Composition and method for evaluating responsiveness of edaravone

Abstract

The composition according to the present disclosure includes edaravone and is used to change the expression level of a gene product in a target. The gene product is a gene product from one or more genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2. The composition is also preferably used as a medicine. The composition is also preferably used for treatment or prevention of a neurodegenerative disease. The neurodegenerative disease is preferably amyotrophic lateral sclerosis (ALS). The present disclosure also provides a method for evaluating responsiveness of edaravone on the basis of an expression level of the gene product or a change in the expression level.

Description

Composition and method for assessing responsiveness to edaravone

[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 herein by reference.

[0002] The present invention relates to a composition, a method for evaluating the possibility of response to edaravone, and the like.

[0003] Edaravone is an organic compound known as 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (CAS number 89-25-8). Edaravone is used medicinally as a neuroprotectant and a therapeutic agent for amyotrophic lateral sclerosis (ALS), a type of neurodegenerative disease (Patent Documents 1 and 2).

[0004] ALS is an intractable disease that begins with initial symptoms such as hand weakness, finger movement disorder, and upper limb fasciculations, and progresses to muscle atrophy, muscle weakness, bulbar paralysis, and muscle fasciculations, eventually leading to respiratory failure. Neurodegenerative diseases such as ALS have been reported to involve, for example, TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 is a protein identified as one of the components found in the brains of patients with frontotemporal lobar degeneration (FTLD) and ALS (see Non-Patent Document 1). The onset and progression of neurodegenerative diseases such as ALS are thought to be due in part to the formation of TDP-43 protein aggregates.

[0005] Japanese Patent Publication No. 5-31523 U.S. Patent No. 6,933,310

[0006] Arai et al., Biochem Biophys Res Commun. 351(3):602-11 (2006)

[0007] The present inventors have found that exposure of a subject to edaravone causes changes in the expression level of a gene product of a specific gene, and have also found that this change in expression level can be a useful indicator, such as a biomarker, for exploring responsiveness to edaravone and susceptibility to an indicated disease.

[0008] The present disclosure encompasses inventions related to compositions. In one embodiment, the composition is used to alter the expression level of a gene product in a subject. 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.

[0009] The present disclosure also encompasses an invention relating to a method for assessing the likelihood of response to edaravone. In one embodiment, the assessment method comprises a step of assessing whether a subject is likely to be responsive to edaravone based on changes in the expression level of a gene product due to exposure of the subject to edaravone. 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. Other embodiments will be apparent from the description of the present specification and claims.

[0010] According to the present invention, the expression level of a specific gene product can be altered by exposing a subject to edaravone. This allows advantageous effects to be exerted according to the function of the gene, and can be used, for example, to treat or prevent a disease. Furthermore, according to the present invention, the expression variation of a specific gene product can be used to screen for substances that can treat or prevent a disease. Furthermore, according to the present invention, the expression variation of a specific gene product can be used for various types of assessments and predictions, such as the presence or absence of a disease, the degree of its progression, or treatment response.

[0011] FIG. 1 shows fluorescence microscopy images of cells expressing wild-type and mutant TDP-43 proteins when exposed to ethacrynic acid (EA). The lower image in FIG. 1 is an enlarged version of the upper image in FIG. 1. FIG. 2 shows fluorescence microscopy images of cells expressing wild-type and mutant TDP-43 proteins when exposed to ethacrynic acid and edaravone. The lower image in FIG. 2 is an enlarged version of the upper image in FIG. 2. FIG. 3 is a graph showing the cell viability of neurons expressing wild-type and mutant TDP-43 proteins with or without exposure to ethacrynic acid and / or edaravone. FIG. 4 shows an outline of the treatment of experimental groups used in gene product expression analysis. FIG. 5 shows an outline of the procedure for gene product expression analysis. FIG. 6 is a graph showing the distribution of expression in each experimental group. FIGS. 7(a) to (d) are all graphs showing changes in gene product expression depending on the presence or absence of edaravone and / or pathological conditions. Figures 8(a) to (d) are graphs showing the expression fluctuations of another gene product depending on the presence or absence of edaravone and / or pathology. Figures 9(a) to (d) are graphs showing the expression fluctuations of yet another gene product depending on the presence or absence of edaravone and / or pathology. Figures 10(a) and 10(b) are graphs showing the expression fluctuations of yet another gene product depending on the presence or absence of edaravone and / or pathology. Figure 11 is a graph showing the expression fluctuations of yet another gene product depending on the presence or absence of edaravone and / or pathology. Figure 12 is a graph showing the cell viability of neurons expressing wild-type and mutant TDP-43 proteins and neurons not expressing TDP-43 protein, with or without exposure to edaravone. Figure 13 is a graph showing the rate of change in neurite length depending on the presence or absence of exposure to edaravone, in neurons differentiated from iPS cells induced from cells derived from ALS patients and healthy individuals. Figure 14 is a graph showing the rate of change in the number of dead cells with or without exposure to edaravone in neurons differentiated from iPS cells induced from cells derived from ALS patients and healthy individuals. Figure 15 is a fluorescence microscope image showing the intracellular localization of TDP-43 when exposed to edaravone in neurons differentiated from iPS cells induced from cells derived from ALS patients and healthy individuals.

[0012] The present invention will be described below based on its preferred embodiments. The present disclosure encompasses compositions containing edaravone. In one embodiment, the composition may be used as a pharmaceutical or as a non-pharmaceutical, such as a reagent. In either case, the expression level of a gene product of a specific gene in a subject can be varied to obtain advantageous effects depending on the type or expression level of the gene and its gene product. Details of the gene and its gene product will be described below.

[0013] As used herein, "edaravone" encompasses 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one itself, its tautomers, derivatives thereof, and salts, hydrates, or solvates thereof. In one embodiment, edaravone preferably comprises at least one selected from 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, its tautomers, and salts, hydrates, or solvates thereof. That is, edaravone preferably comprises at least one selected from isomers represented by the following structural formulas (1) and (2), and salts, hydrates, or solvates thereof. Edaravone can be produced, for example, by the production method described in European Patent Publication No. 208874.

[0014]

[0015] As used herein, unless otherwise specified, the term "gene product" is used to encompass transcription products, reverse transcription products, and translation products derived from a gene, and may refer to one or more of these products. A gene product may be one or more transcription products, one or more reverse transcription products, one or more translation products, or a combination of one or more transcription products and one or more translation products. Examples of transcription products include various RNAs such as mRNA and non-coding RNA. The transcription product is preferably mRNA, whether mature or not, and more preferably mature mRNA. Examples of reverse transcription products include cDNA. Examples of translation products include proteins produced through transcription and translation. The translation product is preferably a protein, whether or not post-translationally modified.

[0016] In one embodiment, a composition containing edaravone is preferably used as a medicine. That is, in one embodiment, the composition is a pharmaceutical composition containing edaravone as an active ingredient. By using the above-mentioned composition as a medicine, the expression level of a specific gene product in a subject can be changed, and for example, brain function can be maintained or improved at a normal level, or diseases such as neurodegenerative diseases, muscle diseases, vascular disorders, and various inflammatory diseases can be treated or prevented.

[0017] As used herein, the term "subject" is not particularly limited as long as the expression level of a gene product can be measured. "Subject" encompasses humans and non-human animals, as well as samples derived from these animals, regardless of whether they have a disease or not, and regardless of the environment, such as in vivo, ex vivo, or in vitro. Humans and non-human animals are preferably mammals. That is, the subject to which the composition is exposed is preferably a mammal or a sample derived from a mammal. Non-human animals include, for example, rodents such as rats, mice, and guinea pigs; and mammals other than humans, such as monkeys, pigs, dogs, and cats.

[0018] Examples of samples include, but are not limited to, one or more of tissues, cells, and body fluids. Examples of tissues include the brain (e.g., the cerebrum and cerebellum), spinal cord, stomach, pancreas, kidneys, liver, adrenal glands, skin, muscles (e.g., skeletal muscle and smooth muscle), lungs, intestines (e.g., the large intestine and small intestine), heart, and blood vessels. Examples of cells include differentiated cells, progenitor cells, or stem cells that constitute tissues. Examples of brain-derived cells include neural-related differentiated cells such as neurons, glial cells, and astrocytes, as well as neural stem cells. Examples of body fluids include liquid components or extracts thereof, such as cerebrospinal fluid, blood, serum, plasma, saliva, urine, and sweat. These samples can typically be collected from living or dead animals using known methods.

[0019] As other forms of cells, various cultured cells can be used, such as primary cultured cells, immortalized cell lines, or diverse stem cells such as ES cells and iPS cells. In particular, iPS cells may be derived from cells derived from ALS patients or cells that have an ALS risk mutation in the TARDBP gene as a heterozygous mutation. Cells differentiated from the diverse stem cells, such as neurons, are also encompassed by the present disclosure.

[0020] In one embodiment, the composition described above is suitable for use in treating or preventing one or more of neurodegenerative diseases, muscular diseases, vascular disorders, and inflammatory diseases.

[0021] Neurodegenerative diseases include, for example, neurodegenerative diseases accompanied by motor dysfunction and neurodegenerative diseases accompanied by cognitive dysfunction. Neurodegenerative diseases accompanied by motor dysfunction include, for example, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), progressive bulbar palsy, primary lateral sclerosis (PLS), and arthrogryposis multiplex congenita (AMC). Neurodegenerative diseases accompanied by cognitive dysfunction include, for example, Alzheimer's disease and frontotemporal lobar degeneration (FTD). Muscle diseases include, for example, muscular dystrophy. Vascular disorders include, for example, cerebral infarction and cerebral hemorrhage. Inflammatory diseases include, for example, systemic inflammatory diseases such as multiple sclerosis and systemic sclerosis, and local inflammatory diseases such as stomatitis.

[0022] Among these diseases, the above-mentioned composition is preferably used as a pharmaceutical composition to treat or prevent neurodegenerative diseases, more preferably to treat or prevent neurodegenerative diseases accompanied by motor dysfunction, and even more preferably to treat or prevent amyotrophic lateral sclerosis (ALS). As used herein, "treatment" includes suppression, improvement, alleviation, and complete cure of disease progression. As used herein, "prevention" includes prevention of disease onset and prevention of disease recurrence.

[0023] In one embodiment, the composition described above is preferably used for the treatment or prevention of diseases associated with the presence of mutant TDP-43 proteins or abnormal intracellular localization of TDP-43. Examples of such diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS). Examples of mutant TDP-43 proteins include, but are not limited to, C-terminal fragments of wild-type TDP-43 proteins (e.g., amino acid residues 208 to 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, Q343R). Examples of wild-type TDP-43 proteins include the amino acid sequence set forth in Accession number NP_031401.1.

[0024] Regardless of the disease, the use of the above-described composition allows the expression level of a specific gene product to be altered by exposure to edaravone. Cellular dysfunction, cell death, and other cellular damage can be effectively suppressed by the expression level of the gene product or its alteration. As a result, it may be possible to treat or prevent the disease or alleviate the symptoms of the disease. As shown in the examples below, the above-described composition is advantageous in that it can treat or prevent diseases in which mutant TDP-43 proteins may be involved, such as ALS. It is presumed that the presence of mutant TDP-43 proteins causes the formation of TDP-43 protein aggregates within cells, further increasing the likelihood of cellular damage. Therefore, cellular damage may be effectively suppressed by using the above-described composition.

[0025] The edaravone-containing composition of the present disclosure is used to vary the expression level of a specific gene product. As a result of extensive research into the mechanism of action of edaravone, the present inventors have found that the expression level of a gene product of a specific gene varies depending on the presence or absence of exposure to edaravone, as shown in the Examples below. They have also found that the expression levels of these gene products can also vary in specific diseases. Furthermore, they have found that expression variations in the presence of edaravone can inhibit cell damage and, ultimately, aid in the prediction, treatment, or prevention of diseases caused by cell damage. In other words, the present inventors' research has revealed unknown attributes and new uses for the above-mentioned composition, leading to the completion of the present invention.

[0026] As used herein, "fluctuation" means an increase or decrease in the amount of a substance compared to a comparable measured value or reference value, and in addition to the magnitude of the amount or ratio, also encompasses the appearance of the amount from an undetectable state to a detectable state, and the disappearance of the amount from a detectable state to an undetectable state.

[0027] The expression level is determined to have changed if the measured expression level of the gene product in the subject is greater or smaller than the measured expression level of the gene product in the comparison subject or the reference value. The change in expression level may be determined based on the magnitude of the measured value itself, or the magnitude of the ratio calculated from the measured values. The change in expression level may also be determined based on the magnitude of the arithmetic mean or median calculated from multiple measurements or ratios, or may be determined to have changed based on the presence of a statistically significant difference. The change in expression level is evaluated by comparing the expression levels of measurable molecules derived from the same gene between the two experimental groups, provided that the two experimental groups are of the same animal species or are derived from the same animal species. 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 the RNA, and proteins, which are translation products.

