.

[0296] On the other hand, for one or more specific genes, when a change in the expression level of the gene product in the test substance exposure group compared with the normal target is confirmed, it can be determined that the test substance is not a substance capable of treating or preventing neurodegenerative diseases, or the possibility of this is low.

[0297] The gene products of the above-mentioned specific genes can also be used as biomarkers. That is, this embodiment relates to biomarkers.

[0298] As shown in the following examples, the above-mentioned specific genes are those genes that the inventors have newly identified as showing changes in expression levels based on the results of exposure to edaravone and the disease model. Therefore, by using the gene products of specific genes as biomarkers, the presence or absence of neurodegenerative diseases, the progression or severity of the disease, or the possibility of the disease can be easily diagnosed. In addition, by using the gene products as biomarkers, the possibility of promoting the responsiveness or efficacy of edaravone against the disease, or the efficacy of the test substance, can be easily evaluated.

[0299] Specifically, in one embodiment, the biomarker includes gene products of one or more specific genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2. By including multiple types of gene products from two or more genes as biomarkers, the reliability of the assessment or diagnosis can be improved.

[0300] In one embodiment, the above-mentioned biomarkers may consist only of gene products from the above-mentioned specific genes.

[0301] The gene products in the above-mentioned biomarkers may consist of only transcription products, may consist of only translation products, or may consist of a combination of transcription products and translation products.

[0302] The above biomarkers can be used to determine the presence or likelihood of a neurodegenerative disease in a target, or to predict prognosis.

[0303] In another embodiment, the above biomarkers are used to diagnose the prediction of responsiveness to edaravone.

[0304] In yet another embodiment, the above-described biomarkers are used to diagnose responsiveness to a test substance.

[0305] For biomarkers, one or more types of samples described above, more specifically, tissues, cells and body fluids collected from organisms such as animals (regardless of whether edaravone has been administered) and animals (regardless of whether they suffer from a disease) can be used as specimens, and at least one of the transcription product and the translation product in the specimen can be measured using the above-mentioned measurement method. Preferably, this can be performed in vitro.

[0306] The present disclosure also provides a detection kit for detecting the above-mentioned biomarkers. The detection kit includes a detection reagent capable of specifically detecting the gene product of a specific gene. The kit can detect (preferably in vitro) one or more gene products as biomarkers.

[0307] Examples of detection reagents include nucleic acid probes or primers for detecting the transcription products of specific genes. Other examples of detection reagents include antibodies or antibody fragments for detecting the translation products of specific genes, etc. If necessary, for example, fluorescent substances or radioactive nuclides can be used to label the detection reagents.

[0308] In addition, the present disclosure also provides a method for treating a neurodegenerative disease using the above composition. The method of treatment may include, for example, administering the above composition orally or parenterally to a human or non-human animal. When the administration target is a human, the human is preferably a patient suffering from a neurodegenerative disease.

[0309] In one embodiment, the above-mentioned treatment method can be used, for example, using an injection of edaravone or an oral administration preparation as an administration condition of a composition containing edaravone. For the administration conditions of edaravone injection, for example, the administration method described in WO2020 / 091036 can be used, or the administration method of the edaravone preparation used in a clinical setting can be used.

[0310] In addition, the present disclosure also provides use of a substance that causes a change in the expression level of a gene product of the above-mentioned specific gene in the manufacture of a composition for preventing or treating a neurodegenerative disease.

[0311] In addition, the present disclosure also provides a substance for causing a change in the expression level of the gene product of the above-mentioned specific gene. In one embodiment, a substance is also provided for treating a neurodegenerative disease by causing a change in the expression level of the gene product of the above-mentioned specific gene.

[0312] The above-mentioned substance is not particularly limited, and examples thereof include edaravone, a composition containing edaravone, an organic low molecular weight compound, a nucleic acid, a protein, a peptide, an antibody, a natural component, and a test substance discovered using the above-mentioned screening method.

[0313] The present disclosure also provides a method for inhibiting the occurrence or progression of cell damage by causing a change in the expression level of the gene product of the above-mentioned specific gene in the cell.

[0314] In order to cause a change in expression level, for example, a method of contacting cells with a predetermined substance can be used. The substance used for contact is not particularly limited, and examples thereof include edaravone, a composition containing edaravone, an organic low molecular weight compound, a nucleic acid, a protein, a peptide, an antibody, a natural component, and a test substance discovered using the above-mentioned screening method.

[0315] The above embodiments disclosed in this specification can each independently adopt one of the above specific genes, or all of the specific genes, or any combination of two or more specific genes. The following combinations of specific genes are examples and are not limited to these combinations.

[0316] For example, in one embodiment, the above-mentioned specific gene can be one or more selected from KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0317] In addition, for example, in one embodiment, the above-mentioned specific gene may be one or more selected from KAZALD1, SBK1, UBE2L6, NTM, HAUS4, DCTD, ASF1B, FCSK and FAIM2.

[0318] In addition, for example, in one embodiment, the above-mentioned specific gene may be one or more selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B and FCSK.

[0319] Furthermore, for example, in one embodiment, the above-mentioned specific gene may be one or more selected from KAZALD1, SBK1, DCTD and FCSK.

[0320] Furthermore, for example, in one embodiment, the above-mentioned specific gene may be one or more selected from SBK1, DCTD and FCSK.

[0321] Furthermore, for example, in one embodiment, the above-mentioned specific gene may be one or both selected from DCTD and FCSK.

[0322] Regarding the above-mentioned embodiments, this specification also discloses the following embodiments.

[0323] <1> A composition for causing a change in the expression level of a gene product in a target, the composition comprising edaravone, wherein the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0324] <2> according to <1> The composition is used as a medicine containing edaravone as an active ingredient.

[0325] <3> according to <2> The composition is used for treating or preventing neurodegenerative diseases.

[0326] <4> according to <3> The composition, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0327] <5> according to <1> - <4> The composition described in any one of the preceding claims, wherein the target is preferably a mammal or a sample from a mammal, and the mammal is more preferably a human or a rat.

