Composition for preventing or treating degenerative nervous system diseases containing colchicine and metformin as active ingredients

Abstract

The present invention relates to a therapeutic composition for treating, preventing, inhibiting, and improving or slowing down the onset or progression of degenerative nervous system diseases, particularly diseases associated with the aggregate formation of TAR DNA-binding protein 43 (TDP-43), such as amyotrophic lateral sclerosis, using a combination agent containing colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof as active ingredients. Examples of diseases associated with TDP-43 protein include frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encelopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE). and diseases, disorders and abnormalities associated with TDP-43, particularly those associated with TDP-43 aggregates, or TDP-43 proteinopathies. The inventors of the present invention have confirmed that the above-mentioned degenerative diseases can be treated or prevented by using a combination of colchicine and metformin, which are drugs not used in the prior art, as a therapeutic agent for the diseases.

Description

[Technical Field]

[0001] The present invention relates to a therapeutic composition for treating, preventing, inhibiting, and ameliorating or slowing down diseases associated with the aggregate formation of TAR DNA-binding protein 43 (TDP-43), as well as the onset or progression of such conditions, using a combination agent containing colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof as active ingredients. [Background technology]

[0002] Diseases associated with the TDP-43 protein, as described above, are primarily degenerative nervous system diseases, and examples of such degenerative diseases include frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encelopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE), as well as diseases, disorders, and abnormalities associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies.

[0003] The inventors of the present invention have found that the above-mentioned degenerative diseases can be treated or prevented by using a combination of colchicine and metformin, which are drugs not used in the prior art, as a therapeutic agent for the diseases. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 10-2012-0023352 [Patent Document 2] Korean Patent Publication No. 10-2022-0120906 Summary of the Invention [Problem to be solved by the invention]

[0005] It is known that the ultimate cause of degenerative nervous system diseases such as Parkinson's disease, Alzheimer's disease, and stroke is cell death, which leads to neuronal damage and loss. For example, in Parkinson's disease, selective loss of dopaminergic neurons in the substantia nigra of the midbrain leads to dysfunction of the basal ganglia, which are involved in the organization of motor function, resulting in motor dysfunction such as persistent tremor, rigidity, bradykinesia, and postural instability.

[0006] Regarding the above-mentioned degenerative neurological diseases, the inventors of the present invention have confirmed that a combination drug of colchicine and metformin is particularly effective for amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, which is closely related to the TDP-43 protein.

[0007] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a rare degenerative neurological disorder characterized by the gradual degeneration of motor neurons in the central nervous system, resulting in the death of motor neurons. The exact mechanism of Lou Gehrig's disease and research into treatments have yet to be clearly identified. Currently, the only drugs approved by the U.S. FDA for the treatment of Lou Gehrig's disease are riluzole and edaravone. Both drugs are known to extend patients' life expectancy by several months or slow the progression of the disease in the early stages, but there is currently no cure for Lou Gehrig's disease.

[0008] It is reported that approximately 10% of patients with Lou Gehrig's disease have a genetic cause, and in the remaining 90% of patients, it is estimated that the cause is immune, oxidative toxicity, excitotoxicity, environmental factors, or protein aggregation.

[0009] Among these, aggregation of a protein called TDP-43 is observed in over 90% of Lou Gehrig's disease patients, and numerous research results have shown that this phenomenon is an important cause of Lou Gehrig's disease.

[0010] TDP-43 is primarily found in the cell nucleus, but when TDP-43 aggregates accumulate in the cytoplasm, it is known to induce diseases related to the TDP-43 protein. Such abnormal cytoplasmic aggregation of TDP-43 has recently been discovered in the brains of patients with limbic-specific senile TDP-43 encephalopathy (LATE) dementia and Alzheimer's dementia.

[0011] In the present invention, the development of effective treatments for amyotrophic lateral sclerosis (ALS), which is the main target of treatment, has presented great difficulties to the pharmaceutical industry because, as mentioned above, a clear mechanism for inducing ALS disease has not yet been identified.