[0028] When evaluating fluctuations in expression level using the ratio of measured values, for example, the ratio (R2 / R1) of the measured expression level R2 of the gene product in the experimental group to be evaluated to the reference measured value or reference value R1 may be determined to have fluctuated in expression level, for example, 1.01 times or more, for example, 1.10 times or more, for example, 1.30 times or more, for example, 1.50 times or more, for example, 1.70 times or more, or for example, 2.0 times or more (log2 ratio of 1 or more). In this case, the experimental group to be evaluated may also be determined to have fluctuated in such a way that the expression level increases. Alternatively, the expression level may be determined to have fluctuated in such a way that the R2 / R1 ratio is, for example, 0.99 times or less, for example, 0.90 times or less, for example, 0.80 times or less, for example, 0.70 times or less, for example, 0.60 times or less, or for example, 0.50 times or less (log2 ratio of -1 or less). In this case, the experimental group to be evaluated may also be determined to have fluctuated in such a way that the expression level decreases.

[0029] The composition of the present disclosure is preferably used to vary the expression levels of transcription products of one or more genes selected from a group of specific genes discovered through extensive research by the present inventors, or translation products encoded by the transcription products. The transcription products and translation products whose expression levels are varied may be one or more of these.

[0030] Examples of genes whose expression levels change due to edaravone and / or disease include, but are not limited to, the following. For ease of explanation, the following genes will be referred to as human genes unless otherwise specified, but the present disclosure also encompasses orthologs of animal species other than humans. The nucleotide sequences and protein amino acid sequences of these genes can be searched using public databases, etc.

[0031] In the present specification, the group of genes whose expression levels vary due to the presence of edaravone and / or a disease (hereinafter referred to as (A) and (B)) is also collectively referred to as "specific genes." The explanation regarding specific genes applies to all embodiments described in the present specification unless otherwise specified.

[0032] <Specific Genes> Examples of genes whose expression levels are increased by the presence of a disease and the absence of edaravone and decreased by the presence of a disease and edaravone are shown below as (A). In one embodiment, the composition described above is used to reduce the expression levels of any one, any combination of two or more, or all combinations of gene products from the group of genes (A) in a subject with a disease. In one embodiment, the disease is preferably a neurodegenerative disease.

[0033] [Gene group (A)] ・KAZALD1 (Kazal Type Serine Peptidase Inhibitor Domain 1) ・SBK1 (SH3-binding domain kinase 1) ・SCN2A (sodium voltage-gated channel alpha subunit 2) ・UBE2L6 (ubiquitin conjugating enzyme E2 L6) ・ALPL (alkaline phosphatase) ・NTM (neurotrimin)・PTTG1 (regulator of sister chromatid separation; Securin) ・ITGB4 (integrin subunit beta 4) ・HAUS4 (HAUS augmin like complex subunit 4) ・DCTD (dCMP deaminase) ・MT2A (metallothionein 2A) ・ASF1B (anti-silencing function 1B histone chaperone) ・FCSK (fucose kinase) ・MAST1 (microtubule associated serine / threonine kinase 1)

[0034] Furthermore, examples of genes whose expression levels are decreased by the presence of a disease and the absence of edaravone and increased by the presence of a disease and edaravone are shown below as (B). In one embodiment, the composition described above is used for increasing the expression levels of gene products of gene group (B) in a subject having a disease. In one embodiment, the disease described above is preferably a neurodegenerative disease.

[0035] [Gene group (B)] ・FAIM2 (Fas apoptotic inhibitory molecule 2)

[0036] KAZALD1 is a gene encoding a protein belonging to the insulin growth factor (IGF)-binding protein superfamily. Fluctuations in KAZALD1 expression can contribute to cell proliferation and fluctuations in IGF function or expression levels. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Fluctuations in IGF expression may be associated with risk fluctuations for neurodegenerative diseases such as ALS through some mechanism. Therefore, reducing the expression level of KAZALD1 can reduce the risk of developing or progressing neurodegenerative diseases through regulation of IGF activity, which is thought to be useful in improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0037] SBK1 is a gene encoding a serine / threonine protein kinase. Changes in SBK1 expression may contribute to cancer progression and cell proliferation. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Reducing SBK1 expression may be useful in suppressing the formation of TDP-43 protein aggregates due to phosphorylation, thereby suppressing cell injury and improving the potential for treatment and prognosis of neurodegenerative diseases such as ALS.

[0038] SCN2A is a gene encoding a protein belonging to the neuronal voltage-gated sodium channel family in excitatory neurons. Altered SCN2A expression may contribute to the development of epilepsy and developmental disorders. Furthermore, excessive activation of SCN2A function during neuronal cell injury may further exacerbate cell injury. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Reducing SCN2A expression can suppress the onset of cell injury. Furthermore, reducing SCN2A expression is thought to be useful in improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS. In particular, reducing SCN2A expression is thought to be advantageous in that it can suppress SCN2A function during neuronal cell injury, thereby effectively inhibiting the progression of cell injury.

[0039] UBE2L6 is a gene encoding a protein belonging to the ubiquitin-conjugating enzyme family. It is thought that altered expression of UBE2L6 contributes to protein degradation and inhibits autophagy in pathological conditions such as cancer. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Inhibition of autophagy slows the degradation of abnormal proteins, such as TDP-43 protein aggregates, potentially contributing to the progression of the disease. Therefore, reducing the expression level of UBE2L6 can promote autophagy and accelerate the degradation of abnormal proteins. This may be useful in suppressing cell damage and improving the potential for treatment and prognosis prediction for neurodegenerative diseases such as ALS.

[0040] ALPL is a gene encoding a protein belonging to the alkaline phosphatase family. ALPL is highly expressed in axons and dendrites of neurons. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased expression of ALPL may contribute to the onset or progression of neurodegenerative diseases such as ALS through some mechanism within neurons. Therefore, reducing ALPL expression may be useful in suppressing cell injury and improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0041] NTM is a gene encoding a protein belonging to the IgLON family of cell adhesion molecules. Changes in NTM expression may contribute to neurite and axonal outgrowth and cell-cell adhesion. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Autoimmune neurological diseases involving antibodies against the IgLON family can result in the accumulation of phosphorylated tau protein in the thalamus and brainstem tegmentum, resulting in symptoms similar to those of bulbar motor neuron disease. Therefore, reducing the expression level of NTM may be useful in reducing the accumulation of abnormal proteins and the adverse effects associated with their accumulation, thereby improving the possibility of treating and predicting the prognosis of various neurodegenerative diseases, such as bulbar ALS.

[0042] PTTG1 is a gene encoding securin, a protein that contributes to cell mitosis. Changes in PTTG1 expression may contribute to the regulation of cell division and tumorigenesis. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased expression of PTTG1 may contribute to the onset or progression of neurodegenerative diseases such as ALS through some mechanism related to cell division. Therefore, reducing PTTG1 expression may be useful in suppressing cell injury and improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0043] ITGB4 is a gene encoding a protein belonging to the integrin family. Fluctuations in ITGB4 expression can contribute to cell proliferation, adhesion, and migration, as well as tissue formation and repair. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased expression of ITGB4 may contribute to the onset or progression of neurodegenerative diseases such as ALS through some mechanism related to cell proliferation or repair. Therefore, reducing the expression level of ITGB4 is thought to be useful in suppressing the onset of cell injury and improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0044] HAUS4 is a gene encoding a protein that contributes to microtubule formation during mitosis. Fluctuations in HAUS4 expression may contribute to the regulation of cell division and the elongation of axons and dendrites. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased HAUS4 expression has been confirmed in animal models of ALS and may contribute to the onset or progression of neurodegenerative diseases such as ALS. Therefore, reducing HAUS4 expression may be useful in contributing to the formation and maintenance of normal nerve cell function, suppressing cell injury, and improving the potential for treatment and prognosis prediction of neurodegenerative diseases such as ALS.

[0045] DCTD is a gene encoding a protein (dCMP deaminase) that contributes to the deamination of dCMP to dUMP. Changes in DCTD expression may contribute to the regulation of nucleic acid biosynthesis. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased DCTD expression can lead to an imbalance between substrates and products in nucleic acid biosynthesis, increasing the likelihood of DNA mutations during DNA replication. Therefore, reducing DCTD expression may be useful in contributing to the formation and maintenance of normal neuronal function, suppressing cell injury, and improving the potential for treatment and prognosis of neurodegenerative diseases such as ALS.

[0046] MT2A is a gene encoding a protein belonging to the metallothionein family. Changes in MT2A expression may contribute to heavy metal metabolism, including intracellular heavy metal concentration, and apoptosis. Expression levels of this gene product were found to be elevated in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and to be reduced in the presence of edaravone. Increased MT2A expression is thought to be due in part to the occurrence of oxidative stress. Therefore, reducing MT2A expression may be useful in regulating the response to oxidative stress, contributing to the maintenance of normal neuronal function, suppressing cell injury, and improving the potential for treatment and prognosis of neurodegenerative diseases such as ALS.

[0047] ASF1B is a gene encoding a protein belonging to the histone chaperone H3 / H4 family. Fluctuations in ASF1B expression can contribute to cell division, proliferation, and aging. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased expression of ASF1B may contribute to the onset or progression of neurodegenerative diseases such as ALS through some mechanism related to cell division or proliferation. Therefore, reducing the expression level of ASF1B is thought to be useful in suppressing the onset of cell injury and improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0048] FCSK is a gene that belongs to the phosphotransferase family and encodes a protein that accepts alcohol. Fluctuations in FCSK expression may contribute to the metabolism of fructose and mannose. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Increased expression of FCSK may contribute to the onset or progression of neurodegenerative diseases such as ALS through some mechanism related to intracellular glucose metabolism. Therefore, reducing FCSK expression may be useful in suppressing cell injury and improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0049] MAST1 is a gene encoding a serine / threonine protein kinase. Changes in MAST1 expression are primarily related to brain development and may contribute to its expression at postsynapses and synaptic terminals at the neuromuscular junction. It has been found that the expression level of this gene product increases in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and decreases in the presence of edaravone. Reducing MAST1 expression may be useful in suppressing the formation of TDP-43 protein aggregates due to phosphorylation, thereby suppressing cell injury and improving the potential for treatment and prognosis of neurodegenerative diseases such as ALS.

[0050] FAIM2 encodes an anti-apoptotic protein that protects cells from Fas-induced apoptosis. This gene is also known as the rat orthologue protein lifeguard 2-like (LOC102551901). Changes in FAIM2 expression may contribute to cerebellar size, internal granular layer thickness, and Purkinje cell development. The expression level of this gene product was found to be decreased in the presence of mutant TDP-43 proteins, which are a pathological condition of neurodegenerative diseases, and increased in the presence of edaravone. Since apoptosis can be induced upon neuronal injury, FAIM2 expression is thought to be decreased. Therefore, increasing FAIM2 expression may be useful in maintaining neuronal survival and improving the potential for treatment and prognosis prediction of neurodegenerative diseases such as ALS. FAIM2 is also involved in the regulation of autophagy. Therefore, increasing the expression level of FAIM2 may suppress the formation of mutant SOD1 (Superoxide dismutase 1) protein aggregates, which are a pathological condition of neurodegenerative diseases such as ALS, and may promote the degradation of abnormal protein aggregates such as TDP-43 and SOD1. As a result, it is thought to be useful in improving the possibility of treating and predicting the prognosis of neurodegenerative diseases such as ALS.

[0051] The exposure method for the above-mentioned composition is not particularly limited. Examples of exposure methods include administration to a living body and contact with a sample. In vivo administration to humans or non-human animals includes oral administration and parenteral administration. In this case, the edaravone-containing composition is preferably a pharmaceutical composition containing edaravone. Examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous, or intradermal injection, injection into the digestive tract, or inhalation. These administrations may be single or multiple rapid administrations, or continuous administration such as intravenous drip infusion. Contact with a sample can be achieved, for example, using a liquid in which the above-mentioned composition is dissolved or dispersed.

[0052] The above-mentioned composition can be in a solid or liquid state at 1 atmosphere and 20°C depending on the mode of use. When the composition is in a solid form, examples of the dosage form include tablets, capsules, powders, fine granules, granules, suppositories, etc. When the composition is in a liquid form, examples of the dosage form include solutions, syrups, suspensions, injections, drip infusions, etc. The liquid may be a solution containing a solvent or a dispersion containing a dispersion medium.

[0053] The above-described composition may further contain additives, if necessary. These additives are preferably pharmaceutically acceptable. In one embodiment, when additives are contained in the composition, the additives may constitute the remainder of the composition, either alone or in combination.