[0328] <6> according to <1> - <5> The composition of any one of claims 1 to 6, wherein the target is the brain of a mammal or a sample from the brain.

[0329] <7> according to <1> - <6> The composition described in any one of the preceding claims, which is used to reduce the expression level of the gene product of a first gene in a target suffering from a neurodegenerative disease, and / or to increase the expression level of the gene product of a second gene in a target suffering from a neurodegenerative disease, wherein the first gene is one or more selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK and MAST1, and the second gene is FAIM2.

[0330] <8> according to <1> - <7> The composition of any one of the above, wherein the target comprises one or more selected from neuronal cells (neurons), glial cells, astrocytes and neural stem cells.

[0331] <9> according to <1> - <8> The composition of any one of the above, wherein a TDP-43 mutant protein is present in the target.

[0332] <10> A method for assessing the likelihood that a target suffers from a neurodegenerative disease, the method comprising step α: assessing whether the target has the likelihood of suffering from a neurodegenerative disease based on changes in the expression levels of gene products in the target, wherein the gene products are gene products of one or more genes selected from KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0333] <11> according to <10> The method, wherein step α comprises: step α1, which compares the expression level of the gene product in the target with the expression level of the gene product in the normal target; and step α2, which evaluates the target as having a high possibility of suffering from a neurodegenerative disease when the expression level of the gene product between the target and the normal target changes.

[0334] <12> according to <11> The method, wherein step α2 is a step that evaluates the target as having a high possibility of suffering from a neurodegenerative disease when the expression level of the gene product of one or more genes selected from KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK and MAST1 is higher than the expression level of the gene product in the normal target, or when the expression level of the gene product of FAIM2 is lower.

[0335] <13> according to <10> - <12> The method of any one of the preceding claims, wherein the gene is any one of the following (i)-(v):

[0336] (i) one or more selected from KAZALD1, SBK1, HAUS4, DCTD, FCSK and FAIM2, (ii) one or more selected from KAZALD1, SBK1, DCTD and FCSK, (iii) one or more selected from SBK1, DCTD and FCSK, (iv) one or more selected from DCTD and FCSK, and (v) one or more selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B and FCSK.

[0337] <14> according to <10> - <13> The method of any one of the above, wherein the target is the brain of a mammal or a sample from the brain, and the mammal is preferably a human.

[0338] <15> according to <10> - <14> The method of any one of the above, wherein the target comprises one or more selected from neuronal cells (neurons), glial cells, astrocytes and neural stem cells.

[0339] <16> according to <10> - <15> The method of any one of claims 1 to 6, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0340] <17> A method for evaluating the responsiveness of a target to edaravone, the method comprising step β: evaluating whether the target is likely to be responsive to edaravone based on changes in the expression levels of gene products after the target is exposed to edaravone, wherein the gene products are gene products of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2, and the target is preferably a target suffering from or suspected of suffering from a neurodegenerative disease.

[0341] <18> according to <17> The method, wherein step β includes: step β1, which compares the expression level of the gene product in the first target that suffers from or is suspected of suffering from a neurodegenerative disease and has not been exposed to edaravone with the expression level of the gene product in the second target that suffers from or is suspected of suffering from a neurodegenerative disease and has been exposed to edaravone; and step β2, when the expression level of the gene product has changed between the first target and the second target, assessing the target that suffers from or is suspected of suffering from the neurodegenerative disease as having a high possibility of being responsive to edaravone.

[0342] <19> according to <18> The method, wherein the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK and MAST1, and step β2 is a step in which, when the expression level of the gene product in the second target is lower than the expression level of the gene product in the first target, the target suffering from or suspected of suffering from a neurodegenerative disease is assessed as having a high possibility of being responsive to edaravone.

[0343] <20> according to <18> and <19> The method described in any one of the preceding claims, wherein the gene product is the gene product of FAIM2, and step β2 is a step in which, when the expression level of the gene product in the second target is higher than the expression level of the gene product in the first target, the target suffering from or suspected of suffering from a neurodegenerative disease is assessed as having a high probability of being responsive to edaravone.

[0344] <21> according to <17> - <20> The method described in any one of the preceding claims, wherein step β comprises: step β3, which compares the expression level of the gene product in the second target suffering from or suspected of suffering from a neurodegenerative disease and exposed to edaravone with the expression level of the gene product in the normal target; and step β4, which assesses that the target suffering from or suspected of suffering from a neurodegenerative disease is likely to be affected by edaravone when there is no change in the expression level of the gene product between the second target and the normal target.

[0345] <22> according to <17> - <21> The method described in any one of the preceding claims, wherein the step β comprises: a step β3 of comparing the expression level of the gene product in the second target suffering from or suspected of suffering from a neurodegenerative disease and exposed to edaravone with the expression level of the gene product in the normal target; and a step β5 of assessing that the target suffering from or suspected of suffering from a neurodegenerative disease is less likely to be affected by edaravone when a change occurs in the expression level of the gene product between the second target and the normal target.

[0346] <23> according to <17> - <22> The method of any one of claims 1 to 5, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS) and the target is a human or a sample from a human.

[0347] <24> A method for screening for substances capable of treating or preventing neurodegenerative diseases, the method comprising step γ: selecting a test substance as a substance capable of treating or preventing a neurodegenerative disease based on changes in the expression levels of gene products in a target exposed to the test substance, wherein the gene products are gene products of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0348] <25> according to <24> The method, wherein the step γ includes: step γ1, which compares the expression level of the gene product in the target that suffers from or is suspected of suffering from a neurodegenerative disease and has been exposed to a test substance with the expression level of the gene product in the target that suffers from or is suspected of suffering from a neurodegenerative disease and has not been exposed to the test substance and edaravone; and step γ2, which selects the test substance as a substance capable of treating or preventing the neurodegenerative disease when a change occurs in the expression level of the gene product between the target that has been exposed to the test substance and the target that has not been exposed to the test substance and edaravone.