[0012] Riluzole (Rilutek, Sanofi) and edaravone (Radicut, Mitsubishi Tanabe Pharma) are the only approved treatments for ALS to date. Riluzole is a neuroprotective drug that blocks glutamate neurotransmission in the central nervous system and prevents apoptosis (programmed cell death) of motor neurons, while edaravone inhibits oxidative stress caused by free radicals. However, both of these treatments have only been confirmed to alleviate symptoms.

[0013] Therefore, there is a need to develop improved therapies for the treatment of TDP-43 proteopathies such as ALS. The development of such effective therapies would not only contribute to improving the quality of life and lifespan of patients suffering from ALS disease, but would also significantly help reduce the cost burden of such diseases.

[0014] In relation to the TDP-43 proteopathy provided by the present invention, a treatment method using a combination drug containing colchicine and metformin as active ingredients has not yet been studied. [Means for solving the problem]

[0015] The present invention aims to solve the situation where there is a shortage of therapeutic agents for treating amyotrophic lateral sclerosis (ALS) related to TDP-43 proteopathy as described above, and development of new therapeutic agents is a considerable burden due to the lack of clear research on the disease.

[0016] Therefore, the present invention has been completed by continuously studying the therapeutic effects of a combination preparation of colchicine and metformin, which has not been studied as a therapeutic agent for amyotrophic lateral sclerosis in the prior art, and confirming that the combination preparation is sufficiently effective.

[0017] Age-related brain disorders, characterized by pathological protein aggregation in the central nervous system (CNS) and peripheral organs, are one of the leading causes of disability and death worldwide. The most well-characterized aggregate-forming protein in Alzheimer's disease and related disorders is amyloid-β. Other disease-related, aggregation-prone proteins that cause neurodegeneration include, but are not limited to, α-synuclein (αSyn), Huntington's disease, fused in sarcoma (FUS), dipeptide repeat protein (DPR) generated by the aberrant translation of the C9orf72 repeat expansion, superoxide dismutase 1 (SOD1), and TDP-43. Among these, diseases associated with TDP-43 aggregates are commonly referred to as TDP-43 proteopathies, including, but not limited to, ALS and FTD.

[0018] Transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) is a 414-amino acid protein encoded by the TARDBP gene on chromosome 1p36.2 (ALS10). TARDBP is composed of six exons (exon 1 is non-coding; exons 2-6 are protein-coding). TDP-43 belongs to the family of heterogeneous ribonucleoprotein (hnRNP) RNA-binding proteins. TDP-43 contains five functional domains. It consists of two highly conserved hexameric ribonucleoprotein 2 (RNP2) and octameric ribonucleoprotein 1 (RNP1) domains with two RNA recognition motifs (RRM1 and RRM2), a nuclear export signal (NES) and nuclear localization signal (NLS) that enable shuttle between the nucleus and cytoplasm where bound mRNA is transported, and a C-terminal glycine-rich domain that mediates protein-protein interactions.

[0019] TDP-43 is involved in multiple aspects of RNA processing, including transcription, splicing, transport, and stabilization. It is a highly conserved, ubiquitously expressed protein with a strictly autoregulated expression level that shuttles continuously between the nucleus and cytoplasm, but is primarily localized in the nucleus. In 2006, TDP-43 was identified as a protein that accumulates in most cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) with tau-negative, ubiquitin-positive inclusions (termed FTLD-TDP).

[0020] Thirty-eight negative-dominant mutations in TDP-43 were identified in patients with sporadic and familial ALS as well as inherited FTD, primarily located in the glycine-rich domain. TDP-43 is inherently prone to aggregation as revealed by sedimentation analysis, and this tendency is further increased by some ALS-associated TARDBP mutations that link TDP-43 aggregation to clinical disease manifestations.

[0021] In a TDP-43 proteopathy cell model, the colchicine and metformin combination increased autophagy and chaperone activity, reducing the abnormal aggregation of TDP-43 protein or its accumulation in the cytoplasm due to mislocalization, a finding that has not yet been reported.

[0022] Autophagy is known to play an important role in maintaining cellular homeostasis by breaking down unnecessary proteins and abnormal organelles within the cell and reusing them as a new energy source. Furthermore, a deficiency or dysfunction of autophagy is known to be closely related to various human diseases, including brain disorders.