[0054] Any additives commonly used in the art can be used without particular limitation. Examples of additives that can be used include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, pigments, diluents, bases, solubilizers or solubilizers, isotonicity agents, pH adjusters, stabilizers, propellants, and adhesives. These can be used alone or in combination of two or more. Examples of bases or diluents that can be used include water, electrolyte-containing water such as saline, monohydric alcohols having 1 to 3 carbon atoms such as methanol, ethanol, and propanol, and aqueous liquids such as cell culture media. These aqueous liquids can be used alone or in combination of two or more as solvents or dispersion media.

[0055] The content of edaravone in the composition can be varied as appropriate depending on the subject of application, but when administered to a living mammal, for example, it can be set to an amount that can be administered preferably in the range of 0.01 μg / kg body weight to 10 mg / kg body weight per day, regardless of the administration form. In one embodiment, when administered to an adult human, the content of edaravone in the composition can be set to an amount that can be administered preferably in the range of 10 mg to 150 mg, more preferably in the range of 20 mg to 120 mg, and even more preferably in the range of 90 mg to 120 mg per day, regardless of the administration form. The above content can also be set, for example, as a therapeutically effective amount.

[0056] When the composition is exposed to a sample such as a tissue or cell collected from an animal or an in vitro sample, the content of edaravone in the composition, in a liquid form for example, is preferably 0.1 μmol / L to 1000 μmol / L, more preferably 0.5 μmol / L to 500 μmol / L, even more preferably 0.75 μmol / L to 300 μmol / L, still more preferably 10 μmol / L to 300 μmol / L, and still more preferably 50 μmol / L to 250 μmol / L. In any embodiment, the above-mentioned content of edaravone can effectively suppress the occurrence of adverse effects such as cell damage in the subject and the progression of the disease.

[0057] The above has been a description of an embodiment of the edaravone-containing composition, and another embodiment of the present invention will be described below. In the following description, differences from the above-described embodiment will be mainly described, and for details not specifically described, the matters described in this specification will be applied as appropriate as long as the effects of the present invention are achieved.

[0058] In one embodiment, the expression level or fluctuations of the gene product of the above-mentioned specific gene can be used as an index to evaluate whether a subject is likely to have a disease. That is, this embodiment relates to a method for evaluating the likelihood of disease. By employing this evaluation method, early detection of disease and the need for appropriate treatment can be facilitated, which can contribute to improving the quality of life of patients. In one embodiment, the disease that can be evaluated by this method is preferably a neurodegenerative disease, more preferably ALS, and even more preferably ALS accompanied by the presence of a mutant TDP-43 protein or abnormal localization of intracellular TDP-43.

[0059] In the present specification, when describing an embodiment of a method, unless otherwise specified, the method may involve only one step, or multiple steps may be performed simultaneously or in any order. Furthermore, in an embodiment that may include multiple steps, unless otherwise specified, one or more additional steps may be interposed between two steps, two steps may be performed consecutively without any other steps intervening, or two or more steps may be performed simultaneously. Furthermore, as long as the effects of the present invention are achieved, the steps according to each embodiment described herein may be combined as appropriate.

[0060] In detail, this method comprises a step of assessing whether a subject is likely to have a disease based on the expression level or variation of a gene product of one or more specific genes in the subject. The subject to which this method is applied is preferably a mammal or a sample derived from the mammal. More specifically, the subject to which this method is applied is preferably the brain of a mammal or a sample derived from the brain. Furthermore, the subject to which this method is applied can preferably use a sample collected from a living or dead mammal, and more preferably the mammal is a human or a non-human animal. In one embodiment, this method can also be performed as a method to assist in the detection of a disease in a subject, and the disease is preferably a neurodegenerative disease. In one embodiment, this method can also be performed under in vitro conditions.

[0061] In one embodiment, the method includes a step A1 of measuring the expression level of a gene product of a specific gene in a subject. Specifically, in step A1, the expression level of a transcription product or a translation product encoded by one or more specific genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2 is measured. In this method, one or more gene products of the specific genes can be used as an indicator or biomarker for assessing the susceptibility to a disease such as a neurodegenerative disease. Measurement of the expression level may be performed in vitro or in vivo.

[0062] When quantifying the expression level of a gene product by targeting a transcription product, for example, RNA is extracted from the sample to be measured by a standard method, and if necessary, cDNA, which is a reverse transcription product, is obtained, and then measurement can be performed by methods well known in the art, such as Northern blot, RT-PCR, nucleic acid array, RNA-seq, etc. Furthermore, if necessary, the measured value may be corrected based on the expression level of housekeeping genes such as GAPDH and β-actin.

[0063] When quantifying the expression level of a gene product by targeting the translation product (i.e., protein), the measurement can be performed using, for example, an immunological method well known in the art, such as ELISA, flow cytometry, Western blotting, or immunohistochemical staining, using the sample to be measured or a solution thereof. If necessary, the measured value may be corrected based on the protein expression level of the housekeeping gene described above. Other methods for quantifying the translation product include mass spectrometry, high-performance liquid chromatography, gas chromatography, NMR analysis, or a combination thereof. Further methods for measuring the translation product include, for example, measuring the activity of the translation product and considering the activity measurement as the expression level, or administering a ligand that specifically binds to the translation product to the body.

[0064] In one embodiment, the method includes, in addition to step A1, step B1 of assessing whether the subject is likely to have a disease based on the measured expression level of the gene product or its fluctuation. To assess the possibility of disease in step B1, for example, a method including at least one of the following steps B11, B12, and B13 can be employed. In one embodiment, the method can include, for example, performing at least one of steps B12 and B13 after step A1. In one embodiment, the method can include, for example, performing step B11 before or after performing step A1, or simultaneously with step A1, and then performing steps B12 and B13 in this order.

[0065] Step B11 is a step of measuring the expression level of a gene product in a control group, i.e., a normal subject, i.e., a healthy animal not affected by a disease, or a sample derived from such an animal. The expression level of the gene product in the control group can also be used as a reference value. Step B12 is a step of comparing the expression level of the gene product in the normal subject (control group) with the expression level of the gene product in the subject to be evaluated. Step B13 is a step of comparing the normal subject (control group) with the subject to be evaluated, and evaluating that the subject to be evaluated is likely to have a disease if a change in the expression level of one or more specific genes is confirmed. Evaluation of the change in expression level is preferably performed based on the presence or absence of a statistically significant difference, in terms of improving evaluation accuracy. Comparison of expression levels between experimental groups can be performed by comparing transcription products derived from the same gene or translation products derived from the same gene under identical or deemed-identical experimental conditions.

[0066] Specifically, as shown in the examples below, the expression levels of one or more of the above-mentioned specific genes belonging to gene group (A) may be elevated compared to normal subjects due to the presence of a disease (e.g., a neurodegenerative disease). Therefore, if the expression levels of one or more genes belonging to gene group (A) are elevated compared to those in normal subjects, it can be assessed that the subject to be evaluated or the animal from which the subject was obtained is highly likely to have a disease (e.g., a neurodegenerative disease). On the other hand, if the expression levels of one or more of the above-mentioned specific genes belonging to gene group (A) are reduced or equivalent to those in normal subjects, it can be assessed that the subject to be evaluated or the animal from which the subject was obtained is less likely to have a disease (e.g., a neurodegenerative disease). From the perspective of improving the accuracy of the evaluation of the likelihood of disease and making it easier to exclude the possibility of diseases other than the disease being evaluated, it is preferable to target two or more of the specific genes and compare the expression levels of each gene product for evaluation. It is more preferable to target all of the above-mentioned specific genes and compare the expression levels of each gene product for evaluation.

[0067] Furthermore, among the above-mentioned specific genes, the expression levels of genes belonging to gene group (B) may be reduced compared to normal subjects due to disease. Therefore, for genes belonging to gene group (B), if the expression level of the gene product is reduced compared to the expression level in normal subjects, it can be evaluated that the subject to be evaluated or the animal from which the subject was obtained is likely to have a disease (e.g., neurodegenerative disease). On the other hand, for gene group (B), if the expression level of the gene product is increased or equivalent to the expression level in normal subjects, it can be evaluated that the subject to be evaluated or the animal from which the subject was obtained is unlikely to have a disease (e.g., neurodegenerative disease). From the perspective of further improving the accuracy of the evaluation, it is preferable to target all genes belonging to gene groups (A) and (B) and compare the expression levels of each gene product for evaluation.

[0068] Alternatively, or in addition, the possibility of the presence or absence of a disease can be evaluated based on a cutoff value for the expression level of a gene product, which is set as a reference value or threshold. For example, if the expression level of one or more of the specific genes described above is higher than the cutoff value (gene group (A)) or lower than the cutoff value (gene group (B)), the subject of evaluation or the animal from which the subject was obtained can be evaluated as having a high probability of having a disease (e.g., a neurodegenerative disease). The "cutoff value" can be, for example, an expression level that satisfies at least one of sensitivity and specificity at a predetermined level when the presence or severity of a disease is evaluated using that value as a reference. The cutoff value can also be the expression level of a gene product in a healthy animal or healthy individual. The cutoff value can be set appropriately depending on the type of disease and the level of strictness of the evaluation. Furthermore, in order to evaluate the severity of a disease, multiple cutoff values ​​can be set to stratify the possibility of disease.

[0069] Another embodiment of the present invention will be described below. In the following description, differences from the above-described embodiment will be mainly described. For details that are not specifically described, the matters described in this specification will be applied as appropriate as long as the effects of the present invention are achieved.

[0070] In one embodiment, the expression level or variation thereof of the gene product of the above-mentioned specific gene can be used as an index to evaluate whether a subject is likely to be responsive to edaravone. That is, this embodiment relates to a method for evaluating the possibility of response to edaravone. This method allows for earlier and easier evaluation and prediction of the effect of edaravone on a disease.

[0071] The subject to which this method is applied is preferably a subject suffering from or suspected of suffering from a neurodegenerative disease. The subject to which this method is applied is more preferably a patient with a neurodegenerative disease or a mammal that serves as a disease model for such a disease, or a sample derived from such a mammal. In this case, the mammal is more preferably a human or an animal other than a human. More specifically, the subject to which this evaluation method is applied is preferably a sample collected from a patient, or the brain of a mammal or a sample derived from the brain. Furthermore, the disease to which this method is applied is preferably a neurodegenerative disease, more preferably ALS, and even more preferably ALS accompanied by the presence of a mutant TDP-43 protein or abnormal localization of intracellular TDP-43.

[0072] In particular, this method preferably uses subjects with or suspected of having a disease and who have been exposed to edaravone. This method includes a step of evaluating whether or not a subject is likely to be responsive to edaravone based on variations in the expression levels of gene products of one or more specific genes in the subject. In one embodiment, this method can also be used as a method to assist in predicting whether or not a subject (e.g., a subject with or suspected of having a neurodegenerative disease) will effectively respond to treatment with edaravone. In one embodiment, this method can also be performed under in vitro conditions. Hereinafter, in this embodiment, the experimental group to which subjects with or suspected of having a disease and who have been exposed to edaravone belong is also referred to as the "exposed-disease group."

[0073] In one embodiment, the method includes a step A2 of measuring the expression levels of gene products of specific genes in subjects with or suspected of having a disease and who have been exposed to edaravone (exposed-disease group). That is, in this step, the expression levels of one or more specific genes selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2 are measured in subjects with or suspected of having a disease and who have been exposed to edaravone. The expression levels of the transcription products or translation products can be measured using the same measurement methods as in the above-mentioned embodiments.

[0074] Whether or not a subject used in this method has or is suspected of having a disease can be evaluated, for example, by employing the method according to the above-described embodiment. Additionally, or instead of this, when evaluating a neurodegenerative disease as one aspect of the disease, the presence of a TDP-43 mutant protein or abnormal localization of intracellular TDP-43 in the subject can also be used to evaluate whether or not the subject has or is suspected of having a neurodegenerative disease. The presence of a TDP-43 mutant protein or abnormal localization of intracellular TDP-43 can be determined, for example, by subjecting a sample such as a tissue or cell to the above-described immunological method. Alternatively, for example, an animal or a sample thereof that has been evaluated as having or is suspected of having a neurodegenerative disease by performing electromyography or the like on a living animal, including a human, can be used.

[0075] The subject may be exposed to edaravone by in vivo administration, for example, or by contacting a sample collected from an animal not exposed to edaravone under in vitro conditions. When edaravone is administered in vivo, for example, a sample collected from a living body such as a human patient or an animal after administration may be used to measure the expression level. In this case, the sample is treated as a subject exposed to edaravone. In either case, edaravone itself may be used for exposure to edaravone, or the composition according to the above-mentioned embodiment may be used.