[0349] <26> according to <24> or <25> The method, wherein the step γ includes: step γ3, which compares the expression level of the gene product in the target suffering from or suspected of suffering from a neurodegenerative disease and exposed to a test substance with the expression level of the gene product in a normal target that has not been exposed to the test substance and edaravone; and step γ4, which selects the test substance as a substance capable of treating or preventing the neurodegenerative disease when there is no change in the expression level of the gene product between the target that has been exposed to the test substance and the target that has not been exposed to the test substance but has been exposed to edaravone.

[0350] <27> according to <25> or <26> The method, wherein the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK and MAST1, and step γ2 is a step of selecting the test substance as a substance capable of treating or preventing a neurodegenerative disease when the expression level of the gene product in the target that has been exposed to the test substance is lower than the expression level of the gene product in the target that has not been exposed to the test substance and edaravone.

[0351] <28> according to <25> - <27> The method described in any one of the preceding claims, wherein the gene product is the gene product of FAIM2, and step γ2 is a step of selecting the test substance as a substance capable of treating or preventing a neurodegenerative disease when the expression level of the gene product in the target that has been exposed to the test substance is higher than the expression level of the gene product in the target that has not been exposed to the test substance and edaravone.

[0352] <29> according to <24> - <28> The method described in any one of the preceding claims, wherein the step γ comprises: a step γ5 of comparing the expression level of the gene product in the target that suffers from or is suspected of suffering from a neurodegenerative disease and has been exposed to a test substance with the expression level of the gene product in the target that suffers from or is suspected of suffering from a neurodegenerative disease and has not been exposed to the test substance but has been exposed to edaravone; and a step γ6 of assessing that the test substance has a high possibility of being a substance capable of treating or preventing the neurodegenerative disease when there is no change in the expression level of the gene product between the target that has been exposed to the test substance and the target that has not been exposed to the test substance but has been exposed to edaravone.

[0353] <30> according to <24> - <29> The method described in any one of the preceding claims, wherein step γ includes: step γ5, which compares the expression level of the gene product in the target that suffers from or is suspected of suffering from a neurodegenerative disease and has been exposed to a test substance with the expression level of the gene product in the target that suffers from or is suspected of suffering from a neurodegenerative disease and has not been exposed to the test substance but has been exposed to edaravone; and step γ7, when a change occurs in the expression level of the gene product between the target that has been exposed to the test substance and the target that has not been exposed to the test substance but has been exposed to edaravone, assessing that the test substance has a low possibility of being a substance capable of treating or preventing the neurodegenerative disease.

[0355] <31> according to <24> - <30> The method of any one of claims 1 to 6, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0356] <32> according to <24> - <31> The method of any one of claims 1 to 5, wherein the target is a human or a sample from a human.

[0357] <33> A biomarker for diagnosing a neurodegenerative disease or for diagnosing responsiveness to edaravone, the biomarker comprising a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2, preferably a gene product of one or more genes selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B and FCSK.

[0358] <34> A detection kit comprising a detection reagent capable of specifically detecting gene products of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2, for detecting one or more of the gene products as biomarkers for diagnosing neurodegenerative diseases or diagnosing responsiveness to edaravone.

[0359] <35> A method for treating a neurodegenerative disease, the method comprising administering to a patient suffering from a neurodegenerative disease a <1> to <9> The steps of any one of the compositions described above.

[0360] <36> Use of a substance that causes a change in the expression level of a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2, for preparing a composition for preventing or treating a neurodegenerative disease.

[0361] <37> A method for inhibiting the occurrence or progression of cell damage, the method comprising a step of causing a change in the expression level of a gene product of one or more genes selected from the group consisting of KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2 in a cell.

[0362] <38> Edaravone or a pharmaceutical composition containing edaravone, which is used to treat or prevent neurodegenerative diseases, such as ALS, by causing changes in the expression levels of gene products of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

[0363] <39> according to <1> to <37> The composition, method, biomarker, detection kit or use of any one of the above, wherein the gene is selected from one, any combination of two or more or all of KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2, preferably, the gene is selected from one, any combination of two or more or all of KAZALD1, SBK1, UBE2L6, NTM, HAUS4, DCTD, ASF1B, FCSK and FAIM2, more preferably, the gene is selected from one, any combination of two or more or all of KAZALD1, SBK1, DCTD and FCSK, even more preferably, the gene is selected from one, any combination of two or all of SBK1, DCTD and FCSK, and most preferably, the gene is selected from one or both of DCTD and FCSK. [Example]

[0364] Hereinafter, the present invention will be described in further detail based on examples. However, the scope of the present invention is not limited to these examples.

[0365] [Example 1: Preparation of disease model cells]

[0366] (1): Cell culture and differentiation

[0367] As a target, 1464R adult rat neural stem cell line (hereinafter also referred to as "1464R cell line" or "undifferentiated cell line") was used. 1464R cell line was established using the method described in Neuropathology 2014, 34, 83-98.

[0368] The cell lines were cultured in a non-adherent state in a 10 cm culture dish treated with poly-2-hydroxyethyl methacrylate in a maintenance medium at 37°C and 5% CO2, and subculture was performed twice a week.

[0369] A maintenance medium was prepared containing the following components at their final concentrations in Neurobasal Plus medium (manufactured by Thermo Fisher Scientific).

[0370] Typical neurosphere-forming 1464R cells were isolated. The isolated 1464R cells were dispersed and maintained in culture.

[0371] <Additives in Maintenance Medium>

[0372] 2 mM L-glutamine (manufactured by Thermo Fisher Scientific)

[0373] 2% v / v B-27 supplement (manufactured by Thermo Fisher Scientific)

[0374] 10 ng / mL fibroblast growth factor (FGF)-2 (manufactured by Sigma)

[0375] 10 ng / mL epidermal growth factor (EGF) (manufactured by Sigma)

[0376] 50 units / mL penicillin and 50 μg / mL streptomycin (manufactured by Thermo Fisher Scientific)

[0377] Next, the 1464R cell line was cultured in a poly-L-lysine treated culture dish in an adherent state using a differentiation medium as shown below. The culture conditions for cell differentiation were 37°C, 5% CO2, and 4 days. Through this step, the 1464R cell line differentiated into neuronal cells (neurons). In addition, the 1464R cell line was partially differentiated into glial cells. In the following description, the differentiated 1464R cell line is also collectively referred to as a "differentiated cell line".