[0023] Meanwhile, chaperones, which are involved in preventing protein misfolding and suppressing neuronal damage and death, are well known as a defense mechanism against protein aggregation and have been considered an important target in the treatment of neurodegenerative diseases.

[0024] The inventors of the present invention have first discovered that a combination composition of colchicine and metformin can effectively inhibit the abnormal aggregation of TDP-43 protein, a major etiological factor in neurodegenerative diseases, and have also confirmed that the composition of the present invention exhibits the ability to selectively induce chaperones and autophagy associated with degenerative neurological diseases.

[0025] Based on these results, an object of the present invention is to provide a combination composition of colchicine and metformin for treating or preventing amyotrophic lateral sclerosis such as Lou Gehrig's disease associated with the TDP-43 protein.

[0026] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative nervous system brain diseases such as amyotrophic lateral sclerosis (ALS) represented by Lou Gehrig's disease, as well as frontotemporal dementia, amyotrophic lateral sclerosis, Alexander disease, argyrophilic grain dementia, Guan's ALS (parkinsonism-dementia complex), limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, facial hemifacial atrophy, Alzheimer's disease, Down's syndrome, British familial dementia, Huntington's disease, Machado-Joseph disease, hippocampal sclerosis dementia, inclusion body myositis, oculopharyngeal muscular dystrophy with rimmed vacuoles, muscle fiber radiculopathy, traumatic brain injury, dementia with Lewy bodies, and Parkinson's disease, which are diseases associated with TDP-43 protein aggregation.

[0027] The technical problem that the present invention aims to solve is not limited to the problem mentioned above, and various problems related to the problem will be clearly understood by a person having ordinary skill in the technical field to which the present invention pertains from the following description.

[0028] To achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative nervous system brain diseases such as amyotrophic lateral sclerosis represented by Lou Gehrig's disease, Parkinson's disease, Alzheimer's disease or Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, senile dementia caused by loss of nerve cells, stroke, post-traumatic stress disorder, and amnesia, which comprises colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof as the main active ingredient.

[0029] The present invention also provides a pharmaceutical composition for enhancing the therapeutic effect of the above diseases, which comprises colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof as main active ingredients. The pharmaceutical composition for enhancing the therapeutic effect is conceptually the same as a therapeutic adjuvant for preventing and treating Lou Gehrig's disease, which is the target disease of the present invention.

[0030] The pharmaceutical composition of the present invention is prepared in the form of a pharmaceutical composition for treating or preventing degenerative nervous system diseases, further comprising a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier includes a non-naturally occurring carrier. Specifically, the pharmaceutical composition can be formulated into powder, pills, granules, tablets, capsules, liquid preparations, emulsifiable concentrates, suspensions, emulsions, syrups, aerosols, topical preparations, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, suppositories, and sterile injections by conventional methods.

[0031] The pharmaceutical composition provided by the present invention further comprises, in addition to a suitable pharmacologically acceptable carrier, one or more ingredients selected from the group consisting of melatonin, creatine, magnesium, coenzyme Q10, vitamin D, vitamin B, vitamin E, L-serine, L-carnitine, and N-acetylcysteine, which are effective against degenerative nervous system diseases.

[0032] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level is determined by factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, and sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present invention used, the duration of treatment, drugs used in combination with or concomitantly with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can also be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy with the minimum amount without causing side effects.

[0033] The dosage of the pharmaceutical composition provided by the present invention can be determined by a person skilled in the art, taking into consideration the purpose of use, the degree of toxicity of the disease, the age, weight, sex, medical history of the patient, or the type of substance used as an active ingredient, but it is important to administer an amount that will achieve the maximum effect with the minimum amount without causing side effects, taking all of the above factors into consideration. [Effects of the Invention]

[0034] The pharmaceutical composition of the present invention, which contains colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof as active ingredients, inhibits the abnormal aggregation and mislocalization of TDP-43 protein through the synergistic effect of the two ingredients, and as a result, can effectively prevent or treat degenerative nervous system diseases such as Lou Gehrig's disease.