[0076] The exposure amount of edaravone to a subject can be appropriately changed depending on 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 to 10 mg / kg body weight per day. In one embodiment, when administered to an adult human, the exposure amount can be set to an amount that can be administered preferably in the range of 10 mg to 150 mg per day, more preferably in the range of 20 mg to 120 mg, and even more preferably in the range of 90 mg to 120 mg per day, regardless of the administration form. For in vivo administration, for example, the above-mentioned oral administration and / or parenteral administration methods can be used.

[0077] When edaravone is exposed to samples such as tissues or cells collected from animals or in vitro samples, the exposure concentration is preferably 0.1 μmol / L to 1000 μmol / L, more preferably 0.5 μmol / L to 500 μmol / L, even more preferably 0.75 μmol / L to 300 μmol / L, even more preferably 10 μmol / L to 300 μmol / L, and even more preferably 50 μmol / L to 250 μmol / L. The exposure time of edaravone to the sample can be appropriately changed depending on the purpose. For example, the exposure time can be preferably 1 hour to 120 hours, more preferably 12 hours to 60 hours, provided that the concentration is as described above.

[0078] In this method, to further improve the accuracy of the evaluation, the subject may be exposed to a stress-inducing agent simultaneously with, before, or after exposure to edaravone. Examples of stress-inducing agents include compounds that induce stress in the exposed subject, making it more susceptible to cytotoxicity. Examples of such compounds include one or more of endoplasmic reticulum stress agents, osmotic stress agents, and oxidative stress agents. These are preferably used in an in vitro environment. Examples of endoplasmic reticulum stress agents include thapsigargin, tunicamycin, and dithiothreitol. Examples of osmotic stress agents include sorbitol and sodium chloride. Examples of oxidative stress agents include arsenite salts such as ethacrynic acid and sodium arsenite, and quinone compounds such as tert-butylhydroquinone. When a stress inducer is contained, the content of the stress inducer can be appropriately changed depending on the type and combination of compounds, but can 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. The exposure time of the stress inducer can be set to the same range as the exposure time of edaravone.

[0079] In one embodiment, the method includes, in addition to step A2, step B2 of evaluating whether the subject is likely to be responsive to edaravone based on the measured fluctuations in expression levels of the gene products. To evaluate the possibility of responsiveness to edaravone in step B2, for example, a method including at least one of the following steps B21, B22, and B23 can be employed. In one embodiment, the method can include, for example, at least one of steps B22 and B23 after step A2. In one embodiment, the method can include, for example, step B21 before or after step A2, or simultaneously with step A2, and then steps B22 and B23 can be performed in this order.

[0080] Step B21 is a step of measuring the expression level of a gene product in a non-exposed disease group, using subjects who have or are suspected to have a disease and have not been exposed to edaravone. From the viewpoint of improving the accuracy of the evaluation, it is preferable that the subjects in the non-exposed disease group and the subjects in the exposed disease group are identical except for the presence or absence of exposure to edaravone. Step B22 is a step of comparing the expression level of a gene product in subjects belonging to the non-exposed disease group with the expression level of a gene product in the evaluation subjects (exposed disease group). Step B23 is a step of comparing the non-exposed disease group with the exposed disease group, and evaluating that the subject is likely to be edaravone responsive when a change in the expression level of the gene product of one or more specific genes is confirmed. The evaluation of the change in expression level may be performed by setting an arbitrary threshold value or based on a statistically significant difference. From the viewpoint of improving the accuracy of the evaluation, it is preferable that the evaluation of the change in expression level be performed based on the presence or absence of a statistically significant difference.

[0081] Specifically, the expression levels of one or more of the gene products of one or more of the above-mentioned specific genes belonging to gene group (A) are increased in the presence of a disease (e.g., a neurodegenerative disease) compared to normal subjects, and can be reduced by exposure to edaravone compared to the presence of the disease (e.g., a neurodegenerative disease) alone. Therefore, for one or more genes in gene group (A), if the expression levels of the gene products in the exposed-disease group are lower than those in the non-exposed-disease group, it can be evaluated that a subject with or suspected of having a disease (e.g., a neurodegenerative disease) is highly likely to be responsive to edaravone. On the other hand, if the expression levels of the gene products of gene group (A) in the exposed-disease group are higher than or equivalent to those in the non-exposed-disease group, it can be evaluated that a subject with or suspected of having a disease (e.g., a neurodegenerative disease) is less likely to be responsive to edaravone. From the viewpoint of improving the accuracy of the evaluation of edaravone responsiveness, it is preferable to target multiple specific genes and compare the expression levels of each gene product for evaluation. It is more preferable to target all of the above-mentioned specific genes and compare the expression levels of each gene product for evaluation.

[0082] Furthermore, among the specific genes described above, the expression levels of the gene products of genes belonging to gene group (B) are reduced in the presence of a disease (e.g., a neurodegenerative disease) compared to normal subjects, and can be increased by exposure to edaravone compared to the presence of the disease (e.g., a neurodegenerative disease) alone. Therefore, for genes belonging to gene group (B), if the expression level of the gene product in the exposed disease group is higher than that in the non-exposed disease group, it can be assessed that subjects with or suspected of having a disease (e.g., a neurodegenerative disease) are highly likely to be responsive to edaravone. On the other hand, for gene group (B), if the expression level of the gene product in the exposed disease group is lower than or equivalent to that in the non-exposed disease group, it can be assessed that subjects with or suspected of having a disease (e.g., a neurodegenerative disease) are less likely to be responsive to edaravone. From the viewpoint of further improving the accuracy of the assessment, it is preferable to target all genes belonging to gene groups (A) and (B) and compare the expression levels of each gene product for assessment.

[0083] From the viewpoint of more accurately evaluating the possibility of responsiveness to edaravone, it is also preferable to further use normal subjects, i.e., healthy animals not affected by disease, or samples derived from such animals, as a control group and compare the expression levels of the gene products in the subjects to be evaluated. That is, in this embodiment, it is preferable to compare using two or three experimental groups from among an exposed disease group, a non-exposed disease group, and a normal group. The expression levels of the gene products of the above-mentioned specific genes are increased or decreased in the presence of disease compared to normal subjects, and may further be increased or decreased by exposure to edaravone. Based on such useful expression profiles, this embodiment can evaluate the possibility of a therapeutic effect of edaravone administration or assist in effective prediction of therapeutic responsiveness.

[0084] In detail, step B2 in this embodiment preferably includes at least one of the following steps B24, B25, and B26. In one embodiment, the method may, for example, perform at least one of steps B25 and B26 after step A2. In one embodiment, the method may, for example, perform step B24 before or after performing step A2, or simultaneously with step A2, and then perform steps B25 and B26 in this order.

[0085] Step B24 is a step of measuring the expression level of gene products in normal subjects (control group). The normal subjects are preferably not exposed to edaravone. Step B25 is a step of comparing the expression level of gene products in subjects with or suspected of having a disease and exposed to edaravone (exposed-disease group) with the expression level of gene products in normal subjects (control group). Step B26 is a step of evaluating that, for one or more specific genes, if no change in the expression level of gene products in subjects in the exposed-disease group is confirmed compared with the expression level of gene products in normal subjects, the subject with or suspected of having a disease is likely to be responsive to edaravone and to be highly effective with edaravone. When evaluated in this way, it can also be evaluated that the exposure dose or frequency of edaravone in the evaluation subject is likely to be appropriate.

[0086] On the other hand, when a variation in the expression level of a gene product of a subject in the disease-exposed group is confirmed compared with the expression level of the gene product of a normal subject for one or more specific genes, the subject can be evaluated as possibly not having edaravone responsiveness, or as possibly having low edaravone responsiveness and low edaravone efficacy, or as having high edaravone responsiveness and very high edaravone efficacy. When evaluated in this way, it can also be evaluated that the exposure amount or frequency of edaravone in the subject may be excessive or insufficient, depending on the increase or decrease in the expression level or the degree thereof.

[0087] Taking gene group (A) as an example, if the expression level of the gene product in the disease-exposed group is lower than that in the disease-unexposed group and higher than that in normal subjects (control group), it can be evaluated that the subject may have a low edaravone responsiveness or may have insufficient edaravone exposure dose or exposure frequency. Based on such evaluation, for example, a step of determining whether or not appropriate treatment of the subject is necessary may be further performed to monitor the efficacy of edaravone treatment or assist in optimizing the treatment. Examples of such necessity include an increase or decrease in the exposure dose or exposure frequency of edaravone, or the implementation of treatment other than edaravone exposure. In either case, the evaluation of the change in expression level may be performed by setting an arbitrary threshold value or based on statistical significance. It is preferable to evaluate the change in expression level based on the presence or absence of statistical significance in terms of improving the evaluation accuracy.

[0088] In another embodiment of the method for evaluating edaravone response possibility, for example, step B2 in this embodiment preferably includes steps B21 and B24, as well as the following steps B27 and B28. In one embodiment, the method may, for example, perform step A1, step B21, and step B24 simultaneously or in any order, and then perform steps B27 and B28 in this order.

[0089] Step B27 is a step of comparing the expression level of a gene product of a specific gene in a normal subject (control group) with the expression level of the gene product of a specific gene in a non-exposed disease group. Step B28 is a step of evaluating, when it is determined that the expression level varies between the two groups, that the evaluation subject in the non-exposed disease group or the patient or animal from which the subject was obtained is likely to respond to treatment with edaravone. That is, this embodiment is an example of a method for distinguishing the possibility of response to edaravone using the expression level of a gene product of a specific gene that is increased or decreased due to disease compared to a healthy subject as a biomarker. In one embodiment, this method can also be used as a method to assist in predicting whether a subject with or suspected of having a disease will effectively respond to treatment with edaravone.

[0090] When the control group and the non-exposed disease group are compared, if the expression level of one or more genes in the gene group (A) in the non-exposed disease group is higher than that in the normal group, it can be evaluated that the subject has a disease (e.g., a neurodegenerative disease) or is suspected of having a disease (e.g., a neurodegenerative disease) is highly likely to be responsive to edaravone. On the other hand, when the control group and the non-exposed disease group are compared, if the expression level of the gene products in the gene group (A) in the non-exposed disease group is lower than or equivalent to that in the normal group, it can be evaluated that the subject has a disease (e.g., a neurodegenerative disease) or is suspected of having a disease is low in possibility of being responsive to edaravone.

[0091] Similarly, when a comparison is made between a control group and a non-exposed disease group, and the expression level of the gene product in the non-exposed disease group is lower than that in the normal group, it can be assessed that the subject has a disease (e.g., a neurodegenerative disease) or is suspected of having a disease, and that the subject is highly likely to be responsive to edaravone. On the other hand, when a comparison is made between a control group and a non-exposed disease group, and the expression level of the gene product in the non-exposed disease group is higher than or equivalent to that in the normal group, it can be assessed that the subject has a disease (e.g., a neurodegenerative disease) or is suspected of having a disease, and that the subject is highly unlikely to be responsive to edaravone.

[0092] Another embodiment of the present invention will be described below. In the following description, differences from the above-described embodiment will be mainly described. For details that are not specifically described, the matters described in this specification will be applied as appropriate as long as the effects of the present invention are achieved.

[0093] In one embodiment, by using the expression level or fluctuations of the gene product of the above-mentioned specific gene as an index, any candidate substance can be selected as a substance that can treat or prevent a neurodegenerative disease. That is, this embodiment relates to a method for screening for substances that can treat or prevent a neurodegenerative disease. This method makes it possible to determine whether an unknown substance is a substance that can treat or prevent a neurodegenerative disease using a method different from conventional methods. As a result, the substance can be screened earlier as a drug candidate, which can contribute to the early development of drugs.

[0094] In detail, the screening method for a substance capable of treating or preventing a neurodegenerative disease includes a step A3 of exposing a subject to a test substance, and a step B3 of selecting the 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 of a specific gene in the subject exposed to the test substance. In this method, edaravone is preferably excluded from the test substances. That is, it is preferable that the subject subjected to substance evaluation in this method is exposed to the test substance but not to edaravone.

[0095] In this method, the subject to be exposed to the test substance is preferably a mammal suffering from or suspected of suffering from a neurodegenerative disease, or a sample derived from a mammal suffering from or suspected of suffering from a neurodegenerative disease, and more preferably the mammal is a human. More specifically, the subject to which this method is applied is preferably the brain of a mammal or a sample derived from the brain. Furthermore, the neurodegenerative disease to be evaluated is preferably ALS, and more preferably ALS accompanied by the presence of a mutant TDP-43 protein or abnormal localization of intracellular TDP-43. The test substance is not particularly limited, and examples include small organic molecules, nucleic acids, proteins, peptides, antibodies, and natural components.

[0096] In step A3, the method for exposing the subject to the test substance is not particularly limited, and can be carried out in vitro or in vivo, for example, using the same method as in the above-mentioned embodiment. When the subject is exposed to the test substance in vitro, for example, the subject can be contacted with a liquid in which the test substance is dissolved or dispersed. Examples of such a liquid include saline, buffer, or culture medium containing the test substance. When the subject is exposed to the test substance in vivo, for example, the test substance itself or a liquid in which the test substance is dissolved or dispersed may be administered to a mammal. The administration method may be oral administration or parenteral administration.