[0378] A differentiation medium was prepared containing the following components at their final concentrations in F-12 medium (manufactured by Thermo Fisher Scientific).

[0379] <Additives in Differentiation Medium>

[0380] 5v / v% fetal bovine serum (manufactured by Moregate)

[0381] 0.5 v / v% N-2 supplement (manufactured by Thermo Fisher Scientific)

[0382] 1v / v% B-27 supplement (manufactured by Thermo Fisher Scientific)

[0383] 1 μmol / L ATRA (manufactured by Sigma)

[0384] 50 units / mL penicillin and 50 μg / mL streptomycin (manufactured by Thermo Fisher Scientific)

[0385] (2): Forced expression of TDP-43 protein

[0386] Subsequently, an in vitro model of neurodegenerative diseases was prepared by forced and overexpression of TDP-43 protein in differentiated cell lines according to the following method.

[0387] After obtaining the differentiated cell line using the method described in (1), the differentiation medium was removed and replaced with infection medium. The infection medium composition was prepared, which contained the following components at their final concentrations in F-12 medium (manufactured by ThermoFisher Scientific) without antioxidants and serum: The culture conditions were maintained at 37°C in a 5% CO2 environment.

[0388] <Additives in infection medium>

[0389] 0.5 v / v% N-2 supplement (manufactured by Thermo Fisher Scientific)

[0390] 1 μmol / L ATRA (manufactured by Sigma)

[0391] 50 units / mL penicillin and 50 μg / mL streptomycin (manufactured by Thermo Fisher Scientific)

[0392] Separately, the following recombinant adenoviral vectors (a) and (b) were prepared according to the method described in Neuropathology 2014, 34, 83-98.

[0393] (a) Wild type: a recombinant adenoviral vector containing cDNA encoding full-length human wild-type TDP-43 tagged with DsRed (AxDsRhWT TDP43 strain; RIKEN DNA Bank Japan; #RDB15499)

[0394] (b) Mutant: a recombinant adenoviral vector containing cDNA encoding the C-terminal fragment of TDP-43 (amino acid sequence number 208-414) tagged with DsRed (AxDsRhWT TDP43 strain; RIKEN DNA Bank Japan; #RDB15500)

[0395] Using vectors (a) and (b), the differentiated cell line in the infection medium was infected with adenovirus at an infection multiplicity of 50 and cultured for 24 hours. As a result, neurons from the 1464R cell line were obtained in which both wild-type and mutant TDP-43 were forcibly expressed in the cells (hereinafter also referred to as disease model cells).

[0396] These disease model cells serve as in vitro models of neurodegenerative diseases in which the localization of TDP-43 expression and the presence of mutant TDP-43 proteins may be involved. An example of such a neurodegenerative disease is ALS.

[0397] [Example 2: Evaluation of cell protection by microscopic observation]

[0398] The disease model cells prepared by the method of Example 1 were used to evaluate whether edaravone (hereinafter also referred to as Eda) inhibits cell damage.

[0399] A 96-well plate treated with poly-L-lysine was used with 8 × 10 4 Cells were inoculated at a concentration of 10 cells / well and cultured and differentiated under the conditions of (1) in Example 1. TDP-43 was then forcibly expressed in the differentiated cells using the method of (2) in Example 1 to obtain disease model cells.

[0400] For disease model cells, a group exposed to EA (final concentration 20 μmol / L) for 24 hours was prepared as an Eda-free group (see Figure 1 ). Separately, as an Eda-containing group, a group exposed to Eda (final concentration 200 μmol / L) for 24 hours and then to EA (final concentration 20 μmol / L) for 24 hours was prepared (see Figure 2 ). Then the cell morphology in each group was observed using a fluorescence microscope.

[0401] Tubulin βIII (indicated by TuJ1 in the figure), an indicator of neuronal differentiation, was fluorescently stained by fixing the cells with 4% paraformaldehyde after 24 hours of EA exposure, treating with TuJ1 antibody at 4°C overnight, and then treating with fluorescent antibody at room temperature for 15 minutes. The cell nuclei were fluorescently stained with Hoechst33342 using conventional methods. The results are shown in Figure 1 and 2 middle.

[0402] like Figure 1 As shown, in the Eda-free group, it was confirmed that the neuron-specific neurite shape observed by the localization of tubulin βIII had disappeared, indicating that cell damage had occurred.

[0403] On the other hand, Figure 2 As shown, in the Eda-containing group, the neuron-specific neurite shape observed by the localization of tubulin βIII was confirmed to be fully maintained, indicating a cytoprotective effect. The localization of TDP-43 was confirmed by DsRed, and the localization of the cell nucleus was also confirmed to exist in the cell.

[0404] [Examples 3-1 to 3-6, Comparative Example 1 and Reference Example 1: Evaluation of cell protection using a chromogenic substrate as an indicator]

[0405] The disease model cells prepared using the method of Example 1 were used to prepare the following groups: an Eda-containing group (Examples 3-1 to 3-6): the cells were exposed to Eda (final concentration 1-200 μmol / L) for 24 hours and then exposed to EA (final concentration 20 μmol / L) for 24 hours; an Eda-free group (Comparative Example 1): the cells were exposed to EA (final concentration 20 μmol / L) for 24 hours without previous exposure to Eda; and an unexposed group (Reference Example 1): the cells were not exposed to Eda or EA.

[0406] Thereafter, the cells in each group were exposed using a cytotoxicity assay kit (Cell Counting Kit-8, manufactured by DOJINDO) according to the product protocol, and the amount of the generated chromogenic substrate was measured by absorbance at 450 nm.