[0035] The combination preparation provided by the present invention can efficiently enhance the preventive or therapeutic effects of the above diseases even when administered in low doses without using conventional expensive drugs, and therefore will greatly contribute to the promotion of human health and the development of related industries such as health care. [Brief explanation of the drawings]

[0036] [Figure 1] Figure 1 shows the quantification of the mRNA expression (AC) or protein expression (D) of the chaperone protein HSPB8 in human SH-SY5Y cells treated with metformin, colchicine alone, or the metformin / colchicine combination. [Figure 2] Figure 2 shows that TDP-43 aggregation in H2O2-treated human SH-SY5Y cells was quantified using a TDP-43 aggregation kit after treatment with metformin, colchicine alone, or the metformin / colchicine combination. [Figure 3] Figure 3 shows the results of immunofluorescence staining and fluorescence microscopy of TDP-43 aggregation in H2O2-treated human SH-SY5Y cells treated with metformin, colchicine, or the metformin / colchicine combination (A), and the quantification of measured stress granules (SGs) (B). Filter retardation analysis was also used to confirm and quantify TDP-43 aggregation (C). [Figure 4] Figure 4 shows that SH-SY5Y cells transfected with human GFP-TDP-25 were treated with metformin and colchicine alone or in combination. The intracellular localization of TDP-25 was observed by fluorescence microscopy (A), and the expression of insoluble and soluble TDP-43 proteins (B) was monitored, followed by quantification of the degree of TDP-43 aggregation (C). In Figure 4, blue fluorescence indicates DAPI, representing nuclei, and green fluorescence indicates GFP, representing TDP-25. [Figure 5] Figure 5 shows the quantification of the mRNA expression of the autophagy transcription factor TFEB in human SH-SY5Y cells treated with metformin or colchicine alone or with the metformin / colchicine combination. [Figure 6]Figure 6 shows the quantification of autophagy receptor SQSTM1 / p62 mRNA expression in human SH-SY5Y cells treated with metformin or colchicine alone or the metformin / colchicine combination. [Figure 7] Figure 7 shows the quantification of the expression of the autophagy marker MAP1LC3A mRNA in human SH-SY5Y cells when treated with metformin or colchicine alone or with the metformin / colchicine combination. [Figure 8] FIG. 8 shows the quantification of p62 protein expression in human SH-SY5Y cells treated with metformin and colchicine alone or with the metformin / colchicine combination. [Figure 9] FIG. 9 shows the quantification of LC3-II protein expression in human SH-SY5Y cells treated with metformin and colchicine alone or with the metformin / colchicine combination. BEST MODE FOR CARRYING OUT THE INVENTION

[0037] It was confirmed that when a combination preparation containing colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof as active ingredients provided by the present invention was administered to human SH-SY5Y cells, the degree of TDP-43 aggregation was reduced, and based on this, the present invention was completed.

[0038] The present invention will be described in detail below.

[0039] Colchicine, the main active ingredient used in the present invention, is a substance extracted from Colchicum saffron plants. It is mainly used as an alternative drug for gout when non-steroidal anti-inflammatory drugs are not effective in treating gout. It is also used to treat familial Mediterranean fever, pericarditis, Behçet's disease, etc.

[0040] Metformin, another active ingredient used in the present invention, is a biguanide drug that has a blood glucose-improving effect and is primarily used as a diabetes treatment. Although the mechanism of action of metformin is unclear, it is known to inhibit mitochondrial cellular respiration, activate AMPK, and prevent the increase in cAMP concentration induced by glucagon, thereby inhibiting PKA activity and suppressing glucose production.

[0041] The diseases related to the expression of TDP-43 protein described in the present invention include frontotemporal dementia (FTD, e.g., with or without sporadic or familial motor-neuron disease (MND), with progranulin gene (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, and ubiquitin-positive TDP-43 inclusions (FTLDTDP). Frontotemporal lobar degeneration (FTLD), argyrophilic particle disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, those with TARDBP mutations, those with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy, facial hemifacial atrophy, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, British familial dementia, polyglutamine disorders (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; This includes dementia and radiculopathy with hippocampal sclerosis (sporadic inclusion body myositis, inclusion body myositis with mutations in valosin-containing protein (VCP); also Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with rimmed vacuoles, muscle fiber radiculopathy with mutations in the myotilin (MYOT) gene or the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB) or Parkinson's disease (PD).