[0097] In step B3, the expression level of a gene product of a specific gene is measured, and the test substance is determined to be a substance capable of treating or preventing a neurodegenerative disease based on the fluctuation of the measured expression level. To determine that the test substance is a substance of interest in step B3, for example, a method including at least one of the following steps B31, B32, and B33 can be employed. In one embodiment, for example, this method can include at least one of steps B32 and B33 after step A3. In one embodiment, this method can include, for example, performing step B31 before or after performing step A3, or simultaneously with step A3, and then performing steps B32 and B33 in this order.

[0098] Step B31 is a step of measuring the expression level of a gene product of a specific gene in a non-exposed group, which is a group of subjects not exposed to the test substance and edaravone and having or suspected of having a neurodegenerative disease. 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 subjects contacted with the test substance (the group exposed to the test substance). Step B33 is a step of comparing the non-exposed group with the exposed group, and when a change in the expression level of the gene product of one or more specific genes is confirmed, determining that the test substance is a substance capable of treating or preventing a neurodegenerative disease and selecting the substance. The change in expression level may be determined by setting an arbitrary threshold value or based on a statistically significant difference. The change in expression level is preferably determined based on the presence or absence of a statistically significant difference in terms of improving the accuracy of the determination.

[0099] The expression levels of one or more of the specific genes belonging to gene group (A) among the above-mentioned specific genes may increase due to the presence of a neurodegenerative disease. A test substance with efficacy similar to that of edaravone may reduce the expression levels of these gene products in a subject. Therefore, if the expression levels of one or more of these specific genes in the exposed group are lower than those in the unexposed group, the test substance can be determined to be a substance capable of treating or preventing a neurodegenerative disease. On the other hand, if the expression levels of one or more of the specific genes belonging to gene group (A) in the exposed group are higher than or equivalent to those in the unexposed group, the test substance can be determined to be either not capable of treating or preventing a neurodegenerative disease or to have a low possibility of doing so.

[0100] The expression levels of gene products of genes belonging to gene group (B) among the above-mentioned specific genes can be reduced by the presence of a neurodegenerative disease. Furthermore, a test substance with efficacy similar to that of edaravone can increase the expression levels of the gene products in a subject. Therefore, if the expression level of the gene product of the gene in the exposed group is higher than that in the non-exposed group, the test substance can be determined to be a substance capable of treating or preventing a neurodegenerative disease. On the other hand, if the expression level of the gene product of a gene belonging to gene group (B) in the exposed group is lower than or equivalent to that in the non-exposed group, the test substance can be determined to be not a substance capable of treating or preventing a neurodegenerative disease, or to have a low possibility of doing so. From the perspective of improving the accuracy and reproducibility of test substance determination and performing screening more effectively, it is preferable to compare the expression levels of each gene product of multiple specific genes and make the determination, and more preferably to compare and determine all of the above-mentioned specific genes.

[0101] From the viewpoint of more accurately assessing the degree of contribution of the test substance to the treatment or prevention of a disease, step B3 preferably includes step B35 of further using subjects not exposed to the test substance but exposed to edaravone as an edaravone-exposed group and comparing the expression level of the gene product with that in the test substance-exposed group. That is, it is preferable to compare the expression level in each subject using three experimental groups: a test substance-exposed group, an edaravone-exposed group, and a non-exposed group. That is, in this embodiment, the non-exposed group can be used as a negative control, and the edaravone-exposed group can be used as a positive control. The subjects used in these three experimental groups are preferably mammals with or suspected of having a neurodegenerative disease, or samples derived from mammals with or suspected of having a neurodegenerative disease. More specifically, it is preferable that the subjects to which this assessment method is applied are mammalian brains or brain-derived samples. In this case, it is preferable to prepare groups with the same concentrations of the test substance and edaravone to be exposed, in order to improve the accuracy and reproducibility of the evaluation.

[0102] In one embodiment of the method, step B34 involves measuring the expression level of a gene product of a specific gene in the edaravone-exposed group. When step B34 is performed, step B34 may be performed before step B35. In one embodiment, the method may perform step A3, step B31, and step B34 simultaneously or in any order, and then perform (a) step B32 and step B33 in this order, and / or (b) step B35. When both step groups (a) and (b) are performed, at least step B32 may be performed before step B33 and step B35, and step B33 and step B35 may be performed in any order or simultaneously.

[0103] Step B35 is a step of comparing the expression levels of gene products in the test substance-exposed group with the expression levels of gene products in subjects not exposed to the test substance but exposed to edaravone. The expression levels of specific genes can increase or decrease due to the presence of a neurodegenerative disease. A test substance with efficacy similar to that of edaravone can increase or decrease the expression levels of gene products of specific genes. Therefore, if no change in the expression levels of gene products of one or more specific genes is observed in subjects exposed to the test substance compared to the expression levels of gene products in subjects exposed to edaravone, the test substance can be selected as a substance capable of treating or preventing neurodegenerative diseases. Furthermore, the test substance can also be determined to be highly likely to be a substance with efficacy equivalent to that of edaravone in treating or preventing neurodegenerative diseases.

[0104] On the other hand, when a change in the expression level of one or more specific genes in the test substance-exposed group is confirmed compared to the expression level of the gene product in the edaravone-exposed group, it can be determined that the test substance is unlikely to be a substance that can treat or prevent neurodegenerative diseases.Furthermore, it can be determined that the test substance does not have the same efficacy as edaravone in treating or preventing neurodegenerative diseases, or is unlikely to have the same efficacy.

[0105] Furthermore, for example, for one or more specific genes belonging to gene group (A), if the expression level of the gene product in the test substance-exposed group is lower than that in the non-exposed group and higher than that in the edaravone-exposed group, it can be evaluated that the efficacy of the test substance is likely to be less than that of edaravone. For specific genes belonging to gene group (B), if the expression level of the gene product in the test substance-exposed group is higher than that in the non-exposed group and lower than that in the edaravone-exposed group, it can be evaluated that the efficacy of the test substance is likely to be less than that of edaravone. Based on such evaluation, measures to improve efficacy can be taken, for example, by changing the test substance used or increasing or decreasing the exposure dose or frequency of exposure to the test substance. In either case, it is preferable to evaluate the change in expression level based on the presence or absence of a statistically significant difference, in terms of improving the accuracy of the evaluation.

[0106] In one embodiment, determining that the test substance is the substance of interest in step B3 can be done, for example, by comparing the expression level results in the test substance exposure group with the expression level results in normal subjects. Specifically, in one embodiment, step B3 preferably includes at least one of the following steps B36, B37, and B38. In one embodiment, the method may, for example, perform at least one of steps B37 and B38 after step A3. In one embodiment, the method may, for example, perform step B36 before or after step A3, or simultaneously with step A3, and then perform steps B37 and B38 in this order. In one embodiment, the method may employ a combination of two or all of the above-described steps B31, B32, and B33, or a combination of the above-described steps B34 and B35.

[0107] Step B36 is a step of measuring the expression level of the gene product of a specific gene in a control group of normal subjects not exposed to the test substance or edaravone. Step B37 is a step of comparing the expression level of the gene product in normal subjects (control group) with the expression level of the gene product in subjects with or suspected of having a neurodegenerative disease who have been contacted with the test substance (test substance exposure group). Step B38 is a step of comparing the normal subjects (control group) with the exposure group, and determining that the test substance is a substance capable of treating or preventing a neurodegenerative disease if no change in the expression level of the gene product is confirmed for one or more specific genes, and selecting the substance. The determination of the change in expression level may be performed by setting an arbitrary threshold value or based on a statistically significant difference. The determination of the change in expression level is preferably performed based on the presence or absence of a statistically significant difference in terms of improving the accuracy of the determination.

[0108] The expression levels of one or more of the specific genes described above may increase or decrease due to the presence of a neurodegenerative disease. Therefore, if a test substance is likely to be a substance capable of treating or preventing a neurodegenerative disease, the expression levels of one or more of these specific genes may increase or decrease in the group exposed to the test substance. Therefore, if no changes in the gene products of one or more specific genes are observed in the group exposed to the test substance compared to the expression levels in normal subjects, the test substance can be determined to be a substance capable of treating or preventing a neurodegenerative disease. On the other hand, if changes in the gene products of one or more specific genes are observed in the group exposed to the test substance compared to the expression levels in normal subjects, the test substance can be determined to be either not a substance capable of treating or preventing a neurodegenerative disease or to have a low likelihood of doing so.

[0109] The gene products of the above-mentioned specific genes can also be used as biomarkers. That is, this embodiment relates to biomarkers. As shown in the Examples below, the above-mentioned specific genes were newly discovered by the present inventors as genes that exhibit variations in expression levels based on the results of edaravone exposure and pathological models. Therefore, by using the gene products of the specific genes as biomarkers, it is possible to easily diagnose the presence or absence of a neurodegenerative disease, or the degree of progression or severity of the disease, or the possibility of such a disease. Furthermore, by using the gene products as biomarkers, it is possible to easily evaluate the possibility of contributing to the responsiveness and efficacy of edaravone against a disease, or the efficacy of a test substance.

[0110] Specifically, in one embodiment, the biomarker comprises one or more gene products from specific genes, including KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2. By including multiple gene products derived from two or more genes as a biomarker, the reliability of evaluation or diagnosis can be improved. In one embodiment, the biomarker may consist solely of gene products derived from the specific genes. The gene products in the biomarker may consist solely of transcription products, translation products, or a combination of transcription and translation products.

[0111] The biomarkers described above can be used to determine whether a subject has a neurodegenerative disease, or the likelihood of such a disease, or to predict the prognosis. In another embodiment, the biomarkers described above are used for diagnosing the prediction of responsiveness to edaravone. In yet another embodiment, the biomarkers described above are used for diagnosing the responsiveness to a test substance. The biomarkers can be measured by using the above-mentioned samples, more specifically, one or more of tissues, cells, and body fluids collected from living organisms such as animals with or without edaravone administration or animals with or without disease, as specimens, and measuring at least one of transcription products and translation products in the specimens by the above-mentioned measurement method. This can be preferably performed in vitro.

[0112] 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. This kit can detect one or more of the gene products as biomarkers, preferably in vitro. Examples of the detection reagent include nucleic acid probes or primers for detecting transcription products of specific genes. Other examples of the detection reagent include antibodies or antibody fragments for detecting translation products of specific genes. The detection reagent may be labeled, for example, with a fluorescent substance or a radionuclide, as needed.

[0113] The present disclosure also provides a method for treating a neurodegenerative disease using the above-described composition. For example, the treatment method may involve orally or parenterally administering the above-described composition to a human or non-human animal. When the subject of administration is a human, the human is preferably a patient with a neurodegenerative disease. In one embodiment, the above-described treatment method may employ, for example, administration conditions using an edaravone injection or oral administration agent as the edaravone-containing composition. For example, administration conditions for an edaravone injection may employ the administration method described in WO2020 / 091036 or an administration method using an edaravone formulation used in clinical practice.

[0114] The present disclosure also provides use of a substance that alters the expression level of a gene product of the above-mentioned specific gene for producing a composition for preventing or treating a neurodegenerative disease. The present disclosure also provides a substance for use in altering the expression level of a gene product of the above-mentioned specific gene. In one embodiment, a substance for use in treating a neurodegenerative disease by altering the expression level of a gene product of the above-mentioned specific gene is also provided. The above-mentioned substance is not particularly limited, and examples include edaravone, an edaravone-containing composition, a small organic molecule compound, a nucleic acid, a protein, a peptide, an antibody, a natural component, or a test substance discovered by the above-mentioned screening method.

[0115] The present disclosure also provides a method for suppressing the onset or progression of cell damage by varying the expression level of the gene product of the specific gene in a cell. Examples of methods for varying the expression level include contacting the cell with a specific substance. The substance used for contact is not particularly limited, and examples include edaravone, an edaravone-containing composition, a low-molecular-weight organic compound, a nucleic acid, a protein, a peptide, an antibody, a natural component, or a test substance discovered by the screening method described above.

[0116] Each of the above-described embodiments disclosed herein may independently employ one or all of the above-described specific genes, or any combination of two or more of them. Note that the following combinations of specific genes are exemplary and are not limited to these. For example, in one embodiment, the above-described specific gene may be one or more of the following specific genes selected from KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2. Also, for example, in one embodiment, the above-described specific gene may be one or more of the following specific genes selected from KAZALD1, SBK1, UBE2L6, NTM, HAUS4, DCTD, ASF1B, FCSK, and FAIM2. For example, in one embodiment, the specific gene may be one or more of the specific genes selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B, and FCSK. For example, in one embodiment, the specific genes may be one or more of the specific genes selected from KAZALD1, SBK1, DCTD, and FCSK. For example, in one embodiment, the specific genes may be one or more of the specific genes selected from SBK1, DCTD, and FCSK. For example, in one embodiment, the specific genes may be one or two of the specific genes selected from DCTD and FCSK.