[0407] Using this kit, the more viable cells present, the more chromogenic substrate is produced, resulting in a higher absorbance value at 450nm. Therefore, a higher absorbance at 450nm indicates a greater number of viable cells. The results are shown in Figure 3 middle.

[0408] Figure 3 The data shown in are expressed as mean ± standard error of the mean (SEM, each n = 4). Figure 3 In the table, the symbol "##" indicates that there is a statistically significant difference (determined by Student t test) compared with Comparative Example 1 at p < 0.01. Figure 3 In the figure, the symbol "*" indicates that compared with Comparative Example 1, p<0.05 has a statistically significant difference, and the symbol "**" indicates that compared with Comparative Example 1, p<0.01 has a statistically significant difference (both determined by Williams multiple comparison test).

[0409] like Figure 3 As shown, in the groups containing Eda (Examples 3-1 to 3-6), the absorbance at 450 nm increased in an Eda concentration-dependent manner compared to the group not exposed to Eda (Comparative Example 1), confirming that the number of viable cells was higher. This suggests that cell damage was suppressed in the presence of Eda. In addition, the present inventors confirmed that even when the disease model cells prepared by the method of Example 1 were exposed to Eda and EA at the same time for 24 hours, the same viable cell number could be obtained. Figure 3 Similar results were shown.

[0410] [Example 4: Exploration and evaluation of biomarkers]

[0411] The disease model cells prepared using the method of Example 1 were subjected to RNA-seq analysis to identify biomarkers that can be used as indicators of disease progression, evaluation of treatment effects, and / or selection of candidate compounds.

[0412] In this evaluation, a 6-well plate treated with poly-L-lysine was used with 1 × 10 6 The cells were seeded at a density of 10 cells / well and cultured under the conditions of (1) of Example 1, and the differentiated cells were used.

[0413] Below and Figure 4 Experimental groups (1) to (4) of this example are shown together.

[0414] (1) Group 1 (control group; Figure 4 7-11 ): After preparing the differentiated cell line using the method of (1) in Example 1, the cells were further cultured in a differentiation medium without Eda and EA for 48 hours. This experimental group does not force the expression of TDP-43, but simulates healthy animals, including humans.

[0415] (2) Group 2 (the group exposed only to Eda; Figure 4 and "Group 2" in Figures 7-11): After preparing the differentiated cell line using the method of Example 1 (1), the cells were cultured in a differentiation medium containing Eda (final concentration 50 μmol / L) but without EA for 48 hours. This experimental group does not force the expression of TDP-43, but simulates the situation in which healthy animals, including humans, are exposed to Eda.

[0416] (3) Group 3 (TDP-43+EA exposure group; Figure 4 and "Group 3" in Figures 7-11): After preparing disease model cells expressing TDP-43, the cells were cultured in a differentiation medium without Eda but containing EA (final concentration 20 μmol / L) for 24 hours. This experimental group forced the expression of TDP-43 and simulated a situation in which the target had a neurodegenerative disease and was not treated.

[0417] (4) Group 4 (TDP-43+EA+Eda exposure group; Figure 4 and "Group 4" in Figures 7-11): After preparing disease model cells expressing TDP-43, the cells were cultured in a differentiation medium containing Eda (final concentration 50 μmol / L) but not EA for 24 hours. Then, EA (final concentration 20 μmol / L) was further added, and the cells were further cultured for an additional 24 hours. This experimental group simulates a situation in which Eda is administered to a target suffering from a neurodegenerative disease or is exposed to Eda.

[0418] After completion of the culture, cell pellets containing 1×10 6 or more cells were obtained from experimental groups (1)-(4). According to the product instructions, total RNA was extracted from these cell pellets using NucleoSpin RNA (manufactured by Macherey-Nagel).

[0419] After that, all extracted RNA was confirmed to be of high quality using TapeStation and Bioanalizer RNA6000 Nano Chip (manufactured by Agilent), with an RNA concentration of 2 ng / μL or more and a total RNA amount of 50 ng or more.

[0420] Using the extracted total RNA (1.0 ng) as a template, double-stranded cDNA was synthesized by the SMART method using SMART-Seq v4 Ultra (registered trademark) Low Input RNA Kit for Sequencing (manufactured by Clontech) according to the product instructions.

[0421] After that, 13 cycles of PCR were performed to amplify the synthesized double-stranded cDNA according to the product instructions. Then, the PCR products were purified by the magnetic bead method using AMPure XP (manufactured by Beckman Coulter).

[0422] Using the Nextera XT DNA Library Prep Kit and Nextera XT Index Kit v2 (manufactured by Illumina), the obtained PCR products (double-stranded cDNA) were labeled, and the two ends of the fragmented cDNA were ligated to the adapter sequences according to the product instructions. After that, 12 cycles of PCR amplification were performed on the ligated cDNA (0.2 ng) using the primers provided in the kit according to the product instructions. Then, the PCR products of all samples were pooled into a library to avoid any adverse effects that may occur during the sequencing process.

[0423] The PCR products obtained from the experimental groups were processed through several steps, as Figure 5 shown.

[0424] <RNA sequencing (see Figure 5 )>

[0425] Next, the sequence of the cDNA library was determined using the NovaSeq 6000 system (NovaSeq 6000 S4 kit, NovaSeq Xp4 flow cell kit, and NovaSeq control software (version 1.6.0); all manufactured by Illumina) according to the product instructions.

[0426] Base calls and quality scores were calculated from the cycle-based call files using Real-Time Analysis (version 3.4.4; manufactured by Illumina). The base call files were converted to FASTQ files using bcl2fastq2 software (version 2.20, manufactured by Illumina).

[0427] <Comparison-Expression Analysis (see Figure 5 )>

[0428] Next, the type and abundance of genes were identified from the 150 base sequence information contained in the FASTQ file mentioned above using DRAGEN Bio-IT platform software (version 3.6.3, manufactured by Illumina) with reference to the rat genome assembly (version 6.0, top level) and transcript annotation (release 101) obtained from the Ensembl database.

[0429] Based on the alignment with the rat genome and the transcript annotation from the Ensembl database, the RNA expression level (TPM) of transcript i measured from the cDNA library of the experimental group was calculated according to the following equation (A).