[0042] The inventors of the present invention have confirmed through various experiments that when colchicine and metformin are used in combination as a reagent treatment, abnormal aggregation of TDP-43 protein is reduced.

[0043] In one embodiment of the present invention, when SH-SY5Y cells, a human neuroblastoma cell line, were administered colchicine alone, metformin alone, or a combination of colchicine and metformin, the amount of inhibition of TDP-43 protein aggregation was compared. The greatest reduction in protein aggregation was observed when colchicine and metformin were administered in combination.

[0044] Furthermore, in one embodiment of the present invention, when colchicine and metformin were administered in combination to the cell line, the abnormal aggregation of TDP-43 induced by oxidative stress was further suppressed than when either drug was administered alone.

[0045] Furthermore, in one embodiment of the present invention, co-administration of colchicine and metformin in a TDP-43 aggregation cell model showed a significant inhibition of TDP-43 aggregation compared to administration of either drug alone. These results indicated that co-administration of colchicine and metformin inhibited abnormal TDP-43 aggregation by increasing the expression of cellular autophagy marker genes and chaperone proteins.

[0046] From the above results, it was confirmed that the pharmaceutical composition provided by the present invention increases the expression of chaperone genes and autophagy genes, and suppresses the abnormal aggregation and mislocalization of TDP-43 protein in diseases mediated by abnormal aggregation of TDP-43 protein or its accumulation in the cytoplasm due to mislocalization, and thus the present inventors have completed the present invention.

[0047] The term "combined preparation" as used herein means the pharmaceutical composition provided by the present invention.

[0048] The composition of the present invention may further contain suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, and may be formulated into powders, pills, granules, tablets, capsules, liquid preparations, emulsifiable concentrates, suspensions, emulsions, syrups, aerosols, topical preparations, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations, suppositories, and sterile injections by conventional methods, and preferably formulated into unit-dose preparations suitable for oral administration.

[0049] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level is determined by factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time and route of administration, and excretion rate of the drug, the duration of treatment, concurrently administered drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy with the minimum amount without causing side effects, which can be easily determined by one of ordinary skill in the art.

[0050] The pharmaceutical composition of the present invention can be administered to an individual via various routes. The administration route can be, for example, oral, rectal, intravenous, intramuscular, intraperitoneal, subcutaneous, intranasal, transdermal, intrauterine or intracerebral injection. The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, as well as several related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease.

[0051] In order to facilitate understanding of the present invention, preferred examples are presented below. However, the following examples are provided merely to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0052] Example 1 Whether combined treatment with metformin and colchicine has a synergistic effect on increasing the expression of the chaperone protein HSPB8 SH-SY5Y cells, a human neuroblastoma cell line, were treated with metformin (0, 0.1, 0.5, 1, 2, 5 mM) or colchicine (0, 1, 10, 100, 500, 1000 nM) at various concentrations. After 24 hours, the mRNA expression of the chaperone protein HSPB8 (Heat Shock Protein Family B (Small) Member 8) was observed. The results are shown in Figure 1.

[0053] Treatment with metformin or colchicine alone increased HSPB8 mRNA expression by approximately two-fold, whereas treatment with the combined use of metformin (5 mM) and colchicine (10, 100, 500, 1000 nM) increased HSPB8 mRNA expression by more than 20-fold in a colchicine concentration-dependent manner (Figures 1A-1C).

[0054] Since we confirmed that the combined treatment of metformin and colchicine had an additive effect on HSPB8 mRNA expression, we fixed the concentrations of metformin (5 mM) and colchicine (1000 nM) and observed HSPB8 protein expression. While metformin or colchicine treatment alone showed only slight changes in HSPB8 protein expression, the combined treatment of metformin and colchicine demonstrated an additive effect, increasing HSPB8 protein expression by approximately 8-fold (Figure 1D, and in each figure, ** P<0.01, * P<0.05 compared with normal cell group; ## P<0.01 compared with the colchicine alone group).