[0117] In addition to the above-described embodiments, the present specification further discloses the following embodiments.

[0118] <1> A composition for varying the expression level of a gene product in a subject, the composition comprising edaravone, and 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 <1>, which comprises edaravone as an active ingredient and is used as a medicine.

[0119] <3> The composition according to <2>, which is used for treating or preventing a neurodegenerative disease. <4> The composition according to <3>, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). <5> The composition according to any one of <1> to <4>, wherein the subject is preferably a mammal or a sample derived from a mammal, and the mammal is more preferably a human or a rat. <6> The composition according to any one of <1> to <5>, wherein the subject is a mammalian brain or a sample derived from a brain. <7> The composition according to any one of <1> to <6>, which is used to reduce the expression level of a gene product of a first gene in a subject with a neurodegenerative disease and / or to increase the expression level of a gene product of a second gene in a subject with 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. <8> The composition according to any one of <1> to <7>, wherein the subject comprises one or more selected from nerve cells (neurons), glial cells, astrocytes, and neural stem cells. <9> The composition according to any one of <1> to <8>, wherein the subject has a mutant TDP-43 protein.

[0120] <10> A method for assessing the susceptibility of a subject to a neurodegenerative disease, comprising step α of assessing whether the subject is likely to have a neurodegenerative disease based on a variation in the expression level of a gene product in the subject, wherein the gene product is a gene product of one or more genes selected from KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2. <11> The method according to <10>, wherein step α comprises step α1 of comparing the expression level of the gene product in the subject with the expression level of the gene product in a normal subject, and step α2 of assessing that the subject is likely to have a neurodegenerative disease when the expression level of the gene product varies between the subject and the normal subject. <12> The method according to <11>, wherein step α2 is a step of assessing that the subject is likely to have 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 high, or when the expression level of the gene product of FAIM2 is low, compared to the expression level of the gene product in the normal subject. <13> The method according to any of <10> to <12>, wherein the gene is any of the following (i) to (v): (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 two selected from DCTD and FCSK; (v) one or more selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B, and FCSK.

[0121] <14> The method according to any one of <10> to <13>, wherein the subject is a mammalian brain or a sample derived from a brain, and the mammal is preferably a human. <15> The method according to any one of <10> to <14>, wherein the subject comprises one or more cells selected from nerve cells (neurons), glial cells, astrocytes, and neural stem cells. <16> The method according to any one of <10> to <15>, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0122] <17> A method for evaluating a subject's possibility of responding to edaravone, comprising a step β of evaluating whether the subject is likely to be responsive to edaravone based on a change in expression level of a gene product due to exposure of the subject 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, and the subject is preferably a subject suffering from or suspected of having a neurodegenerative disease. <18> The method according to <17>, wherein the step β comprises: a step β1 of comparing an expression level of the gene product in a first subject who has or is suspected of having a neurodegenerative disease and has not been exposed to edaravone with an expression level of the gene product in a second subject who has or is suspected of having a neurodegenerative disease and has been exposed to edaravone; and a step β2 of assessing that the subject who has or is suspected of having a neurodegenerative disease is highly likely to be responsive to edaravone, when the expression level of the gene product varies between the first subject and the second subject.

[0123] <19> The method according to <18>, 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 wherein step β2 is a step of assessing that the subject having or suspected of having a neurodegenerative disease is likely to be responsive to edaravone if the expression level of the gene product in the second subject is lower than the expression level of the gene product in the first subject. <20> The method according to either <18> or <19>, wherein the gene product is a gene product of FAIM2, and wherein step β2 is a step of assessing that the subject having or suspected of having a neurodegenerative disease is likely to be responsive to edaravone if the expression level of the gene product in the second subject is higher than the expression level of the gene product in the first subject.

[0124] <21> The method of any of <17> to <20>, wherein the step β comprises: a step β3 of comparing the expression level of the gene product in a second subject having or suspected of having a neurodegenerative disease and having been exposed to edaravone with the expression level of the gene product in a normal subject; and a step β4 of evaluating, when there is no difference in the expression level of the gene product between the second subject and the normal subject, that the subject having or suspected of having a neurodegenerative disease is likely to be more responsive to edaravone. <22> The method of any of <17> to <21>, wherein the step β comprises: a step β3 of comparing the expression level of the gene product in a second subject having or suspected of having a neurodegenerative disease and having been exposed to edaravone with the expression level of the gene product in a normal subject; and a step β5 of evaluating, when there is a difference in the expression level of the gene product between the second subject and the normal subject, that the subject having or suspected of having a neurodegenerative disease is likely to be less responsive to edaravone. <23> The method according to any one of <17> to <22>, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), and the subject is a human or a sample derived from a human.

[0125] <24> A method for screening for a substance capable of treating or preventing a neurodegenerative disease, comprising a step γ 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 in a subject exposed 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. <25> The method according to <24>, wherein the step γ comprises: a step γ1 of comparing the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and exposed to the test substance, with the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and not exposed to the test substance and edaravone; and a step γ2 of selecting the test substance as a substance capable of treating or preventing a neurodegenerative disease when the expression level of the gene product varies between the subject exposed to the test substance and the subject not exposed to the test substance and edaravone. <26> The method according to <24> or <25>, wherein the step γ comprises: a step γ3 of comparing the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and having been exposed to a test substance, with the expression level of the gene product in a normal subject not exposed to the test substance and edaravone; and a step γ4 of selecting the test substance as a substance capable of treating or preventing a neurodegenerative disease when the expression level of the gene product does not change between the subject exposed to the test substance and a subject not exposed to the test substance but exposed to edaravone.

[0126] <27> The method according to <25> or <26>, 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 the 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 subject exposed to the test substance is lower than the expression level of the gene product in a subject not exposed to the test substance and edaravone. <28> The method according to any one of <25> to <27>, wherein the gene product is a gene product of FAIM2, and the 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 a subject exposed to the test substance is higher than the expression level of the gene product in a subject not exposed to the test substance and edaravone.

[0127] <29> The method according to any one of <24> to <28>, wherein the step γ comprises: a step γ5 of comparing the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and exposed to a test substance, with the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and not exposed to the test substance but exposed to edaravone; and a step γ6 of evaluating that the test substance is highly likely to be a substance capable of treating or preventing a neurodegenerative disease, when the expression level of the gene product does not change between the subject exposed to the test substance and the subject not exposed to the test substance but exposed to edaravone. <30> The method according to any one of <24> to <29>, wherein the step γ comprises: a step γ5 of comparing the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and exposed to a test substance, with the expression level of the gene product in a subject having or suspected of having a neurodegenerative disease and not exposed to the test substance but exposed to edaravone; and a step γ7 of evaluating that the test substance is unlikely to be a substance capable of treating or preventing a neurodegenerative disease, when the expression level of the gene product varies between the subject exposed to the test substance and the subject not exposed to the test substance but exposed to edaravone.

[0128] <31> The method according to any one of <24> to <30>, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). <32> The method according to any one of <24> to <31>, wherein the subject is a human or a sample derived from a human.

[0129] <33> A biomarker for diagnosing a neurodegenerative disease or diagnosing responsiveness to edaravone, 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 one or more genes selected from KAZALD1, SBK1, UBE2L6, NTM, DCTD, ASF1B, and FCSK.

[0130] <34> A detection kit for detecting one or more gene products selected from KAZALD1, SBK1, SCN2A, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2 as biomarkers for diagnosing a neurodegenerative disease or diagnosing responsiveness to edaravone, the detection kit comprising a detection reagent capable of specifically detecting 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. <35> A method for treating a neurodegenerative disease, the method comprising the step of administering the composition according to any one of <1> to <9> to a patient with a neurodegenerative disease. <36> Use of a substance that alters 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 the manufacture of a composition for the prevention or treatment of a neurodegenerative disease. <37> A method for suppressing the onset or progression of cell damage, comprising the step of altering 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 in a cell.

[0131] <38> Edaravone or a pharmaceutical composition containing edaravone for use in treating or preventing neurodegenerative diseases such as ALS by varying 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. <39> The composition, method, biomarker, detection kit, or use according to any one of <1> to <37>, wherein the gene is one, any combination of two or more, or all selected from KAZALD1, SBK1, UBE2L6, ALPL, NTM, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, MAST1, and FAIM2. The gene is preferably one, any combination of two or more, or all selected from KAZALD1, SBK1, UBE2L6, NTM, HAUS4, DCTD, ASF1B, FCSK, and FAIM2. The gene is more preferably one, any combination of two or more, or all selected from KAZALD1, SBK1, DCTD, and FCSK. The gene is even more preferably one, any combination of two, or all selected from SBK1, DCTD, and FCSK. The gene is even more preferably one or two selected from DCTD and FCSK.

[0132] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0133] Example 1: Preparation of disease model cells (1): Cell culture and differentiation The 1464R adult rat neural stem cell line (hereinafter also referred to as the "1464R cell line" or "undifferentiated cell line") was used as a subject. The 1464R cell line was established using the method described in Neuropathology 2014, 34, 83-98. The cell line was cultured in a non-adherent state in maintenance medium using a 10 cm petri dish treated with poly-2-hydroxyethyl methacrylate. The culture conditions were 37°C, 5% CO ２The cells were subcultured twice a week under a 5% CO2 environment. The maintenance medium was prepared by adding the following components to Neurobasal Plus medium (Thermo Fisher Scientific) at final concentrations. 1464R cells, which form typical neurospheres, were isolated. The isolated 1464R cells were dispersed and maintained in culture.

[0134] <Additional components in maintenance medium> 2 mM L-glutamine (manufactured by Thermo Fisher Scientific) 2 v / v% B-27 supplement (manufactured by Thermo Fisher Scientific) 10 ng / mL fibroblast growth factor (FGF)-2 (manufactured by Sigma) 10 ng / mL epidermal growth factor (manufactured by Sigma) 50 units / mL penicillin and 50 μg / mL streptomycin (manufactured by Thermo Fisher Scientific)

[0135] Next, the 1464R cell line was cultured in an adherent state on a poly-L-lysine-treated petri dish using the differentiation medium shown below. The culture conditions for cell differentiation were 37°C, 5% CO ２ , for 4 days. Through this process, the 1464R cell line was differentiated into nerve cells (neurons). Furthermore, a portion of the 1464R cell line differentiated into glial cells. In the following explanation, the differentiated 1464R cell line will be collectively referred to as "differentiated cell line." The differentiation medium was prepared by adding the following components to F-12 medium (manufactured by Thermo Fisher Scientific) at final concentrations:

[0136] <Additional components in differentiation medium> 5 v / v% fetal bovine serum (manufactured by Moregate) 0.5 v / v% N-2 supplement (manufactured by Thermo Fisher Scientific) 1 v / v% B-27 supplement (manufactured by Thermo Fisher Scientific) 1 μmol / L ATRA (manufactured by Sigma) 50 units / mL penicillin and 50 μg / mL streptomycin (manufactured by Thermo Fisher Scientific)

[0137] (2): Forced Expression of TDP-43 Protein Subsequently, an in vitro model of neurodegenerative disease was prepared by forcibly and excessively expressing TDP-43 protein in a differentiated cell line according to the following method. After obtaining the differentiated cell line by the method of (1), the differentiation medium was removed and replaced with an infection medium. The infection medium was prepared by adding the following components to final concentrations in antioxidant- and serum-free F-12 medium (manufactured by Thermo Fisher Scientific). The culture conditions were 37°C, 5% CO ２ The environment was maintained.

[0138] <Additional components in infection medium> 0.5 v / v% N-2 supplement (manufactured by Thermo Fisher Scientific) 1 μmol / L ATRA (manufactured by Sigma) 50 units / mL penicillin and 50 μg / mL streptomycin (manufactured by Thermo Fisher Scientific)

[0139] Separately, the following recombinant adenoviral vectors (a) and (b) were prepared according to the method described in Neuropathology 2014, 34, 83-98: (a) Wild-type: A recombinant adenoviral vector carrying a cDNA encoding full-length human wild-type TDP-43 labeled with DsRed (AxDsRhWTTDP43 strain; RIKEN DNA Bank Japan; #RDB15499); (b) Mutant: A recombinant adenoviral vector carrying a cDNA encoding a C-terminal fragment of TDP-43 (amino acid sequence numbers 208 to 414) labeled with DsRed (AxDsRhWTTDP43 strain; RIKEN DNA Bank Japan; #RDB15500).