[0430] In the following equation (A), "m_i" represents the number of reads of transcript i, and "l_i" represents the base length of transcript i.

[0431] TPM_i=10 6 ×(m_i / l_i) / (Σ_i[(m_i / l_i)])...(A)

[0432] Differential expression analysis was performed using DESeq2 (two-group comparison package) in statistical analysis software R (version 3.6.0) to calculate the log2 ratio of RNA expression levels and perform significance tests. The log2 ratio is a value expressed as "log2 ([expression level of the first measurement target] / [expression level of the second measurement target])".

[0433] Significance testing was performed based on p-values ​​calculated by the Wald test using multiple testing correction based on the Benjamini-Hochberg method (BH method).

[0434] In this analysis, genes with an absolute log2 ratio of 1 or more (expression change rate of more than 2.0 times or less than 0.50 times) between any two groups and a p-value of 0.01 or less by the Wald test were identified as "genes with changed expression levels" (differentially expressed genes; DEGs). DEGs include up-regulated genes and down-regulated genes.

[0435] <Gene Ontology Analysis (see Figure 5 )>

[0436] Gene ontology analysis was performed by associating DEG with the ontology terms obtained from the BioMart database. These associations were performed by considering that the p value was 0.05 or less ontology terms were significantly associated in the hypergeometric distribution test based on the multiple test correction of the BH method. Gene ontology classification related to DEG was performed, which focused on biological function, structural composition and molecular function, and extracted important ontology terms.

[0437] <Expression regulation pathway analysis (see Figure 5 )>

[0438] DEGs were subjected to expression regulation pathway analysis using software (Ingenuity Pathway Analysis, manufactured by QIAGEN). The association between the expression pathways defined in the software and DEGs was performed by considering those with p values ​​calculated by Fisher's exact test with multiple testing correction based on the BH method as 0.05 or less as significant.

[0439] The analysis results in this example are shown in Figure 6 middle.

[0440] Figure 6 The results of principal component analysis of expression changes in RNA-seq data obtained from experimental groups (each N=3) are shown. The X-axis represents the first principal component, the Y-axis represents the second principal component, and their respective contributions are expressed in percentages.

[0441] Principal component analysis was performed by excluding genes with TPM variance values ​​of 1 or less in all samples.

[0442] like Figure 6 As shown, the RNA-seq data of samples from the same experimental group are very close to each other, indicating that the RNA-seq data are of good quality.

[0443] Comparison of the experimental groups revealed that Groups 3 and 4 had significantly different gene expression profiles compared to Group 1.

[0444] Furthermore, it was found that although both groups 3 and 4 expressed TDP-43, their gene expression profiles were significantly different.

[0445] On the other hand, it was found that Group 1 and Group 2 had similar gene expression profiles regardless of the presence or absence of Eda exposure.

[0446] Table 1 below shows the results of identifying differentially expressed genes (DEGs) between experimental groups based on group gene expression levels. In Table 1, "xxx / yyy" (wherein xxx and yyy independently represent any integer) means that the number of up-regulated genes is xxx, and the number of down-regulated genes is yyy.

[0447]

[0448] From the differentially expressed genes in the experimental groups, genes that met the following criteria a or b were identified, and their expression change rates are shown in Figures 7-10.

[0449] Criterion a: Compared with Group 1, the expression level in Group 3 was significantly upregulated, and compared with Group 3, the expression level in Group 4 was significantly downregulated.

[0450] Criterion b: Compared with Group 1, the expression level in Group 3 was significantly down-regulated, and compared with Group 3, the expression level in Group 4 was significantly up-regulated.

[0451] A gene that meets any of the above criteria means that its expression level changes with the occurrence of the disease (corresponding to group 3) compared with the healthy state (corresponding to group 1), and its expression level is restored to a level equivalent to or close to normal by treating the disease (corresponding to group 4).

[0452] Therefore, the expression levels of these genes can be used as important biomarkers, for example, by using changes in expression levels as indicators to: assess or predict the likelihood of the presence or absence of a disease and the extent of disease progression; assess or predict the likelihood of treatment responsiveness; select appropriate samples for use as disease models; or select candidate compounds in the drug discovery process.

[0453] The genes shown in Figures 7 to 11 are all rat genes that meet the above criteria and have human homologs.

[0454] For example, Alpl (rat ortholog of human ALPL) shown in Figure 8(a) is a gene encoding alkaline phosphatase. Alkaline phosphatase is an enzyme present in extracellular fluid (e.g., blood), and therefore, it is useful, for example, to assess disease progression or treatment effectiveness, or to assist in such predictions in a minimally invasive manner by collecting extracellular fluid (e.g., blood).

[0455] Tables 2 and 3 below show the results of gene ontology analysis, as well as differentially expressed genes, their expression change magnitude (TPM), expression change log2 ratio, and p-value.

[0456] In addition, Table 4 below shows the expression change log2 ratios and p-values ​​of other differentially expressed genes, which are rat genes that meet the above criteria a and b. All genes listed in Table 4 are included in the specific genes of the present disclosure.

[0457] In Table 4, "up-regulated genes" are examples of genes whose expression levels are decreased by the presence of the disease and increased by the presence of the disease and edaravone. These genes are included in the above-mentioned gene set (B).

[0458] In Table 4, "down-regulated genes" are examples of genes whose expression levels are increased by the presence of the disease and decreased by the presence of the disease and edaravone. These genes are included in the above-mentioned gene set (A).

[0459] For ease of description, the results of Mt2A and Alpl genes listed in Tables 2 and 3 are re-listed in Table 4. The present inventors performed quantitative PCR on Mt2A and Alpl and confirmed that expression profiles similar to those based on RNA-seq were obtained.