[0055] Example 2 Whether combined treatment with metformin and colchicine suppresses abnormal aggregation of TDP-43 induced by oxidative stress SH-SY5Y cells, a human neuroblastoma cell line, were treated with metformin (0, 0.1, 0.5, 1, 2, 5 mM) or colchicine (0, 1, 10, 100, 500, 1000 nM) at various concentrations. After 24 hours, the cells were treated with 1 mM hydrogen peroxide (H2O2) for 1 hour to induce oxidative stress.

[0056] The degree of TDP-43 aggregation in each sample was then measured. It was observed that 1 mM hydrogen peroxide (H2O2) significantly induced abnormal aggregation of TDP-43, while metformin alone had no significant effect on the TDP-43 aggregation induced by H2O2 (Figure 2A), and colchicine alone also had a slight effect on this (Figure 2B).

[0057] However, we confirmed that the combined treatment of metformin and colchicine significantly suppressed hydrogen peroxide-induced TDP-43 aggregation compared to treatment with either drug alone. In particular, in the group treated with the highest concentration of colchicine (1000 nM) and metformin, the aggregation was suppressed to almost the same level as normal cells (Figure 2C).

[0058] The results shown in Figure 2 were confirmed again using microscopy and cell protein analysis (Figure 3). Hydrogen peroxide (H2O2) significantly induced TDP-43 aggregation in the cytoplasm, and while metformin or colchicine treatment alone did not inhibit TDP-43 aggregation in the cytoplasm, combined treatment with metformin and colchicine inhibited TDP-43 aggregation at the level of normal cells (Figures 3A and 3B). This result was confirmed again by the filter retardation assay experiment shown in Figure 3C (in the figure, ** P<0.01 compared with normal cell group; ## P<0.01, # P<0.05 compared with the hydrogen peroxide group; $$ P<0.01, $ P<0.05 compared with the hydrogen peroxide + colchicine alone group).

[0059] Example 3 Whether combined treatment with metformin and colchicine inhibits TDP-43 aggregation in a TDP-43 aggregation-cell model To construct a model of TDP-43 protein aggregation in SH-SY5Y cells, a human neuroblastoma cell line, GFP-TDP-25 plasmid was transfected into the cells, and the transfected GFP-TDP-25 (GFP: green fluorescent protein) was confirmed to be present in the cytoplasm.

[0060] Treatment of the cells with metformin or colchicine alone did not affect the abnormal cytoplasmic accumulation of TDP-25, but treatment with the combined use of metformin and colchicine suppressed the accumulation of TDP-25 in the cytoplasm (Figure 4A).

[0061] Furthermore, in a TDP-43 aggregation model using the GFP-TDP-25 plasmid, we confirmed that treatment with metformin (5 mM) or colchicine (1000 nM) alone slightly reduced the expression of insoluble TDP-43, but treatment with the combined use of metformin and colchicine significantly reduced the expression of insoluble TDP-43 (Figures 4B and 4C). ** P<0.01, * P<0.05 compared with normal cell group; ## P<0.01 compared with the colchicine alone group).

[0062] Example 4 Whether combined treatment of metformin and colchicine has a synergistic effect on increasing cellular autophagy Human SH-SY5Y cells, a human neuroblastoma cell line, were treated with metformin (0, 0.1, 0.5, 1, 2, 5 mM) or colchicine (0, 1, 10, 100, 500, 1000 nM) at various concentrations. After 24 hours, the mRNA expression of the autophagy master transcription factor TFEB (Figure 5), the autophagy receptor SQSTM1 / p62 (Figure 6), and the autophagy marker MAP1LC3A (Figure 7) was monitored.

[0063] We found that metformin alone had no effect on mRNA expression of TFEB, SQSTM1 / p62, and MAP1LC3A genes, while colchicine alone increased expression by approximately 2-3 fold. In contrast, combined treatment with metformin (5 mM) and colchicine (10, 100, 500, and 1000 nM) had a 10-fold or greater additive effect on gene expression (Figures 5, 6, and 7).