[0140] Using both vectors (a) and (b), a differentiated cell line in infection medium was infected with adenovirus at a multiplicity of infection of 50 and cultured for 24 hours. This resulted in 1464R cell line-derived neurons (hereinafter also referred to as disease model cells) in which both wild-type and mutant TDP-43 were forcibly expressed intracellularly. These disease model cells are an in vitro model of neurodegenerative diseases that may be related to the localization of TDP-43 expression or the presence of mutant TDP-43 proteins. Examples of such neurodegenerative diseases include ALS.

[0141] Example 2: Evaluation of cell protection by microscopic observation Using the disease model cells prepared by the method of Example 1, it was evaluated whether edaravone (hereinafter also referred to as Eda) could suppress cell damage.

[0142] A poly-L-lysine treated 96-well plate was used, and 8 × 10 ４ The cells were seeded at a cell density of 100 cells / well and cultured under the conditions described in Example 1(1) to differentiate. Then, TDP-43 was forced to be expressed in the differentiated cells by the method described in Example 1(2), to obtain disease model cells.

[0143] For this disease model cell line, an Eda-free group was prepared by exposing the cells to EA (final concentration 20 μmol / L) for 24 hours (see Figure 1). Separately, an Eda-containing group was prepared by exposing the cells to Eda (final concentration 200 μmol / L) for 24 hours, followed by EA (final concentration 20 μmol / L) for 24 hours (see Figure 2). Cell morphology in each group was observed under a fluorescent microscope. Tubulin βIII (represented by TuJ1 in the figure), an indicator of neuronal differentiation, was stained by fixing the cells with 4% paraformaldehyde after 24 hours of EA exposure, treating them with TuJ1 antibody overnight at 4°C, and then treating them with a fluorescent antibody at room temperature for 15 minutes. Cell nuclei were fluorescently stained using Hoechst 33342 staining in the usual manner. The results are shown in Figures 1 and 2.

[0144] As shown in Figure 1, in the Eda-free group, the neuron-specific process morphology observed by the localization of tubulin βIII disappeared, confirming the occurrence of cell damage. On the other hand, as shown in Figure 2, in the Eda-containing group, the neuron-specific process morphology observed by the localization of tubulin βIII was fully maintained, confirming the cytoprotective effect. The localization of TDP-43 in the cell nucleus, as confirmed by DsRed, was also confirmed to be present within the cells.

[0145] Examples 3-1 to 3-6, Comparative Example 1, and Reference Example 1: Evaluation of cell protection using a chromogenic substrate as an index Disease model cells prepared by the method of Example 1 were exposed to Eda (final concentration 1 to 200 μmol / L) for 24 hours and then to EA (final concentration 20 μmol / L) for 24 hours to prepare an Eda-containing group (Examples 3-1 to 3-6), an Eda-free group (Comparative Example 1) that was exposed to EA (final concentration 20 μmol / L) for 24 hours without exposure to Eda, and an Eda- and EA-unexposed group (Reference Example 1).

[0146] Each group of cells was then exposed to a cytotoxicity assay kit (Cell Counting Kit-8, DOJINDO) according to the manufacturer's protocol, and the amount of chromogenic substrate produced was measured by measuring the absorbance at 450 nm. The kit measured the amount of chromogenic substrate produced as the number of viable cells increased, resulting in a higher absorbance at 450 nm. Therefore, a higher absorbance at 450 nm indicates a higher number of viable cells. The results are shown in Figure 3.

[0147] All data shown in Figure 3 are expressed as mean ± standard error of the mean (SEM, n = 4 for each). In Figure 3, the symbol "##" indicates a statistically significant difference of p < 0.01 compared to Comparative Example 1 (Student t-test). In Figure 3, the symbol "*" indicates a statistically significant difference of p < 0.05 compared to Comparative Example 1, and the symbol "**" indicates a statistically significant difference of p < 0.01 compared to Comparative Example 1 (both according to Williams' multiple comparison test).

[0148] As shown in Figure 3, the absorbance at 450 nm increased in an Eda concentration-dependent manner in the Eda-containing groups (Examples 3-1 to 3-6) compared to the Eda-unexposed group (Comparative Example 1), confirming a higher number of viable cells. This indicates that cell damage was suppressed in the presence of Eda. The present inventors confirmed that similar results to those shown in Figure 3 were obtained even after simultaneously exposing the disease model cells prepared by the method of Example 1 to Eda and EA for 24 hours.

[0149] Example 4: Search for and evaluation of biomarkers RNA-seq analysis was performed using the disease model cells prepared by the method of Example 1 to discover biomarkers that could serve as indicators for disease progression, evaluation of therapeutic effects, and / or selection of candidate compounds.

[0150] In this evaluation, a poly-L-lysine-treated 6-well plate was used, and 1 × 10 ６ The cells were seeded at a cell density of 100 cells / well, cultured under the conditions of Example 1(1), and differentiated.

[0151] The experimental groups (1) to (4) of this example are shown below with reference to FIG. 4. (1) Group 1 (control group; designated "Group 1" in FIG. 4 and FIGS. 7 to 11): After preparing differentiated cell lines by the method of Example 1(1), they were further cultured for 48 hours in a differentiation medium lacking Eda and EA. This experimental group was not forced to express TDP-43 and mimics healthy animals, including humans. (2) Group 2 (Eda-only exposure group; designated "Group 2" in FIG. 4 and FIGS. 7 to 11): After preparing differentiated cell lines by the method of Example 1(1), they were cultured for 48 hours in a differentiation medium containing Eda (final concentration 50 μmol / L) but lacking EA. This experimental group was not forced to express TDP-43 and mimics the conditions of healthy animals, including humans, exposed to Eda.

[0152] (3) Group 3 (TDP-43 + EA exposure group; designated "Group 3" in Figures 4 and 7 to 11): Disease model cells expressing TDP-43 were prepared and then cultured for 24 hours in a differentiation medium containing Eda but not EA (final concentration 20 μmol / L). This experimental group was forced to express TDP-43, simulating the condition of subjects suffering from a neurodegenerative disease and not receiving treatment. (4) Group 4 (TDP-43 + EA + Eda exposure group; designated "Group 4" in Figures 4 and 7 to 11): Disease model cells expressing TDP-43 were prepared and then cultured for 24 hours in a differentiation medium containing Eda (final concentration 50 μmol / L) but not EA. EA (final concentration 20 μmol / L) was then added and the cells were further cultured for 24 hours. This experimental group mimics the conditions in which subjects suffering from a neurodegenerative disease are administered or exposed to Eda.

[0153] After the culture was completed, 1 x 10 cells were cultured from each of the experimental groups (1) to (4). ６ More than 100 cell pellets were obtained. Total RNA was extracted from these cell pellets using NucleoSpin RNA (Macherey-Nagel) according to the manufacturer's protocol. The extracted RNA was then analyzed using a TapeStation and a Bioanalyzer RNA 6000 Nano chip (Agilent) to confirm that the RNA concentration was 2 ng / μL or higher, the total RNA amount was 50 ng or higher, and the RNA was of high quality.

[0154] Using the extracted total RNA (1.0 ng) as a template, double-stranded cDNA was synthesized by the SMART method using the SMART-Seq v4 Ultra® Low Input RNA Kit for Sequencing (Clontech) according to the product protocol. The synthesized double-stranded cDNA was then amplified by 13 cycles of PCR according to the product protocol. The PCR product was then purified by magnetic bead purification using an AMPureXP (Beckman Coulter).

[0155] The resulting PCR products (double-stranded cDNA) were tagmented using the Nextera XT DNA Library Prep Kit and Nextera XT Index Kit v2 (Illumina) according to the product's protocol, and adapter sequences were ligated to both ends of the fragmented cDNA. The ligated cDNA (0.2 ng) was then amplified by 12 cycles of PCR using the primers provided with the kit according to the product's protocol. The PCR products from all samples were then pooled into a single library to avoid potential adverse effects during sequencing.

[0156] The PCR products obtained from each experimental group were processed in several steps, as shown in Figure 5. <RNA Sequencing (see Figure 5)> Next, the cDNA libraries were sequenced using the NovaSeq6000 system (NovaSeq6000 S4 Reagent Kit, NovaSeq Xp4 Lane Kit, and NovaSeq Control Software (version 1.6.0) (all from Illumina)) according to the manufacturer's protocol. Base calls and quality scores were calculated using real-time analysis (version 3.4.4; Illumina) according to the cycle-based call file. The base call file was converted to a FASTQ file using bcl2fastq2 software (version 2.20; Illumina).

[0157] <Alignment - Expression Analysis (see Figure 5)> Next, using DRAGEN Bio-IT platform software (version 3.6.3, Illumina), the types of genes and their abundances were identified from the 150-base sequence information included in the FASTQ file, with reference to the rat genome assembly (version 6.0, top level) and transcript annotation (release 101) obtained from the Ensembl database.

[0158] The RNA representation (TPM) of transcript i measured from the cDNA library of each experimental group was calculated based on the alignment to the rat genome and transcript annotation in the Ensembl database using the following formula (A): where "m_i" represents the number of reads of transcript i and "l_i" represents the base length of transcript i. TPM_i = 10 ６ × (m_i / l_i) / (Σ_i [(m_i / l_i)]) ... (A)

[0159] Expression variation analysis was performed using DESeq2 (a two-group comparison package) in the 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 expressed as "log2 ([expression level of the first measured object] / [expression level of the second measured object])." Significance tests were performed based on the p-value calculated by the Wald test with multiple testing correction based on the Benjamini-Hochberg method (BH method). In this analysis, genes with an absolute value of the log2 ratio of 1 or greater (expression variation ratio of 2.0-fold or greater or 0.50-fold or less) and a p-value of 0.01 or less by the Wald test between any two groups were identified as "genes with altered expression levels" (DEGs). DEGs include both upregulated and downregulated genes.

[0160] <Gene Ontology Analysis (see Figure 5)> Gene ontology analysis was performed by associating DEGs with ontology terms obtained from the BioMart database. These associations were performed by determining significant ontology terms with a p-value of 0.05 or less in a hypergeometric distribution test with multiple testing correction based on the BH method. Gene ontology terms associated with DEGs were classified based on biological function, component, and molecular function, and significant ontology terms were extracted.

[0161] <Expression Regulatory Pathway Analysis (see Figure 5)> Analysis of expression regulatory pathways of DEGs was performed using software (Ingenuity Pathway Analysis, manufactured by QIAGEN). The association between the expression pathways defined in the software and DEGs was determined to be significant when the p-value calculated by Fisher's exact test with multiple testing correction based on the BH method was 0.05 or less.

[0162] The analysis results of this example are shown in Figure 6. Figure 6 shows the results of principal component analysis of expression fluctuations in the RNA-seq data obtained from each experimental group (N = 3 for each). The X-axis represents the first principal component, and the Y-axis represents the second principal component, with the contribution rate of each component expressed as a percentage. Note that the principal component analysis was performed excluding genes with a TPM variance of 1 or less for all samples.

[0163] As shown in Figure 6, the RNA-seq data for samples from the same experimental group were all close to each other, and the quality of the RNA-seq data was determined to be good. Comparing the experimental groups, it was found that the gene expression profiles of Groups 3 and 4 were clearly different from those of Group 1. Furthermore, it was found that the gene expression profiles of Groups 3 and 4 were clearly different, despite the expression of TDP-43. On the other hand, it was found that the gene expression profiles of Groups 1 and 2 were similar, regardless of the presence or absence of Eda exposure.

[0164] The differentially expressed genes (DEGs) between the experimental groups were identified based on the gene expression levels in each group, and the results are shown in Table 1. In Table 1, "xxx / yyy" (xxx and yyy each independently represent any integer) indicates that the number of genes with increased expression is xxx and the number of genes with decreased expression is yyy.

[0165]

[0166] Of the genes whose expression levels varied in each experimental group, we focused on genes that met the following criteria a or b, and the expression levels are shown in Figures 7 to 11. Criterion a: Expression is significantly increased in Group 3 compared to Group 1, and expression is significantly decreased in Group 4 compared to Group 3. Criterion b: Expression is significantly decreased in Group 3 compared to Group 1, and expression is significantly increased in Group 4 compared to Group 3.

[0167] A gene that meets any of the above criteria means that its expression level fluctuates with the onset of disease (corresponding to Group 3) compared to that in a healthy state (corresponding to Group 1), and that its expression level returns to a level equivalent to or close to normal by treatment of the disease (corresponding to Group 4). Therefore, the expression levels of these genes can be used as indicators, for example, to evaluate or predict the possibility of the presence or absence of a disease and its progression, evaluate or predict the possibility of treatment response, select samples that can be appropriately used as disease models, or serve as useful biomarkers for selecting candidate compounds in drug discovery.