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472] [Table 4]

[0473]

[0474] a) Significantly upregulated in group 4 compared with group 3 (adjusted p-value ≤ 0.01 and / or log2 fold change ≥ 1)

[0475] b) Significantly downregulated in group 4 compared to group 3 (adjusted p-value ≤ 0.01 and / or log2 fold change ≤ -1)

[0476] [Examples 5-1 and 5-2, Comparative Example 2, and Reference Examples 2 and 3: Evaluation of cell protection using a chromogenic substrate as an indicator (2)]

[0477] The differentiated cell line prepared using the method of Example 1 ( Fig.12A group not exposed to Eda (Reference Example 2) and a group exposed to Eda for 96 hours (final concentration 100 μmol / L) (Reference Example 3) were prepared.

[0478] In addition, the disease model cells ( Fig.12 A group not exposed to Eda (Comparative Example 2) and a group exposed to Eda for 96 hours (final concentration of 50 or 100 μmol / L) were prepared (Examples 5-1 and 5-2).

[0479] All of this is done without the inclusion of EAs.

[0480] Afterwards, similar to Example 3-1, the cells of each group were exposed using a cytotoxicity assay kit according to the product instructions, and the amount of the generated chromogenic substrate was measured by the absorbance at 450 nm. The results are shown in Fig.12 middle.

[0481] Fig.12 The data shown in are expressed as mean ± standard error of the mean (SEM, each n = 4). Fig.12 In the table, the symbol "##" indicates that there is a statistically significant difference compared with Comparative Example 2 at p < 0.01. Fig.12 In the table, the symbol "**" indicates that there is a statistically significant difference compared with Comparative Example 2 at p < 0.05. Fig.12 In the figure, the symbol "$" indicates that there is a statistically significant difference compared with Reference Example 2 at p<0.05 (both determined by Student's t-test).

[0482] like Fig.12 As shown, in the groups in which TDP-43 was present and Eda was contained (Examples 5-1 to 5-2), a higher number of living cells was confirmed compared to the group in which TDP-43 was present and not exposed to Eda (Comparative Example 2). This suggests that cell damage occurs in the presence of TDP-43 even in the absence of EA, and Eda inhibits cell damage caused by TDP-43.

[0483] [Example 6: Preparation of disease model cells (2)]

[0484] Human induced pluripotent stem cells (iPS) are induced from cells derived from ALS patients and cells derived from healthy individuals using conventional methods. Using cells derived from ALS patients, it is known that the cells have ALS risk mutations as heterozygous mutations in the TARDBP gene. Each iPS cell is differentiated into motor neurons (hereinafter also referred to as "neurons") using conventional methods. Neurons differentiated from iPS cells induced by cells derived from ALS patients are referred to as "neurons derived from ALS patients", and neurons differentiated from iPS cells induced by cells derived from healthy individuals are referred to as "neurons derived from healthy individuals". For the long-term preservation of cells, if necessary, neurons are cryopreserved 2 to 7 days after differentiation treatment. Neurons that have been cryopreserved are also referred to as "frozen neurons".

[0485] Subsequently, neurons were cultured using 98-well culture plates (Corning, 3548) under the conditions shown below. Before cell inoculation, the culture plates were coated in advance. Specifically, 0.02% poly-L-ornithine solution (Sigma-Aldrich, P4957) was added to the wells of the culture plates and kept stationary in a 37°C 5% CO2 incubator for 2 hours. Thereafter, the wells were washed, 20 mg / mL laminin solution (Thermo Fisher Scientific, 23017015) was used and kept stationary in a 37°C 5% CO2 incubator for another 2 hours. Afterwards, the frozen neurons were thawed using conventional methods, and the thawed neurons were seeded into the wells at 20,000 cells / well, and the neurons were cultured in the presence of a culture medium having the following composition.

[0486] The culture medium used was a mixture having the following composition.

[0487] ·DMEM / F12 (Thermo Fisher Scientific, 21331-020): 50% v / v

[0488] Neurobasal medium (Thermo Fisher Scientific, 21103-049): 50% v / v

[0489] Glutamax supplement (Thermo Fisher Scientific, 35050061): 1% v / v

[0490] Penicillin-streptomycin (10000 units / mL) (Thermo Fisher Scientific, 15140-148): 0.5% v / v

[0491] Component N1 (Elixirgen): 3% v / v

[0492] Component A (Elixirgen): 0.1% v / v

[0493] Component D4 (Elixirgen): 0.1% v / v

[0494] Component P (Elixirgen): 0.05% v / v

[0495] [Example 7: Evaluation of cell damage by live cell imaging]

[0496] A treatment was performed in which a medium in which a dimethyl sulfoxide (DMSO) solution of an evaluation compound (edaravone) was dissolved (final concentration: edaravone 30 μM, DMSO 0.03% v / v) was contacted with neurons after 7 days of culture, and the occurrence of cell damage was evaluated over time. Separately, a group in which no evaluation compound was added to the medium (a group in which DMSO at the same concentration was added) was also prepared.

[0497] Cell injury was assessed by live cell imaging using IncuCyte S3 (Sartorius) and Cytotox (Sartorius, 4846) according to the attached protocol. Axon length per unit area (mm / mm) was analyzed over time. 2 ) and the number of dead cells (unit: counts / image) and used them as evaluation indicators. The smaller the axon length value and the greater the number of dead cells, the more severe the cell damage.

[0498] <Evaluation of cell damage based on axon length (1)>

[0499] Fig.13 The results of calculating the rate of change in axon length of each experimental group from the start of evaluation compound treatment to the time of analysis (24 hours) are shown, expressed as a ratio, in which the DMSO group without evaluation compound was set to 100. The smaller the value on the vertical axis, the greater the reduction in axon length over time, indicating that cell damage has occurred. Fig.13 The experimental groups in are as follows.

[0500] ALS cells-DMSO: A group using neurons derived from ALS patients, and the culture medium does not contain edaravone

[0501] ALS cells-edaravone: A group using neurons derived from ALS patients, with edaravone in the culture medium

[0502] Healthy cells-DMSO: This group used neurons derived from healthy individuals, and the culture medium did not contain edaravone

[0503] Healthy cells-edaravone: A group using neurons derived from healthy individuals, with edaravone in the culture medium

[0504] When edaravone treatment was applied to neurons derived from healthy individuals and neurons derived from ALS patients, axon length in neurons derived from ALS patients increased significantly compared to the DMSO-treated group, indicating a protective effect on axons. In contrast, no significant changes in axon length were observed in neurons derived from healthy individuals.