[0064] To confirm the effects of metformin and colchicine on autophagy in the TDP-43 aggregation model, SH-SY5Y cells transfected with GFP-TDP-25 plasmid were treated with metformin (5 mM) and colchicine (1000 nM) alone or in combination. The increased p62 protein expression was significantly suppressed by the combined treatment with metformin and colchicine compared to the treatment with metformin or colchicine alone (Figure 8). Furthermore, LC3-II expression was significantly increased by the combined treatment with metformin and colchicine compared to the treatment with metformin or colchicine alone (Figure 9). ** P<0.01, * P<0.05 compared with normal cell group; ## P<0.01, # P<0.05 compared with the colchicine alone group).

[0065] As can be seen from the above results, abnormal aggregation of TDP-43 protein was hardly reduced by treatment with metformin or colchicine alone, but the combined administration of colchicine and metformin significantly inhibited the aggregation of the protein, indicating that the two drugs exhibit a clear synergistic effect and can effectively prevent or treat amyotrophic lateral sclerosis, such as Lou Gehrig's disease.

[0066] The above description of the present invention is for illustrative purposes only, and those skilled in the art can easily modify the present invention into other specific forms without changing the technical idea or essential features of the present invention. The above-described embodiments are merely illustrative in all respects and are not limiting. [Industrial Applicability]

[0067] The present invention is industrially applicable.

Claims

1. A pharmaceutical composition for preventing or treating a degenerative nervous system disease, said pharmaceutical composition comprising colchicine or a pharmaceutically acceptable salt thereof and metformin or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating a degenerative nervous system disease, comprising:

2. The degenerative neurological disease is a disease mediated by abnormal aggregation of TDP-43 protein or accumulation in the cytoplasm due to mislocalization of TDP-43 protein. The pharmaceutical composition for preventing or treating a degenerative nervous system disease according to claim 1.

3. The disease mediated by abnormal aggregation of TDP-43 protein or accumulation in the cytoplasm due to mislocalization of TDP-43 protein is any one selected from the group consisting of frontotemporal dementia, amyotrophic lateral sclerosis, Alexander disease, argyrophilic grain dementia, Guam ALS-parkinsonism-dementia complex, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, facial hemifacial atrophy, Alzheimer's disease, Down syndrome, British familial dementia, Huntington's disease, Machado-Joseph disease, hippocampal sclerosis dementia, inclusion body myositis, oculopharyngeal muscular dystrophy with rimmed vacuoles, muscle fiber radiculopathy, traumatic brain injury, dementia with Lewy bodies, and Parkinson's disease. The pharmaceutical composition for preventing or treating a degenerative nervous system disease according to claim 2.

4. The pharmaceutical composition increases chaperone gene expression, increases autophagy gene expression, and suppresses the abnormal aggregation and mislocalization of TDP-43 protein in diseases mediated by cytoplasmic accumulation due to abnormal aggregation or mislocalization of TDP-43 protein. The pharmaceutical composition for preventing or treating a degenerative nervous system disease according to claim 1.

5. The pharmaceutical composition is formulated into any one of preparation forms selected from oral, rectal, intravenous, intramuscular, intraperitoneal, subcutaneous, intranasal, intrauterine, intracerebrovascular, and transdermal administration. The pharmaceutical composition for preventing or treating a degenerative nervous system disease according to claim 1.

6. The pharmaceutical composition is prepared in any one dosage form selected from the group consisting of powder, pill, granule, tablet, capsule, liquid preparation, emulsion, suspension, emulsion, syrup, aerosol, topical preparation, sterile aqueous solution, non-aqueous solvent, lyophilized preparation, suppository, and sterile injection. The pharmaceutical composition for preventing or treating a degenerative nervous system disease according to claim 1.

7. The pharmaceutical composition further comprises one or more ingredients selected from the group consisting of melatonin, creatine, magnesium, coenzyme Q10, vitamin D, vitamin B, vitamin E, L-serine, L-carnitine, and N-acetylcysteine. The pharmaceutical composition for preventing or treating a degenerative nervous system disease according to claim 1.

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