[0168] All of the genes shown in Figures 7 to 11 are rat genes that meet the above criteria and have human homologs. As an example, Alpl (the rat orthologue of human ALPL) shown in Figure 8(a) is a gene encoding alkaline phosphatase. Because alkaline phosphatase is an enzyme present extracellularly in blood and other tissues, sampling extracellular fluids such as blood offers the advantage of enabling minimally invasive evaluation of disease progression and therapeutic efficacy, as well as aiding in such prediction.

[0169] Tables 2 and 3 below show the results of the gene ontology analysis, as well as differentially expressed genes, their differential expression levels (TPM), log2 ratios of differential expression, and p-values.

[0170] Table 4 below shows the log2 expression variation ratios and p-values ​​for other differentially expressed rat genes that meet the above-mentioned criteria a and b. All genes listed in Table 4 are included in the specific genes of the present disclosure. In Table 4, "Upregulated genes" are examples of genes whose expression levels are decreased by the presence of disease and increased by the presence of disease and edaravone, and these genes are included in the above-mentioned gene group (B). In Table 4, "Downregulated genes" are examples of genes whose expression levels are increased by the presence of disease and decreased by the presence of disease and edaravone, and these genes are included in the above-mentioned gene group (A). For ease of explanation, Table 4 also lists the results for the Mt2A and Alpl genes listed in Tables 2 and 3. The inventors performed quantitative PCR on Mt2A and Alpl and confirmed that expression profiles similar to those based on RNA-seq were obtained.

[0171]

[0172]

[0173]

[0174] Examples 5-1 to 5-2, Comparative Example 2, and Reference Examples 2 and 3: Evaluation of Cell Protection Using a Chromogenic Substrate as an Indicator (2) Using the differentiated cell line prepared by the method of Example 1 (shown as "TDP-43(-)" in Figure 12), an Eda-unexposed group (Reference Example 2) and an Eda-exposed group for 96 hours (final concentration 100 µmol / L) (Reference Example 3) were prepared. Furthermore, using the disease model cells prepared by the method of Example 1 (shown as "TDP-43(+)" in Figure 12), an Eda-unexposed group (Comparative Example 2) and an Eda-exposed group for 96 hours (final concentration 50 or 100 µmol / L) (Examples 5-1 to 5-2) were prepared. All of these experiments were performed without EA.

[0175] Thereafter, similarly to Example 3-1, the cells of each group were exposed to the cytotoxicity measurement kit according to the product protocol, and the amount of the produced chromogenic substrate was measured by absorbance at 450 nm. The results are shown in Figure 12.

[0176] All data shown in Figure 12 are expressed as mean ± standard error of the mean (SEM, n = 4 for each). In Figure 12, the symbol "##" indicates a statistically significant difference of p < 0.01 compared to Comparative Example 2. In Figure 12, the symbol "**" indicates a statistically significant difference of p < 0.05 compared to Comparative Example 2. In Figure 12, the symbol "$" indicates a statistically significant difference of p < 0.05 compared to Reference Example 2 (all based on Student's t-test).

[0177] 12, it was confirmed that the number of viable cells was higher in the groups containing Eda in the presence of TDP-43 (Examples 5-1 and 5-2) than in the group not exposed to Eda in the presence of TDP-43 (Comparative Example 2). This indicates that cell damage occurred in the presence of TDP-43 even in the absence of EA, and that Eda suppressed cell damage caused by TDP-43.

[0178] Example 6: Preparation of Disease Model Cells (2) Human induced pluripotent stem (iPS) cells were induced from cells derived from an ALS patient and cells derived from a healthy individual using standard methods. The ALS patient-derived cells were known to have a heterozygous ALS risk mutation in the TARDBP gene. Each iPS cell was differentiated into a motor neuron (hereinafter simply referred to as "neuron") using standard methods. Neurons differentiated from iPS cells induced from ALS patient-derived cells are simply referred to as "ALS patient-derived neurons," while neurons differentiated from iPS cells induced from healthy individual-derived cells are simply referred to as "healthy individual-derived neurons." For long-term cell storage, neurons were sometimes frozen 2 to 7 days after differentiation, as needed. These frozen neurons are also referred to as "frozen neurons."

[0179] Next, neurons were cultured in a 98-well culture plate (Corning, 3599) under the following conditions. The culture plate was coated before cell seeding. Specifically, 0.02% poly-L-ornithine solution (Sigma-Aldrich, P4957) was added to each well of the culture plate, and the plate was incubated at 37°C, 5% CO ２ After leaving the plate in an incubator for 2 hours, each well was washed and incubated with 20 mg / mL laminin solution (Thermo Fisher Scientific, 23017015) at 37°C, 5% CO ２ The cells were then allowed to stand in the incubator for another 2 hours, after which the frozen neurons were thawed by a conventional method, and the thawed neurons were seeded into each well at 20,000 cells / well and cultured in the presence of a culture medium having the following composition:

[0180] The culture medium used was a mixture of the following composition:・DMEM / F12 (Thermo Fisher Scientific, 21331-020) : 50% v / v ・Neurobasal Medium (Thermo Fisher Scientific, 21103-049) : 50% v / v ・Glutamax Supplement (Thermo Fisher Scientifc, 35050061) : 1% v / v ・Penicillin-Streptomycin (10000 units / mL) (Thermo Fisher Scietific, 15140-148) : 0.5% v / v ・Component N1 (Elixirgen) : 3% v / v ・Component A (Elixirgen) : 0.1% v / v ・Component D4 (Elixirgen) : 0.1% v / v ・Component P (Elixirgen) : 0.05% v / v

[0181] Example 7: Evaluation of cytotoxicity by live cell imaging The culture medium containing a solution of the evaluation compound (edaravone) in dimethyl sulfoxide (DMSO) (final concentration: edaravone 30 μM, DMSO 0.03% v / v) was contacted with neurons after 7 days of culture, and the occurrence of cytotoxicity was evaluated over time. Separately, a group in which the culture medium did not contain the evaluation compound (a group to which DMSO at the same concentration was added) was also prepared.

[0182] Cell damage was evaluated by live cell imaging using IncuCyte S3 (Sartorius) and Cytotox (Sartorius, 4846) according to the attached protocol. Neurite length per unit area (unit: mm / mm ２ The neurite length and the number of dead cells (unit: Count / image) were analyzed over time and used as evaluation indices. A smaller neurite length and a larger number of dead cells indicate the occurrence of cell damage.

[0183] <Evaluation of cell damage based on neurite length (1)> The rate of change in neurite length in each experimental group from the start of treatment with the evaluation compound to the time of analysis (24 hours) was calculated as a ratio such that the DMSO group containing no evaluation compound was set to 100. The results are shown in Figure 13. The smaller the value on the vertical axis, the shorter the neurite length over time, indicating the occurrence of cell damage. The experimental groups in Figure 13 are as follows.

[0184] ALS cells-DMSO: A group using neurons derived from ALS patients, with no edaravone in the culture medium. ALS cells-Edaravone: A group using neurons derived from ALS patients, with edaravone in the culture medium. Healthy cells-DMSO: A group using neurons derived from healthy individuals, with no edaravone in the culture medium. Healthy cells-Edaravone: A group using neurons derived from healthy individuals, with edaravone in the culture medium.

[0185] When neurons derived from healthy individuals and ALS patients were treated with edaravone, the neurite length in the ALS patient-derived neurons was significantly greater than that in the DMSO-treated group, demonstrating a protective effect on neurites, whereas no significant change in neurite length was observed in neurons derived from healthy individuals.

[0186] <Evaluation of cell damage based on the number of dead cells (2)> The rate of change in cell death in each experimental group from the start of treatment with the evaluation compound to the time of analysis (24 hours) was calculated as a ratio such that the rate for the DMSO-treated group not containing the evaluation compound is 100. The results are shown in Figure 14. The larger the value on the vertical axis, the greater the number of dead cells over time, indicating the occurrence of cell damage. The experimental groups in Figure 14 are as follows.

[0187] ALS cells-DMSO: A group using neurons derived from ALS patients, with no edaravone in the culture medium. ALS cells-Edaravone: A group using neurons derived from ALS patients, with edaravone in the culture medium. Healthy cells-DMSO: A group using neurons derived from healthy individuals, with no edaravone in the culture medium. Healthy cells-Edaravone: A group using neurons derived from healthy individuals, with edaravone in the culture medium.

[0188] When neurons derived from healthy individuals and neurons derived from ALS patients were treated with edaravone, the number of neuronal deaths in the ALS patient-derived neurons was significantly lower than in the DMSO-treated group, demonstrating a protective effect against neuronal death. In contrast, no significant change in the number of neuronal deaths was observed in neurons derived from healthy individuals.

[0189] Example 8: Evaluation of TDP-43 localization The intracellular localization of TDP-43 polypeptide was evaluated by immunofluorescence staining of neurons. After culture, neurons were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). The culture medium was maintained in a 5% fetal bovine serum (FBS), 0.1% Triton X-ray diffraction pattern. ＴＭAfter blocking with PBS containing β-TDM-X, a dilution of primary antibodies containing anti-TDP-43 and anti-β-III tubulin antibodies was added and incubated overnight at 4°C. The anti-β-III tubulin antibody was used to visualize neuronal cell bodies. The following day, after washing with PBS, a dilution of secondary antibodies conjugated with Alexa dye was added and incubated at room temperature for 1 hour. Further nuclear staining was performed, followed by washing with PBS and imaging 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. The results of the intracellular localization of TDP-43 polypeptide are shown in Figure 15. The experimental groups in Figure 15 are as follows:

[0190] ALS cells-DMSO: A group using neurons derived from ALS patients, with no edaravone in the culture medium. ALS cells-Edaravone: A group using neurons derived from ALS patients, with edaravone in the culture medium. Healthy cells-DMSO: A group using neurons derived from healthy individuals, with no edaravone in the culture medium. Healthy cells-Edaravone: A group using neurons derived from healthy individuals, with edaravone in the culture medium.

[0191] Regarding the intracellular localization of TDP-43 polypeptide, the nuclear and cytoplasmic TDP-43 fluorescence intensity and the cytoplasmic / nuclear TDP-43 intensity ratio in ALS cells and healthy cells are shown in Figure 15. Values ​​are shown as a ratio such that the DMSO group is 100. When healthy subject-derived neurons and ALS patient-derived neurons were treated with edaravone, the cytoplasmic and cytoplasmic / nuclear TDP-43 intensity ratios were significantly reduced in the ALS patient-derived neurons after 24 hours, indicating that the abnormal localization of TDP-43 observed in ALS pathology was suppressed. Furthermore, no such reduction in the cytoplasmic / nuclear TDP-43 intensity ratio was observed in healthy subject-derived neurons.

[0192] These results newly demonstrated that edaravone, an approved drug for ALS, inhibits cell damage in neurons derived from ALS patients and improves the abnormal localization of intracellular TDP-43. Abnormal localization of TDP-43 is a pathological condition commonly observed in the pathology of ALS, a heterogeneous disease, and the effect of correcting the abnormal localization of TDP-43 suggests the possibility of efficacy in a wide range of ALS patients. Furthermore, this drug may contribute to beneficial effects in the treatment or prevention of diseases associated with the abnormal localization of TDP-43, not limited to ALS. These findings are new findings made clear by the completion of the present invention, and these results indicate that edaravone may be a suitable method for treating or preventing diseases associated with the abnormal localization of TDP-43.

Claims

1. A method for evaluating the responsiveness of a subject to edaravone, The process includes evaluating whether the subject may be responsive to edaravone based on changes in the expression level of gene products caused by exposure to edaravone, The method wherein the gene product is the gene product of one or more genes selected from NTM, MAST1, SCN2A, KAZALD1, SBK1, UBE2L6, ALPL, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, and FAIM2.

2. A method for evaluating the likelihood of developing a target neurodegenerative disease, The process includes evaluating whether a subject may have a neurodegenerative disease based on changes in the expression levels of gene products in the subject, The method wherein the gene product is the gene product of one or more genes selected from NTM, KAZALD1, SBK1, UBE2L6, DCTD, ASF1B, and FCSK.

3. A biomarker for the diagnosis of neurodegenerative diseases, comprising the gene product of one or more genes selected from NTM, KAZALD1, SBK1, UBE2L6, DCTD, ASF1B, and FCSK.

4. A biomarker for diagnosing edaravone responsiveness, comprising the gene product of one or more genes selected from NTM, MAST1, SCN2A, KAZALD1, SBK1, UBE2L6, ALPL, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, and FAIM2.

5. A detection kit comprising a detection reagent capable of specifically detecting the gene product of one or more genes selected from NTM, MAST1, SCN2A, KAZALD1, SBK1, UBE2L6, ALPL, PTTG1, ITGB4, HAUS4, DCTD, MT2A, ASF1B, FCSK, and FAIM2, for detecting one or more of the gene products as a biomarker for the diagnosis of neurodegenerative diseases or for the diagnosis of edaravone responsiveness.