[0505] <Evaluation of cell damage based on the number of dead cells (2)>

[0506] Fig.14 The results of calculating the change rate of cell death in each experimental group from the start of evaluation compound treatment to the time of analysis (24 hours) are shown, expressed as a ratio, in which the DMSO-treated group without evaluation compound was set to 100. The larger the value on the vertical axis, the greater the number of dead cells over time, indicating that cell damage has occurred. Fig.14 The experimental groups in are as follows.

[0507] ALS cells-DMSO: A group using neurons derived from ALS patients, and the culture medium does not contain edaravone

[0508] ALS cells-edaravone: A group using neurons derived from ALS patients, with edaravone in the culture medium

[0509] Healthy cells-DMSO: This group used neurons derived from healthy individuals, and the culture medium did not contain edaravone

[0510] Healthy cells-edaravone: A group using neurons derived from healthy individuals, with edaravone in the culture medium

[0511] When edaravone treatment was applied to neurons derived from healthy individuals and neurons derived from ALS patients, the number of dead neurons in neurons derived from ALS patients was significantly reduced compared to the DMSO-treated group, indicating a protective effect against neuronal cell death. In contrast, no significant changes in the number of dead neurons were observed in neurons derived from healthy individuals.

[0512] [Example 8: TDP-43 localization assessment]

[0513] The intracellular localization of TDP-43 polypeptide was evaluated by immunofluorescence staining of neurons. Cultured neurons were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). The cells were fixed with 5% fetal bovine serum (FBS) and 0.1% Triton. TMAfter blocking with -X PBS, a dilution of primary antibodies containing anti-TDP-43 antibody and anti-β-III tubulin antibody was added and incubated overnight at 4°C. Anti-β-III tubulin antibody was used to visualize neuronal cell bodies. The next day, after washing with PBS, a dilution of secondary antibodies conjugated with Alexa dye was added and incubated at room temperature for 1 hour. After further nuclear staining, the cells were washed with PBS and imaged under a fluorescence microscope. The captured images were analyzed using Matlab (Mathworks), and the fluorescence intensity of TDP-43 in the nuclear and cytoplasmic regions was calculated respectively. The results of intracellular localization of TDP-43 polypeptide are shown in Fig.15 middle. Fig.15 The experimental groups in are as follows.

[0514] ALS cells-DMSO: A group using neurons derived from ALS patients, and the culture medium does not contain edaravone

[0515] ALS cells-edaravone: A group using neurons derived from ALS patients, with edaravone in the culture medium

[0516] Healthy cells-DMSO: This group used neurons derived from healthy individuals, and the culture medium did not contain edaravone

[0517] Healthy cells-edaravone: A group using neurons derived from healthy individuals, with edaravone in the culture medium

[0518] Regarding the intracellular localization of TDP-43 polypeptide, the nuclear and cytoplasmic TDP-43 fluorescence intensities and the cytoplasmic / nuclear TDP-43 intensity ratios of ALS cells and healthy cells are shown in Fig.15 The values ​​are expressed as ratios, with the DMSO group set to 100. When edaravone treatment was applied to neurons derived from healthy individuals and neurons derived from ALS patients, the cytoplasmic TDP-43 intensity and cytoplasmic / nuclear TDP-43 intensity ratio in neurons derived from ALS patients were significantly reduced after 24 hours, indicating that the abnormal localization of TDP-43 observed in ALS pathology was suppressed. In addition, this effect of reducing the cytoplasmic / nuclear TDP-43 intensity ratio was not observed in neurons derived from healthy individuals.

[0519] From the above results, it is newly proved that the drug edaravone, which has long been approved for use in ALS, has the effect of inhibiting neuronal cell damage from ALS patients and improving the abnormal intracellular localization of TDP-43. Abnormal localization of TDP-43 is a common pathological state in the pathological state of ALS (which is a heterogeneous disease), and the demonstration of its effect in correcting abnormal localization of TDP-43 shows the possibility that it has a therapeutic effect on ALS patients in general. In addition, it is not limited to ALS, and it can play a beneficial effect in the treatment or prevention of diseases related to abnormal localization of TDP-43. These are new discoveries that have become clear by completing the present invention, and the results show that edaravone can be a suitable treatment or prevention method for diseases related to abnormal localization of TDP-43.

Claims

1. A composition for causing a change in the expression level of a gene product in a target, the composition comprising edaravone, wherein the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

2. The composition according to claim 1, which is used as a medicament containing edaravone as an active ingredient.

3. The composition according to claim 2, which is used for treating or preventing neurodegenerative diseases.

4. The composition of claim 3, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

5. The composition according to any one of claims 1-4, wherein the target is a mammal or a sample from a mammal.

6. A method for evaluating the responsiveness of a target to edaravone, the method comprising The step of evaluating whether the target is likely to be responsive to edaravone based on a change in the expression level of the gene product caused by exposure of the target to edaravone, wherein The gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

7. A method for assessing the likelihood that a target suffers from a neurodegenerative disease, the method comprising A step of evaluating whether the target has a likelihood of suffering from a neurodegenerative disease based on a change in the expression level of a gene product in the target, wherein The gene product is a gene product of one or more genes selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B and FCSK.

8. A method for screening for substances capable of treating or preventing neurodegenerative diseases, the method comprising the step of selecting a test substance as a substance capable of treating or preventing a neurodegenerative disease based on a change in the expression level of a gene product caused by exposure of a target to the test substance, wherein the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1 and FAIM2.

9. A biomarker for diagnosing a neurodegenerative disease, the biomarker comprising The gene products of one or more of the genes KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B and FCSK.

10. A biomarker for diagnosing edaravone responsiveness, the biomarker comprising a The gene products of one or more of KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2.

Images